Proteins Associated with the Intracellular Signalling Tail of the
Calcium-Sensing Receptor and Their Impact on Receptor Function
By
Aaron Magno, B.Sc (Honours)
This thesis is presented for the Degree of Doctor of Philosophy of the University of Western Australia
School of Medicine and Pharmacology
2008
Preface
The experimental work contained within this thesis was conducted in the Department of
Endocrinology and Diabetes, Sir Charles Gairdner Hospital and the Western Australian
Institute for Medical Research, University of Western Australia, under the supervision
of Associate Professor Thomas Ratajczak and Doctor Bryan Ward. All experimental
work presented in this thesis was performed by myself, except for where expressly
stated.
Aaron Magno, B.Sc (Honours)
i
Acknowledgements
I must first express my gratitude to my supervisor Assoc Prof Thomas Ratajczak for
providing me the opportunity to undertake my PhD.
My thanks must also go to my co-supervisor, Dr Bryan Ward, for his guidance and
support throughout the years.
I’d like to express my appreciation to both the members of the Ratajczak lab who have
been a part of my journey since the beginning Dr Rudi Allan, Dr Carmel Cluning and
Dr Danny Mok and the PhD students who have joined more recently, Ajanthy
Arulpragasam and Sarah Rea.
To the Honours students, Bernadette Pederson and Shelby Chew, who have come
through and furthered the CaR studies, I say thankyou.
I must acknowledge the individuals who have provided their expertise, materials and
insight to assist me with my project. Dr Evan Ingley, who provided the yeast two-hybrid
library and advice on examining the identified clones. Assoc Prof Arthur Conigrave, Ed
Nemeth and Donald Ward for supplying the HEK293-CaR stables. The team from
CMCA, Dr Paul Rigby, Kathy Heel-Miller and Tracey Lee-Pullen, for their assistance
with microscopy and cell sorting. Dr Fiona Pixley for her guidance regarding the
cytosketal studies. Dr Kendall Walker for her assistance with the baculoviral
expression. Dr Michael Way for his gift of the testin antibody and Suszanne Brown for
her help with statistical analysis.
I also recognise the financial support provided by Kidney Health Australia and the
National Health and Medical Research Council throughout my time as a student.
Finally, I would like to thank my Mother for eternal support and patience.
ii
Abstract The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to
changes in extracellular calcium and plays an integral role in calcium homeostasis.
Later studies revealed that the CaR was stimulated by not just calcium, but a diverse
range of stimuli and that activation of the receptor regulated a host of different
biological processes. The CaR is linked to these cellular responses via the various
signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have
identified a number of accessory proteins that, through their interaction with the
intracellular tail of the CaR, are able to regulate important functional aspects of the
receptor, including its signalling and degradation. We hypothesised that many more
proteins that bind to the CaR-tail await identification, especially since most of the
previous studies used the yeast two-hybrid system to screen cDNA libraries generated
from tissues that are important to whole body calcium homeostasis, such as the
parathyroid gland and kidney. In order to identify novel binding partners of the CaR,
which may affect its function, particularly in biological processes that might be
unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen
of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular
tail of the human CaR as bait. This screen revealed a large number of “potentially
interacting” clones when plated on selective medium, 130 of which were confirmed as
such using a Lac Z reporter assay.
The aims of this thesis were:
(i) to examine 60 of these “potentially interacting” clones to determine that they were
“true positives” and once confirmed to establish the identity of the interacting proteins
by sequence analysis. Following this, a secondary aim was to establish the region of the
CaR-tail to which these partner proteins bind, using yeast two-hybrid CaR-tail deletion
mapping studies.
(ii) The second aim was to examine in greater detail two of the proteins, filamin A, a
cytoskeletal protein shown previously to interact with the CaR and influence CaR-
mediated cell signalling, and testin, a LIM domain containing, focal adhesion protein
also known to have effects on the cytoskeleton.
This screen revealed a total of seven CaR interacting proteins, namely filamin A,
filamin B, testin, 14-3-3 θ, OS-9, Ubc9 and MPc2. This included six novel CaR binding
iii
partners and an interacting clone of filamin A that was different to that previously
published. This diverse collection of proteins is associated with a variety of functions
that range from the regulation of intracellular signalling, cytoskeletal organisation,
trafficking, degradation, posttranslational modification and transcriptional repression, to
acting as scaffolding proteins. In addition to demonstrating an interaction between these
interacting proteins and the CaR, their binding sites within the CaR-tail were also
mapped using the yeast two-hybrid system. Data from these studies and the known
interaction domains of previously identified CaR binding partners have revealed two
regions of the CaR intracellular tail, 865-922 and 965-986, which appear to be essential
for the interaction of accessory proteins.
The scaffolding protein, filamin A, was previously shown to bind to the CaR-tail and
influence receptor signalling and degradation. However, two distinct library clones
corresponding to filamin A (one isolated directly from the yeast two-hybrid library
screen and one based on the filamin B clone) did not overlap with the previously
identified site of interaction with the CaR. Direct interaction studies performed in vitro
using pulldown assays confirmed that these two regions of filamin A contained novel
sites of direct interaction with the CaR-tail. Sequence alignment between the two CaR
binding domains identified in this study and the previously defined binding domain
revealed a 40 amino acid region that was highly homologous in all three.
The CaR is the first receptor that has been found to interact with testin. Although direct
interaction of CaR and testin was unable to be confirmed due to insolubility of testin
fusion proteins, coimmunoprecipitation and confocal microscopy experiments
demonstrated that the CaR and testin could interact and colocalise in mammalian cells.
Furthermore, the binding of testin was found to occur at the membrane proximal region
of the CaR-tail, a region known to be important for signalling. Mapping studies
indicated that the interaction between the CaR and testin required key residues essential
in maintaining the integrity of the second zinc finger of LIM domain 1, as well as
additional residues of the zinc finger which may also be critical in maintaining its
structure. The overexpression of testin did not alter the level of CaR-mediated ERK
phosphorylation, but was found to enhance the level of CaR-induced Rho kinase
activity. The work of previous studies showing that CaR agonist stimulation of
HEK293-CaR cells caused changes in cell morphology and actin stress fibre assembly,
was replicated, with the additional finding that focal adhesion formation was also
iv
increased upon CaR activation. Surprisingly, unstimulated HEK293-CaR stable cells in
which testin had been knocked down using shRNA technology exhibited the same cell
morphology, actin stress fibre formation and focal adhesion formation as seen in
stimulated wild-type HEK293-CaR cells.
These studies have shown that the CaR is capable of interacting with a much larger
number of accessory proteins than previously known and highlighted two regions of the
receptor’s intracellular tail that contain elements important to partner protein binding.
Further investigations of the interaction between filamin A and the CaR have revealed
multiple sites of interaction suggesting mechanisms by which filamin A may act as a
more versatile scaffolding protein or perhaps a more efficient clamp for the CaR.
Finally, the novel CaR interacting protein, testin, was shown to enhance CaR-induced
Rho signalling and may potentially be involved in CaR-mediated changes to cell
morphology and cytoskeletal reorganisation.
v
Abbreviations
Associated Molecule with SH3 Domain AMSH
Bicinchoninic Acid BCA
Bovine Serum Albumin BSA
Calcium-Sensing Receptor CaR
Calmodulin-Dependent Protein Kinase CaMK
C-Jun NH2 Terminal Kinase JNK
C-Jun NH2 Terminal Kinase Kinase JNKK
Cyclic adenosine monophosphate cAMP
Dimethyl sulphoxide DMSO
Diacylglycerol DAG
Dithiothreitol DTT
Dulbecco’s Modified Eagle Medium DMEM
Endoplasmic Reticulum ER
Endoplasmic Reticulum-Associated Degradation ERAD
Enhanced Green Fluorescent Protein EGFP
Epidermal Growth Factor EGF
EGF Receptor EGFR
Ethylenediaminetetra-acetic acid EDTA
Extracellular Signal Regulated Kinase ERK
Fetal Calf Serum FCS
Familial Hypocalciuric Hypercalcaemia FHH
G-Protein Coupled Receptor GPCR
G Protein Receptor Kinase GRK
Glutathione S-Transferase GST
γ-Aminobutyric AcidB GABAB
Heparin-Binding Epidemral Growth Factor HB-EGF
Hours hr
Horseradish Peroxidase HRP
Inositol Phosphate IP3
Isopropanol β-thiogalactopyranoside IPTG
Lin-11, Isl-1, Mec-3 LIM
Lymphoid Blast Crisis Lbc
Matrix Metalloprotease MMP
Metabotropic Glutamate Receptors mGluR
vi
Minutes min
Mitogen Activated Protein Kinase MAPK
Mouse Tonicity Phosphate Buffered Saline MT-PBS
Neonatal Severe Hyperparathyroidism NSHPT
Nickel-Nitriloacetic acid Ni-NTA
Optical Density OD
Parathyroid Hormone PTH
Parathyroid Hormone-Related Protein PTHrP
Phenylmethylsulphonylflouride PMSF
Phorbol Myrisate Acetate PMA
Phosphate Buffered Saline PBS
Phosphatidyl Inositol 3 Kinase PI3K
Phosphatidyl inositol 4,5-bisphosphonate PIP2
Phospholipase A2 PLA2
Phospholipase C PLC
Phospholipase D PLD
Polymerase Chain Reaction PCR
Prickle, Espinas, Testin PET
Proheparin-Binding Epidemral Growth Factor ProHB-EGF
Protein Kinase C PKC
Protein Kinase A PKA
Receptor-Activity-Modifying Protein RAMP
Reverse Transcriptase Polymerase Chain Reaction RT-PCR
Rho-Guanine Nucleotide Exchange Factor Rho-GEF
Seconds sec
Serum Response Element SRE
Sodium Dodecyl Sulphate SDS
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SDS-PAGE
Transient Receptor Potential Vailloid Group 4 TRPV4
vii
TTaabbllee ooff CCoonntteennttss
Preface i
Acknowledgements ii
Abstract iii
Abbreviations vi
Table of Contents viii
List of Figures xvii
List of Tables xviii
Chapter 1: Introduction
1.1 Discovering the Calcium-Sensing Receptor 1
1.2 The Calcium-Sensing Receptor Gene 2
1.3 The Calcium-Sensing Receptor is a G Protein-Coupled Receptor 2
1.4 Properties of the Calcium-Sensing Receptor 6
1.4.1 Calcium-Sensing Receptor Dimerisation 6
1.5 Calcium-Sensing Receptor Structure 7
1.5.1 The Extracellular Domain 7
1.5.1.1 Bilobed Venus-Flytrap 7
1.5.1.2 Ca2+-Binding Pocket 8
1.5.1.3 Signal Peptide Cleavage Site 9
1.5.1.4 Cysteines 9
1.5.1.5 Peptide Linker 10
15.1.6 N-Linked Glycosylation Sites 10
1.5.2 The Transmembrane Domain 11
1.5.2.1 Membrane Spanning Region 11
1.5.2.2 Intracellular Loops 12
1.5.2.3 Extracellular Loops 13
1.5.2.4 Binding of Allosteric Modulators 13
1.5.3 The Intracellular Tail 14
1.5.3.1 Membrane Proximal Region 14
1.5.3.2 Phosphorylation Sites 16
1.6 Calcium-Sensing Receptor Signalling 17
viii
1.6.1 Calcium-Sensing Receptor Stimuli 17
1.6.1.1 Cations 17
1.6.1.2 Amino Acids 18
1.6.1.3 Pharmacological Agents 18
1.6.1.4 Polyamines 18
1.6.1.5 Polypeptides 19
1.6.1.6 Aminoglycoside Antibiotics 19
1.6.1.7 Ionic Strength 19
1.6.1.8 pH 19
1.6.2 Intracellular Signalling Pathways Regulated by the Calcium-Sensing
Receptor 20
1.6.2.1 Phospholipase Signalling 20
1.6.2.2 Mitogen Activated Protein Kinase Signalling 22
1.6.2.2.1 Extracellular Signal Regulated Kinase 22
1.6.2.2.2 c-Jun NH2 Terminal Kinase 24
1.6.2.2.3 p38 Mitogen Activated Protein Kinase 24
1.6.2.3 Inhibition of Cyclic AMP 25
1.6.2.4 Rho Signalling 25
1.7 The Biological Roles of the Calcium-Sensing Receptor 26
1.7.1 Calcium-Sensing Receptor in the Parathyroid 28
1.7.2 Calcium-Sensing Receptor in the Kidney 28
1.7.3 Calcium-Sensing Receptor in the Gastrointestinal Tract 29
1.7.4 Calcium-Sensing Receptor in Bone 30
1.7.5 Calcium-Sensing Receptor in the Nervous System 31
1.7.6 Calcium-Sensing Receptor in Breast 32
1.7.7 Calcium-Sensing Receptor in Epidermal Cells 33
1.8 Interacting Protein Partners of the Calcium-Sensing Receptor 33
1.8.1 Filamin 33
1.8.2 Potassium Channels 34
1.8.3 Dorfin 35
1.8.4 Associated Molecule with SH3 Domain of STAM (AMSH) 36
1.8.5 Receptor-Activity-Modifying Proteins 36
1.8.6 β-Arrestins 37
1.9 Statement of Aims 38
ix
Chapter 2: Materials and Methods
2.1 Materials 40
2.1.1 Reagents 40
2.1.2 Plasmids 43
2.1.3 Enzymes 43
2.1.4 Cell lines 44
2.1.5 Antibodies 44
2.1.6 Equipment 44
2.1.7 Commercial Suppliers 45
2.2 Methods 46
2.2.1 General Methods 46
2.2.1.1 Tissue Culture Methodology 46
2.2.1.1.1 Maintenance of Cell Lines 46
2.2.1.1.2 Transfection 47
2.2.1.1.3 Lysis of Cultured Mammalian Cells 47
2.2.1.2 Transformation of Competent cells 47
2.2.1.3 Plasmid DNA Preparation 48
2.2.1.4 Quantitation of DNA 49
2.2.1.5 Agarose Gel Electrophoresis 49
2.2.1.6 Purification of DNA 49
2.2.1.6.1 Purification of DNA from Agarose Gels 49
2.2.1.6.2 Purification of DNA Using the QIAquick PCR
Purification Kit 49
2.2.1.7 Ethanol Precipitation of DNA 50
2.2.1.8 Restriction Enzyme Digestion 50
2.2.1.9 Dephosphorylation of 5’-Ends 50
2.2.1.10 Ligations 50
2.2.1.11 Reverse Transcriptase-PCR 51
2.2.1.12 PCRs Using a Proofreading Enzyme 51
2.2.1.13 Site-Directed Mutagenesis 52
2.2.1.14 DNA Sequencing 52
2.2.1.15 Quantification of Protein Concentration Using a BCA
Assay Kit 53
2.2.1.16 Quantification of Protein Concentration Using a Bradford
Assay 53
x
2.2.1.17 Preparation of Gels and Electrophoresis 53
2.2.1.18 Western Blotting 54
2.2.1.19 Densitometry 54
2.2.1.20 Statistical Analysis 55
2.2.2 Identification of Positive Clones from a Yeast Two-Hybrid Library
Screen 55
2.2.2.1 DNA Extraction from Yeast 55
2.2.2.2 Profiling of Plasmids by Restriction Enzyme Digestion 55
2.2.2.3 Plasmid Recovery of Library Clones 56
2.2.2.4 Cotransformation of Bait and Library Plasmids with Yeast
L40 56
2.2.2.5 β-galactosidase Colony Lift Assays 58
2.2.3 Protein Interaction Studies 58
2.2.3.1 Baculoviral Expression and Purification of His-Tagged
CaR-Tail 58
2.2.3.2 Bacterial Expression and Purification of His-Tagged
CaR-Tail 59
2.2.3.3 Bacterial Expression and Purification of Glutathione S- 60
Transferase (GST)-Fusion Proteins
2.2.3.4 Alternate Purification Method for GST-Testin 61
2.2.3.5 Pulldown Assay with His-Tagged CaR-Tail 61
2.2.3.6 Staining of Polyacrylamide Gels 62
2.2.3.7 Coimmunoprecipitation 62
2.2.4 Confocal Microscopy 63
2.2.4.1 Detection of CaR-FLAG by Confocal Microscopy 63
2.2.5 Detection of Signalling Pathway Activity 63
2.2.5.1 ERK Assay 63
2.2.5.2 SRE-Luciferase Assay 64
2.2.6 Generation of the Testin Knockdown HEK293-CaR Stable Cell Line 65
2.2.6.1 Cloning of Knockdown Target Sequence 65
2.2.6.2 Generating the Stable Packaging Cell Line 65
2.2.6.3 Retroviral Infection of HEK293-CaR Stable Cell Lines 65
2.2.6.4 Enrichment of EGFP-positive Cells and Verification of
Testin Knockdown 66
2.2.7 Studies of Morphological and Cytoskeletal Changes 67
xi
Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the
Chapter 3: Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen
3.1 Introduction 70
3.2 Results 72
3.2.1 Verification of Clones 72
3.2.2 Mapping of Verified Interacting Proteins of the CaR 73
3.2.2.1 Filamin A 76
3.2.2.2 Filamin B 76
3.2.2.3 Testin 76
3.2.2.4 14-3-3 θ 80
3.2.2.5 OS-9 80
3.2.2.5 Ubc9 80
3.2.2.6 MPc2 80
3.3 Discussion 85
3.3.1 Filamins 85
3.3.2 Testin 87
3.3.3 14-3-3 θ 92
3.3.4 OS-9 93
3.3.5 Ubc9 95
3.3.6 MPc2 97
Chapter 4: Investigating the Interaction Between the Intracellular Tail of the
Chapter 4: Calcium-Sensing Receptor and Filamin
4.1 Introduction 98
4.2 Results 99
4.2.1 Construction of Filamin A GST-Fusion Proteins for Pulldown
Studies 99
4.2.2 Purification of His-tagged CaR-tail from Insect Cells 101
4.2.3 Pulldown Assays Performed Using His-tagged CaR-tail Purified
from Insect cells 101
4.2.4 Pulldown Assays Performed Using His-tagged CaR-tail Purified
from Bacteria 104
4.3 Discussion 104
xii
Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the
Calcium-Sensing Receptor and Testin and the Implications for Cell Function
5.1 Introduction 110
5.2 Results 111
5.2.1 Calcium-Sensing Receptor and Testin Interaction Studies 111
5.2.1.1 Yeast Two-Hybrid Mapping 111
5.2.1.2 Cloning of Full-Length Human Testin 113
5.2.1.3 Direct Interaction Studies 114
5.2.1.4 Coimmunoprecipitation Studies 114
5.2.2 Colocalisation of Testin and the Calcium-Sensing Receptor 117
5.2.3 The Effects of Testin on Calcium-Sensing Receptor Activated 117
ERK Signalling
5.2.4 The Effects of Testin on Calcium-Sensing Receptor-Mediated
Rho Signalling 120
5.2.5 The Calcium-Sensing Receptor Regulates Changes in Cell
Morphology 122
5.2.6 The Impact of Testin Knockdown on HEK293 Cells Stably
Expressing the Calcium-Sensing Receptor 126
5.3 Discussion 133
5.3.1 The Calcium-Sensing Receptor and Testin Interaction 133
5.3.2 Sites of Interaction Between the Calcium-Sensing Receptor and
Testin Identified in the Yeast Two-Hybrid System 133
5.3.3 Calcium-Sensing Receptor and Testin Interaction Studies 134
5.3.4 The Effects of Testin Binding on Calcium-Sensing Receptor
Regulated Signalling 135
5.3.4.1 Calcium-Sensing Receptor-Mediated ERK Phosphorylation
is Unaffected by Testin Overexpression 135
5.3.4.2 Testin Accentuates Calcium-Sensing Receptor-Mediated
Rho Kinase Activity 136
5.3.5 The Relationship Between Cell Morphology and the Calcium-
Sensing Receptor’s Interaction with Testin 136
xiii
Chapter 6: General Discussion
6.1 The Calcium-Sensing Receptor 139
6.2 Interacting Protein Partners of the Calcium-Sensing Receptor 141
6.3 Interacting Protein Partners of the Calcium-Sensing Receptor Regulate its
Function 141
6.3.1 The Effect of Interacting protein partners on Calcium-Sensing
Receptor Dimerisation 142
6.3.2 The Regulation of Calcium-Sensing Receptor Trafficking by
Interacting Proteins 143
6.3.3 The Regulation of Calcium-Sensing Receptor Degradation by
Interacting Proteins 143
6.3.4 Calcium-Sensing Receptor-Mediated Intracellular Signalling is 144
Directed by Interacting Proteins
6.2.5 The Role of the Calcium-Sensing Receptor and its Binding Partners
in Cell Morphology and Organisation of the Cytoskeleton 146
6.4 Future Studies
6.4.1 Filamin A 147
6.4.2 Filamin B 147
6.4.3 Testin 148
6.5 Conclusions 149
Chapter 7: References 150
Appendices
Appendix 1: Oligonucleotides 172
Appendix 2: Anitbodies and Western Blotting Conditions 174
xiv
LLiisstt ooff FFiigguurreess
Chapter 1: Introduction
Figure 1.1: A comparison of the amino acid sequences of mammalian CaRs 3
Figure 1.2: CaR-mediated signalling pathways 21
Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the
Chapter 3: Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen
Figure 3.1 A schematic representation of the yeast two-hybrid screen using the
LexA system using the intracellular tail of the CaR as bait. 71
Figure 3.2 Profiling of library screen clones by PCR amplification and restriction
enzyme analysis. 74
Figure 3.3 Sites of interaction for the CaR-tail and filamin A identified in the
yeast two-hybrid system. 77
Figure 3.4 Sites of interaction for the CaR-tail and filamin B identified in the
yeast two-hybrid system. 78
Figure 3.5 Sites of interaction between the CaR and testin identified with the
yeast two-hybrid system. 79
Figure 3.6 Sites of interaction between the CaR-tail and 14-3-3q identified in a
yeast two-hybrid library screen. 81
Figure 3.7 Sites of interaction between the CaR and OS-9 identified with the
yeast two-hybrid system. 82
Figure 3.8 Sites of interaction between the CaR-tail and Ubc9 identified in a
yeast two-hybrid library screen. 83
Figure 3.9 Sites of interaction between the CaR-tail and MPc2 identified in a
yeast two-hybrid library screen. 84
Figure 3.10: A comparison of the amino acid sequence of testin from different
mammalian species. 89
Figure 3.11: Comparison of LIM domains. 90
Figure 3.12 The SUMOylation and ubiquitination pathways. 96
xv
Chapter 4: Investigating the Interaction Between the Intracellular Tail of the
Chapter 4: Calcium-Sensing Receptor and Filamin
Figure 4.1: Alignment of the filamin B fragment that binds to the CaR-tail with
its filamin A counterpart. 100
Figure 4.2: Purification of baculoviral His-tagged CaR-tail from Sf21 insect cells. 102
Figure 4.3: In vitro interaction studies between GST-tagged filamin A fragments
and His-tagged CaR-tail purified from insect cells. 103
Figure 4.4: In vitro interaction studies between GST-tagged filamin A fragments
and His-tagged CaR-tail purified from bacteria. 105
Figure 4.5: A comparison of the amino acid sequence of the identified
CaR-binding sites within human filamin A. 107
Figure 4.6 Schematic representations of proposed roles of multiple CaR binding
sites within filamin A. 109
Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the
Chapter 5: Calcium-Sensing Receptor and Testin and the Implications for Cell
Chapter 5: Function
Figure 5.1: An alanine scan of the second zinc-finger of LIM domain 1 of testin
using the yeast two-hybrid system. 112
Figure 5.2 Examination of the expression and solubility of testin fusion proteins. 115
Figure 5.3 Coimmunoprecipitation of CaR-FLAG and EGFP-testin. 116
Figure 5.4: Colocalisation of CaR-FLAG and EGFP-testin in HEK293 cells. 118
Figure 5.5: The effect of testin on ERK phosphorylation in HEK293 cells stably
expressing the CaR following stimulation with extracellular calcium. 119
Figure 5.6: The effect of testin on ERK phosphorylation in HEK293 cells stably
expressing the CaR following stimulation with extracellular calcium in the
presence of an allosteric modulator. 121
Figure 5.7: The effect of testin on Rho kinase activity measured in either wild-type
HEK293 cells or HEK293 cells stably expressing the CaR. 123
Figure 5.8 Effects of magnesium stimulation on the morphology of HEK293 cells
stably expressing the CaR. 124
Figure 5.9: Detection of actin stress fibres and focal adhesions in HEK293 cells
stably expressing the CaR when exposed to different concentrations of
magnesium. 125
Figure 5.10: Detection of testin in lysates from normal HEK293-CaR stable cells
xvi
or those expressing testin knockdown shRNA by Western analysis. 127
Figure 5.11: Rho kinase activity measured in either wild-type HEK293-CaR stable
cells or testin knockdown HEK293-CaR cells. 127
Figure 5.12: Comparative cellular morphology of wild-type and testin knockdown
HEK293 cells stably expressing the CaR. 129
Figure 5.13: Detection of actin stress fibres in wild-type and testin knockdown
HEK293 cells stably expressing the CaR. 130
Figure 5.14: Detection of focal adhesions in wild-type and testin knockdown 131
HEK293 cells stably expressing the CaR.
Chapter 6: General Discussion
Figure 6.1: A simplistic overview of the translation of extracellular stimuli into an
intracellular response by the CaR. 140
xvii
LLiisstt ooff TTaabblleess Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the
Chapter 3: Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen
Table 3.1 Protein interacting partners of the CaR-tail identified in a yeast two-hybrid screen of a haemopoietic cell line library with their comparative binding strengths to the CaR-tail and various CaR-tail truncations. 75 Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the
Chapter 5: Calcium-Sensing Receptor and Testin and the Implications for Cell
Chapter 5: Function
Table 5.1: Observed effects of the knockdown of testin in HEK293 cells stably expressing the CaR. 132
xviii
CChhaapptteerr 11 Introduction
1.1 Discovering the Calcium-Sensing Receptor
The importance of calcium ions in the regulation of physiological functions has been
known since the 19th century, when Sydney Ringer serendipitously discovered that
calcium was essential for the contraction of isolated hearts (Ringer 1883). From that
early discovery, the importance of calcium in biological systems and the necessity for
organisms to tightly regulate calcium homeostasis has been firmly established (Carafoli
2003; Chang and Shoback 2004). Systemic calcium homeostasis is maintained through
the secretion of hormones in response to extracellular calcium and the consequent
actions of these hormones on various tissues to normalise extracellular calcium by
altering the levels of calcium released or absorbed by the affected tissues (Brown 1999).
One such hormone involved in regulating serum calcium levels is the parathyroid
hormone (PTH). Experiments using a radioimmunoassay to detect PTH levels in whole
animals revealed that an increase in serum calcium resulted in a decrease in PTH and
that a decrease in serum calcium caused an increase in PTH levels (Sherwood et al.
1966). The same radioimmunoassay was subsequently used to demonstrate that the
inverse relationship between extracellular calcium and PTH was also observed in
isolated parathyroid glands (Care et al. 1966). It was observed in an
electrophysiological study that extracellular calcium, even in the presence of a calcium
channel blocker, had a depolarising effect on parathyroid cells in a similar manner as
the inverse relationship between extracellular calcium and PTH (Lopez-Barneo and
Armstrong 1983). When dispersed parathyroid cells were exposed to increasing levels
of extracellular calcium, a relative increase in the levels of cytosolic calcium was
detected, which correlated with the suppression of PTH secretion from the treated cells
(Shoback et al. 1983). Further studies revealed that increases in extracellular calcium
increased the production of both inositol phosphate (IP3) and diacylglycerol (DAG),
which are recognised as components of general mechanisms of receptor mediated
intracellular calcium mobilisation (Brown et al. 1987; Kifor and Brown 1988; Shoback
et al. 1988). The data from these studies provided compelling evidence that there was a
receptor at the cell surface of parathyroid cells sensitive to extracellular calcium that
regulated PTH secretion through the mobilisation of intracellular calcium (Nemeth and
Carafoli 1990). In 1993, a receptor exhibiting these traits, the calcium-sensing receptor,
1
was cloned (Brown et al. 1993). The calcium-sensing receptor has two widely used
abbreviations, CaR and CaSR. Throughout this thesis the former will be used.
1.2 The Calcium-Sensing Receptor Gene
The first CaR identified was cloned from a bovine parathyroid gland cDNA library
(Brown et al. 1993). The isolated clone was 5,276 bp long, with an open reading frame
of 3,255 bp that encoded a protein of 1,085 amino acids and when expressed in Xenopus
laevis oocytes displayed a pharmacological profile similar to that observed in
parathyroid cells (Brown et al. 1993). The human CaR equivalent was cloned from
adenomatous parathyroid gland in 1995 and as with the bovine CaR, pharmacological
characteristics resembling those detected in parathyroid cells were observed in Xenopus
laevis oocytes expressing the human CaR (Garrett et al. 1995). The isolated human CaR
cDNA consists of seven exons, the first of which is a 5’-untranslated region while the
other six encode for a protein of 1078 amino acids with 93% identity to the bovine CaR
(Garrett et al. 1995). The identification of a promoter containing TATA and CAAT
boxes and a second GC-rich promoter without a TATA box in the human CaR suggest
that multiple CaR mRNAs may be produced through tissue specific regulation of the
two promoters (Chikatsu et al. 2000). With a known sequence for the human CaR it was
possible to map the CaR gene to human Chromosome 3q13.3-21 using fluorescence in
situ hybridisation (Janicic et al. 1995b). Besides the bovine and human, the CaR has
been found in a wide variety of vertebrates including rat (Riccardi et al. 1995), rabbit
(Butters et al. 1997), mouse (Oda et al. 2000), dog (Skelly and Franklin 2007), chicken
(Diaz et al. 1997), salamander (Cima et al. 1997) and several types of fish (Loretz
2008). Figure 1.1 contains a comparison of the protein sequences of selected
mammalian CaRs.
1.3 The Calcium-Sensing Receptor is a G Protein-Coupled Receptor
After extrapolating the amino acid sequence of the bovine CaR from the isolated cDNA
clone it was found that the CaR shared significant homology to the metabotropic
glutamate receptors (mGluR), which are part of the G protein-coupled receptor (GPCR)
superfamily (Brown et al. 1993). The GPCR superfamily is a diverse group of
membrane bound receptors involved in signal transduction containing 1000 to 2000
members in vertebrates that constitute over 1% of the genome (Bockaert and Pin 1999).
Phylogenetic analysis has been used to classify this superfamily of distinct proteins into
five families; Rhodopsin, Secretin, Adhesion, Glutamate and Frizzled/Taste2
2
Human 1 MAFYSCCWVLLALT-WHTSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Bovine 1 MALYSCCWILLAFSTWCTSAYGPDQRAQKKGDIILGGLFPIHFGV 45 Rat 1 MASYSCCLALLALA-WHSSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Dog 1 MAFHSCSLILLAIT-WCTSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Mouse 1 MAWFGYCLALLALT-WHSSAYGPDQRAQKKGDIILGGLFPIHFGV 44
Human 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89 Bovine 46 AVKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 90 Rat 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPSLLP 89 Dog 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89 Mouse 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89
Loop I
Human 90 NLTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Bovine 91 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 135 Rat 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Dog 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Mouse 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134
Loop II
Human 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Bovine 136 IPSTIAVVGATGSGISTAVANLLGLFYIPQVSYASSSRLLSNKNQ 180 Rat 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Dog 135 IPSTIAVVGATGSGISTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Mouse 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Human 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Bovine 181 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 225 Rat 180 YKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Dog 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Mouse 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Human 225 KFREEAEERDICIDFSELISQYSDEEEIQHVVEVIQNSTAKVIVV 269 Bovine 226 KFREEAEERDICIDFSELISQYSDEEKIQQVVEVIQNSTAKVIVV 270 Rat 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Dog 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Mouse 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Human 270 FSSGPDLEPLIKEIVRRNITGKIWLASEAWASSSLIAMPQYFHVV 314 Bovine 271 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 315 Rat 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Dog 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Mouse 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Human 315 GGTIGFALKAGQIPGFREFLKKVHPRKSVHNGFAKEFWEETFNCH 359 Bovine 316 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 360 Rat 315 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359 Dog 315 GGTIGFALKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359 Mouse 315 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359
Human 360 LQEGAKGPLPVDTFLRGHEESGDRFSNSSTAFRPLCTGDENISSV 404 Bovine 361 LQEGAKGPLPVDTFLRGHEEGGARLSNSPTAFRPLCTGEENISSV 405 Rat 360 LQEGAKGPLPVDTFVRSHEEGGNRLLNSSTAFRPLCTGDENINSV 404 Dog 360 LQEGAKGPLSMDTFLRGHEEGGGRISNSSTAFRPLCTGDENISSV 404 Mouse 360 LQDGAKGPLPVDTFVRSHEEGGNRLLNSSTAFRPLCTGDENINSV 404
Loop III Human 405 ETPYIDYTHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Bovine 406 ETPYMDYTHLRISYNVYLAVYSIAHALQDIYTCIPGRGLFTNGSC 450 Rat 405 ETPYMDYEHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Dog 405 ETPYMDYTHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Mouse 405 ETPYMGYEHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449
Loop IV Human 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494 Bovine 451 ADIKKVEAWQVLKHLRHLNFTSNMGEQVTFDECGDLAGNYSIINW 495 Rat 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494
Dog 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLMGNYSIINW 494 Mouse 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494 Human 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREVPF 539 Bovine 496 HLSPEDGSIVFKEVGYYNVYAKKGERLFINDEKILWSGFSREVPF 540 Rat 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREVPF 539 Dog 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREMPF 539 Mouse 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEGKILWSGFSREVPF 539 Human 540 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACNK 584 Bovine 541 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACDK 585 Rat 540 SNCSRDCQAGTRKGIIEGEPTCCFECVECPDGEYSGETDASACDK 584 Dog 540 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACDK 584 Mouse 540 SNCSRDCQAGTRKGIIEGEPTCCFECVECPDGEYSGETDASACDK 584 Human 585 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 629 Bovine 586 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 630 Rat 585 CPDDFWSNENHTSCIAKEIEFLAWTEPFGIALTLFAVLGIFLTAF 629 Dog 585 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 629 Mouse 585 CPDDFWSNENYTSCIAKEIEFLAWTEPFGIALTLFAVLGIFLTAF 629
TM1
Human 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Bovine 631 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 675 Rat 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Dog 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Mouse 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674
TM2
Human 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Bovine 676 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 720 Rat 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Dog 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Mouse 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719
TM3
Human 720 GLNLQFLLVFLCTFMQIVICVIWLYTAPPSSYRNQELEDEIIFIT 764 Bovine 721 GLNLQFLLVFLCTFMQIVICAIWLNTAPPSSYRNHELEDEIIFIT 765 Rat 720 GLNLQFLLVFLCTFMQILICIIWLYTAPPSSYRNHELEDEIIFIT 764 Dog 720 GLNLQFLLVFLCTFMQIVICVIWLYTAPPSSYRNHELEDEIIFIT 764 Mouse 720 GLNLQFLLVFLCTFMQIVICIIWLYTAPPSSYRNHELEDEIIFIT 764
TM4
Human 765 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Bovine 766 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 810 Rat 765 CHEGSLMALGSLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Dog 765 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Mouse 765 CHEGSLMALGSLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809
TM5
Human 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Bovine 811 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 855 Rat 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Dog 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Mouse 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854
TM6 TM7 Human 855 NKIYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRKRS 899 Bovine 856 NKVYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRQRS 900 Rat 855 NKVYIILFKPSRNTIEEVRSSTAAHAFKVAARATLRRPNISRKRS 899 Dog 855 NKVYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRKRS 899 Mouse 855 NKVYIILFKPSRNTIEEVRSSTAAHAFKVAARATLRRPNISRKRS 899
Human 900 SSLGGSTGSTPSSSISSKSNSEDPFPQ—-PERQKQQQPLALTQQE 942 Bovine 901 SSLGGSTGSTPSSSISSKSNSEDPFPQQQPKRQKQPQPLALSPHN 945 Rat 900 SSLGGSTGSIPSSSISSKSNSEDRFPQ--PERQKQQQPLSLTQQE 942 Dog 900 GSLGGSTGSTPSSSISSKSNSEDPFPQ--PERQKQQQPLALTQRE 942 Mouse 900 SSLGGSTGSNPSSSISSKSNSEDRFPQ--PERQKQQQPLALTQQE 942
Human 943 -QQQQP---LTLPQQQRSQ-QQPRCKQKVIFGSGTVTFSLSFDEP 982 Bovine 946 AQQPQPRPPSTPQPQPQSQ-QPPRCKQKVIFGSGTVTFSLSFDEP 989 Rat 943 –QQQQP---LTLHPQQQQQPQQPRCKQKVIFGSGTVTFSLSFDEP 983 Dog 943 QQPPQP---LTLPPQPQ-----PRCKQKVIFGSGTVTFSLSFDEP 979 Mouse 943 -QQQQP---LTLQPQQQQQPQQPRCKQKVIFGSGTVTFSLSFDEP 983 Human 983 QKNAMAHRNSTHQNSLEAQKSSDTLTRHQPLLPLQCGETDLDLTV 1027 Bovine 990 QKTAVAHRNSTHQTSLEAQKNNDALTKHQALLPLQCGETDSELTS 1034 Rat 984 QKNAMAHRNSMRQNSLEAQRSNDTLGRHQALLPLQCADADSEMTI 1028 Dog 980 QKSAAAPRNSTLQHSLEAQRSPEPPARPQALLPPQGGDTDAELPA 1024 Mouse 984 QKNAMAHRNSMRQNSLEAQKSNDTLNRHQALLPLQCAEADSEMTI 1028 Human 1028 QETGLQGPVGGDQRPEVEDPEELSPALVVSSSQSFVISGGGSTVT 1072 Bovine 1035 QETGLQGPVGEDHQLEMEDPEEMSPALVVSNSRSFVISGGGSTVT 1079 Rat 1029 QETGLQGPMVGDHQPEMESSDEMSPALVMSTSRSFVISGGGSSVT 1073 Dog 1025 QEPGLQGPGGADRRPEMRDPEELSPALVVSSSQSFVISGGGSTVT 1069 Mouse 1029 QETGLQGPMVGDHQPEIESPDEMSPALVVSTSRSFVISGGGSSVT 1073 Human 1073 ENVVNS 1078 Bovine 1080 ENMLRS 1085 Rat 1074 ENVLHS 1079 Dog 1070 ENILHS 1075 Mouse 1074 ENILHS 1079 Figure 1.1: A comparison of the amino acid sequences of mammalian CaRs. The sequences of human, bovine, rat, dog and mouse have been aligned with conserved residues highlighted in black ( X ). The site of signal peptide cleavage has been highlighted in blue ( X ). The 19 conserved cysteines in the extracellular domain have been highlighted in green ( X ). The three predicted sites of phosphorylation in the intracellular tail have been highlighted in yellow ( X ). The loops within the Venus-flytrap are indicated with a red line (―).The regions of the transmembrane domain that span the membrane are indicated with a blue line (―).
(Fredriksson et al. 2003). The Glutamate family, named after the first GPCR identified
of this class, the mGluR, is alternatively known as Family C or 3 and is characterised by
members that have very large extracellular domains (Bockaert and Pin 1999). While the
CaR was the first member of Family C identified that differed from the mGluR, the
number of family members has grown to the point that Family C has been subdivided
into the following six subgroups; mGluRs, CaRs, γ-aminobutyric acidB receptors
(GABAB), pheromone receptors, taste receptors and orphan receptors (Pin et al. 2003).
1.4 Properties of the Calcium-Sensing Receptor
As the receptor’s name suggests, the principal physiological ligand of the CaR is
calcium (Brown et al. 1994). It has been hypothesised that 3 to 5 Ca2+ ions can bind
cooperatively to the CaR, based on the Hill coefficient (Quinn et al. 2004). Detection of
the CaR by Western blotting revealed that the receptor produced three different protein
bands between 100 and 200 kDa that represent different monomeric forms of the CaR
(Bai et al. 1996). The lowest band, at ~120 kDa, is a non-glycosylated form of the CaR
that is expressed at a much lower level than the other two forms and is not always
detected in Western blots (Bai et al. 1996). A doublet of bands equivalent to molecular
masses of ~130-140 and ~150-160 kDa correspond to the immature form of CaR, which
is glycosylated with carbohydrates containing high mannose content, and a mature form
of the receptor, which is glycosylated with complex carbohydrates, respectively (Bai et
al. 1996). Only the mature form of the receptor is expressed at the cell surface (Bai et al.
1998a). Frequently, Western blotting would also detect bands greater than 200 kDa that
were eventually shown to represent dimeric and oligomeric forms of the receptor (Bai et
al. 1998a).
1.4.1 Calcium-Sensing Receptor Dimerisation
Bai et al. demonstrated that the CaR was normally expressed at the cell surface as a
homodimer, although there have been instances where the CaR has been detected in
heterodimeric complexes with another receptor, such as the mGluR (Bai et al. 1998a;
Gama et al. 2001). The endoplasmic reticulum (ER) has been identified as the site of
CaR dimer formation and dimerisation is essential but not sufficient, for the release of
the receptor from the ER (Pidasheva et al. 2006). Although CaR dimerisation occurs
prior to cell surface expression, studies of detergent solubilised CaR indicated that the
receptor undergoes conformational changes after binding to cations that favoured the
6
oxidation of free sulfhydryl groups and promoted CaR dimer formation, suggesting
ligand binding may have a stabilising effect on the receptor dimer (Ward et al. 1998).
1.5 Calcium-Sensing Receptor Structure
As a GPCR the CaR is comprised of the three main structural features of this receptor
family, an extracellular domain, a seven-transmembrane domain and an intracellular tail
(Brown et al. 1993). Studies examining both the biochemical and functional properties
of the CaR have provided insight into how the receptor’s three distinct structural regions
influence its expression, dimerisation and function (Bai 2004; Hu and Spiegel 2007).
Characteristics of the three domains will be discussed below, primarily in relation to the
human CaR.
1.5.1 The Extracellular Domain
As with all the members of GPCR Family C, the CaR contains a very large extracellular
domain, which covers 612 amino acids and includes two clusters of acidic residues at
amino acids 216-251 and 557-611 (Garrett et al. 1995). The importance of the
extracellular domain to the function of the CaR is highlighted by the fact that the
majority of naturally occurring mutations identified in subjects with calcium
homeostatic disorders are located in this domain (Bai 2004). While the extracellular
domain of the CaR can be subdivided into a bilobed Venus-flytrap and a cysteine-rich
domain, it also contains a number of other structural elements which will be discussed
below (Bai 2004).
1.5.1.1 Bilobed Venus-Flytrap
The amino acid sequence of the extracellular domain of the human CaR was aligned
with that of the mGluR and the bacterial periplasmic-binding protein, from which the
extracellular domains of the Family C GPCRs are proposed to be derived, in order to
better understand the properties of this region (Ray et al. 1999). The resultant model
consisting of amino acids 36-513 of the CaR produced a bilobed Venus-flytrap structure
with the N-terminal Lobe I connected to the C-terminal Lobe II by three strands (Ray et
al. 1999). Although the Venus-flytrap structure can exist in two states-opened and
closed, it has been proposed that ligand binding stabilises the closed state and triggers
the transmission of signal from the extracellular to the transmembrane domain
(Parmentier et al. 2002). Both the CaR and mGluR contain four segments within Lobe I
that do not align with the sequence of the bacterial periplasmic-binding protein and are
7
modelled as unstructured loops designated I-IV (Ray et al. 1999). Following the
determination of the three-dimensional crystal structure of the mGluR1 in both the
unliganded and ligand-bound forms, it was noted that Loops I, III and IV connected
regions of secondary structure within the extracellular domain (Kunishima et al. 2000).
Loop I consists of residues 39-67 and based on the proposed structural model connects a
β-sheet to an α-helix (Reyes-Cruz et al. 2001). CaR constructs with deletion of residues
48-59 and 50-59 were expressed at equivalent levels to the wild-type CaR, but had
reduced biological activity, while a CaR construct lacking residues 42-47 was unable to
respond to extracellular calcium and was only expressed as the incompletely processed
130 kDa form that did not reach the cell surface (Reyes-Cruz et al. 2001). Residues 117-
137 form Loop II and deletion of this region severely diminished biological activity of
the receptor with only a very minor fraction of the mutated receptor reaching the cell
surface (Reyes-Cruz et al. 2001). The longest of the loops is Loop III, which covers
residues 356-416 and a deletion construct removing residues 365-385 had no impact on
the receptor’s biological activity and expression (Reyes-Cruz et al. 2001). Loop IV is
the shortest loop spanning only 12 residues between 437 and 449 (Reyes-Cruz et al.
2001). The three CaR constructs with deletions of residues 438-445, 440-444 and 447-
453 all expressed at levels equivalent to wild-type, but all had a lower biological
activity (Reyes-Cruz et al. 2001).
1.5.1.2 Ca2+-Binding Pocket
It has been proposed that like the mGluRs the extracellular domain of the CaR contains
the sites of ligand-binding (Brauner-Osborne et al. 1999; Reyes-Cruz et al. 2001).
Identification of the residues of the CaR involved in Ca2+-binding has been hindered by
the lack of both a crystal structure for the receptor and a method to directly measure
Ca2+-binding to the receptor (Hu and Spiegel 2003). Several groups have generated
models based on the x-ray structures of a related Family C GPCR, the mGluR1, to help
identify the Ca2+-binding pocket (Huang et al. 2007b; Silve et al. 2005). Silve et al.
proposed that residues Ser147, Ser170, Asp190, Gln193, Tyr218, Phe270, Ser296 and
Glu297 were critical to the binding of Ca2+ in the CaR (Silve et al. 2005). In
experiments where these amino acids, with the exception of Glu297, had been mutated
to alanines it had been shown that CaR activity was impaired (Silve et al. 2005; Zhang
et al. 2002)). Glu297 was not mutated to an alanine but instead the mutations E297K
and E297D were made to mimic the naturally occurring mutations detected in patients
with FHH and ADH, respectively (Pollak et al. 1993; Silve et al. 2005). Experiments
8
examining the functionality of these mutant CaRs revealed that the E297K mutation
impaired the function of the CaR, while the E297D mutation enhanced the receptor’s
activity (Silve et al. 2005). In the model proposed by Huang et al. there are three
predicted sites of Ca2+-binding within the extracellular domain (Huang et al. 2007b).
The first site is in lobe 2 and contains the residues Glu224, Glu228, Glu229, Glu231
and Glu232. At the second site, located in lobe 1, the residues Glu378, Glu379, Thr396,
Asp398 and Glu399 have been indicated as key residues involved in Ca2+-binding
(Huang et al. 2007b). Ser147, Ser170, Asp190, Tyr218 and Glu297 are predicted to be
the amino acids that are important to Ca2+-binding at the third site that is positioned in a
crevice between the two lobes. It should be noted that the five key residues for Ca2+-
binding identified at the third site in the model from Huang et al. were also identified as
being critical for Ca2+-binding in the model presented by Silve et al. (Huang et al.
2007b; Silve et al. 2005). Experiments using CaR constructs in which glutamates had
been mutated to isoleucines revealed that mutation of the residues Glu228, Glu229,
Glu224, Glu378, Glu379 and Glu297 impaired CaR function while a mutation of
Glu398 and Glu399 enhanced CaR responsiveness (Huang et al. 2007b). To further
validate the model put forward by Huang et al, fragments of the CaR containing the first
(Gly222-Ile235) and second (Gly383-Ile408) putative Ca2+-binding sites were inserted
into the CD2 protein to be used in metal binding studies (Huang et al. 2007b). These
studies indicated that with the addition of the CaR fragments the CD2 fusion proteins
were capable of binding metal ions, but alanine mutation of residues of the CaR
fragments predicted to be critical for Ca2+-binding led to weaker metal ion binding
(Huang et al. 2007b).
1.5.1.3 Signal Peptide Cleavage Site
A site of signal peptide cleavage was identified at Tyr20 (Figure 1.1) through amino
acid sequencing of the N-terminus of a CaR extracellular domain that had been
expressed and purified from HEK293 cells, a cell line that does not endogenously
express the receptor (Goldsmith et al. 1999).
1.5.1.4 Cysteines
While there is a cluster of cysteines between residues 542 and 598, there are a total of
19 highly conserved cysteines spread throughout the extracellular domain of the CaR
following the signal peptide cleavage site as indicated in Figure 1.1 (Fan et al. 1998).
The expression in HEK293 cells of 19 mutant CaR constructs, in which individual
9
cysteines of the extracellular domain had been mutated to serines, revealed that 14
cysteines (Cys60, Cys101, Cys236, Cys358, Cys395, Cys542, Cys546, Cys561,
Cys562, Cys565, Cys568, Cys582, Cys585 and Cys598), which are conserved in
mGluR, were critical to the cell surface expression and biological activity of the
receptor (Fan et al. 1998). Further studies using CaR mutants with cysteine to serine
substitutions revealed that a single residue change was not sufficient to disrupt the
covalent disulfide bonds which mediate the formation of receptor dimers (Pace et al.
1999; Ray et al. 1999). However, two groups demonstrated that the mutation of two
cysteines, as in the case of the C101S/C239S and C129S/C131S CaR constructs,
eliminated dimerisation (Pace et al. 1999; Ray et al. 1999). A later study confirmed that
the combination of Cys129 and C131S mutations leads to the CaR losing the capacity to
form disulfide bonds, but that this was not sufficient to disrupt dimerisation, suggesting
that the CaR is also able to dimerise through other intermolecular interactions (Zhang et
al. 2001). With the exception of the GABAB receptors, all members of the GPCR
Family C contain nine cysteines that are located near the C-terminal end of the
extracellular domain in a region that has been referred to as the nine-cysteine domain of
family 3 GPCRs (Yu et al. 2004). In the CaR this region has been shown to be critical
for CaR-mediated signalling (Hu et al. 2000).
1.5.1.5 Peptide Linker
Immediately following this cysteine-rich domain there are 14 amino acids, residues
599-612, that have been described as a peptide linker connecting the extracellular
domain to the seven transmembrane domain (Ray et al. 2007). While all members of the
GPCR Family C with a nine-cysteine domain contain a peptide linker that is 14 amino
acids long, there is variability between the sequence of the peptide linker of each
member (Ray et al. 2007). Insertion or deletion of amino acids within the peptide linker
of the CaR had a negative impact on the cell surface expression of the receptor and
abrogated its signalling capacity in response to extracellular calcium (Ray et al. 2007).
The CaR’s cell surface expression and signalling was impaired by the substitution of an
alanine at Leu606 but not by the replacement of the CaR’s peptide linker with the 14
amino acids corresponding to the mGluR’s peptide linker (Ray et al. 2007).
15.1.6 N-Linked Glycosylation Sites
Upon cloning of the human CaR, 11 putative N-linked glycosylation sites, Asn-Xaa-
Ser/Thr, were identified in its extracellular domain, the majority of which are highly
10
conserved amongst species (Garrett et al. 1995; Ray et al. 1998). Indirect evidence from
experiments using CaR constructs with natural occurring mutations showed that
glycosylation of the receptor was necessary for the CaR to be fully biologically active
(Bai et al. 1996). In later studies it was revealed that inhibition of glycosylation of the
CaR by tunicamycin treatment blocked normal cell surface expression of the receptor,
which would impair its biological activity, as seen in the experiments using mutated
CaR constructs (Fan et al. 1997). Glycosylation of eight conserved N-linked
glycosylation sites, Asn90, Asn130, Asn261, Asn287, Asn446, Asn468, Asn488 and
Asn541, was demonstrated to be important for cell surface expression in experiments
using CaR constructs in which the asparagines of predicted N-linked glycosylation sites
had been substituted with glutamines (Ray et al. 1998). Further experiments with
mutant CaR constructs containing multiple asparagine to glutamine substitutions
revealed that glycosylation at a minimum of three sites was necessary for cell surface
expression of the receptor (Ray et al. 1998).
1.5.2 The Transmembrane Domain
1.5.2.1 Membrane Spanning Region
Garrett et al. proposed that the transmembrane domain of the human CaR spans residues
613-862 that include seven hydrophobic regions (labelled TM1-TM7 in Figure 1.1),
which form helices linked by alternating intracellular and extracellular loops, a feature
present in all GPCRs (Garrett et al. 1995). There is currently no clear consensus as to
exactly which amino acids comprise the helices and loops but only an estimate as to
where the helices and loops of the transmembrane region begin and end. Although there
is low sequence homology between the transmembrane domains of Family C GPCRs
and those of the Rhodopsin GPCR Family, there is evidence that there is similarity
between the three dimensional structure of the two families’ transmembrane domains
including the conserved disulfide bond linking the top of TM3 to the second
extracellular loop and the seven highly conserved residues between Family C and the
Rhodopsin Family members that are likely to act in a similar manner in both types of
receptors (Pin et al. 2003). As the transmembrane domains of the two GPCR families
are structurally similar it is likely that the mechanisms of G-protein coupling via the
transmembrane, as outlined by Wess in regards to rhodopsin, are valid for the CaR (Pin
et al. 2003; Wess 1997). It has been hypothesised that ligand binding to the extracellular
domain of a GPCR, leads to conformational changes in TM3 and TM4 that induce the
activation of different types of G protein (Wess 1997). In support of conformational
11
changes to the transmembrane helices leading to CaR activation was the identification
of a mutation in TM7 that leads to a constitutively active receptor (Zhao et al. 1999).
The A843E mutation is currently the only CaR mutation identified that results in
constitutive activation of the receptor and is proposed to alter the conformational state
of the the transmembrane to promote G protein coupling (Zhao et al. 1999). Mutations
such as the A843E that cause constitutive activation have been identified in other
GPCRs, including the L457R mutation identified in TM3 of the luteinizing hormone
receptor (Latronico et al. 1998). In addition to its role in signal transduction, the
transmembrane domain is believed to be involved in receptor dimerisation through
noncovalent interactions (Zhang et al. 2001). The examination of two naturally
occurring truncation mutations of the CaR, P747fs and A877Stop, revealed that dimer
formation was abolished in the P747fs mutant, which lacks TM5, while the A877Stop,
which contains TM5, had the capacity to form a dimer (Pearce et al. 1996; Zhang et al.
2001). A consensus dimerisation motif for noncovalent hydrophobic interactions that
was originally identified in the β2-adrenergic receptor has also been identified in TM5
of the CaR (Hebert et al. 1996).
1.5.2.2 Intracellular Loops
As the intracellular loops of other GPCRs have been implicated in the coupling of
receptors to G proteins, the intracellular loops of the CaR were examined in order to
deduce their importance in G protein-mediated signalling (Chang et al. 2000). A tandem
alanine scan throughout the second and third intracellular loops of the bovine CaR
identifed residues critical to the receptor’s ability to signal (Chang et al. 2000). The
third intracellular loop, which is highly homologous to the third intracellular loop of the
mGluRs, contained more residues involved in CaR signalling than the second
intracellular loop, which has poor homology with its mGluR amino acid counterparts
(Chang et al. 2000). Only the N-terminal portion of the second intracellular loop,
particularly amino acids Leu704 and Phe707, was found to be important to the
signalling capabilities of the CaR (Chang et al. 2000). Mutagenesis of any one of the
three residues of the third intracellular loop - Leu798, Phe802 and Glu804, completely
abrogated CaR signalling, while alanine mutation of any one of the following residues
in the third intracellular loop - Lys794, Arg796, Lys797, Pro799, Asn801, Asn803,
Lys806 and Phe807, resulted in an impairment of CaR signalling (Chang et al. 2000).
12
1.5.2.3 Extracellular Loops
Within the first and second extracellular loops of the CaR are two cysteines, Cys677
and Cys765, which have been identified as residues critical in maintaining the
conformation of the CaR (Ray et al. 2004). Mutation of either cysteine resulted in
incorrect processing of the CaR, which has been hypothesised to be due to the
disruption of disulfide bonds (Ray et al. 2004). Experiments using a chimeric receptor
with the extracellular domain of rhodopsin fused to the transmembrane domain and
intracellular tail of the CaR showed that although this chimeric receptor lacked the
extracellular domain of the CaR it was still expressed at the cell surface and was
capable of responding to extracellular calcium (Hauache et al. 2000). Even though the
chimeric receptor was only able to respond to calcium in the presence of an allosteric
modulator, this data suggested that the transmembrane domain contained additional
sites for calcium binding (Hu et al. 2002). As the extracellular loops are the only parts
of the transmembrane domain exposed to the extracellular environment and contain a
number of acidic residues that may be involved in calcium binding, Hu et al. mutated
the eight acidic residues found in extracellular loops 1-3 to alanines to examine their
possible role in calcium binding (Hu et al. 2002). Substitution of either of the two acidic
residues in extracellular loop 1 - Glu671 or Asp674, did not significantly alter the
biological activity of the CaR (Hu et al. 2002). The individual mutation of three of the
five acidic amino acids in extracellular loop 2 - Asp758, Glu759 and Glu767, to
alanines increased the sensitivity of the CaR to extracellular calcium, while mutations at
Glu755 and Glu 758 had no significant impact on CaR signalling (Hu et al. 2002).
Alanine substitution of the remaining acidic residue in the third extracellular loop,
Glu837, resulted in a mutant CaR that responded to extracellular calcium, albeit with a
lower maximal response compared to the wild-type CaR, but unlike the other seven
mutant CaRs was not potentiated by NPS R-568, a phenylalkylamine (Hu et al. 2002).
Later studies revealed that the negative charge of the glutamate was required for the
interaction between the CaR and phenylalkylamines, leading to the hypothesis that a salt
bridge formed between the glutamate and the positively charged central amine of these
compounds (Hu et al. 2005). These observations led to a series of experiments
attempting to identify the binding site of allosteric modulators.
1.5.2.4 Binding of Allosteric Modulators
Several types of allosteric modulator are known to influence the CaR’s activity, but of
particular interest are compounds referred to as calcimimetics and calcilytics because
13
they act as allosteric agonists and antagonists, respectively (Nemeth et al. 2001; Nemeth
et al. 1998). Homology modelling of the transmembrane domain of the CaR based on
the crystal structure of rhodopsin predicted that the residues Phe668, Arg680, Phe684
and Glu837 were important for the binding of phenylalkylamines to the CaR (Miedlich
et al. 2004). It was shown experimentally that all four of these residues were involved in
the binding of a calcilytic, NPS 2143, but only Phe668, Phe684 and Glu837 were
essential for binding of the calcimimetic NPS R-568 (Miedlich et al. 2004). A separate
group, also applying the rhodopsin structure to model the CaR transmembrane domain,
demonstrated experimentally that while the binding sites of calcimimetics and
calcilytics overlap they are not identical (Petrel et al. 2004). It has also been reported
that structurally different calcimimetics and calcilytics interact with specific sets of
residues in the second and third extracellular loops that share some commonality, but
are distinct (Hu et al. 2006; Petrel et al. 2004).
1.5.3 The Intracellular Tail
Unlike the extracellular and the transmembrane domains, very few naturally occurring
mutations have been identified in the 216 amino acids that comprise the intracellular tail
of the CaR (Ray et al. 1997). The first mutation identified in the intracellular tail was an
Alu sequence insertion at codon 877 (Janicic et al. 1995a). In 2000 Lienhardt et al
identified a large in-frame deletion, S895-V1075, while Carling et al identified the first
point mutation, F881L, located in the intracellular tail of the CaR (Carling et al. 2000;
Lienhardt et al. 2000). In addition to these mutations, the cytoplasmic tail of the CaR
contains three polymorphisms, A986S, G990R and Q1011E (Heath et al. 1996). As can
be seen in Figure 1.1 the intracellular tail of the CaR is the least conserved domain
between mammalian species and the divergence in sequence is even greater in
comparison to non-mammalian CaRs (Loretz et al. 2004). However, there remain
portions of the intracellular tail that are highly homologous between species including a
membrane proximal region spanning residues 863-925 and a region comprised of amino
acids 960-984. The former has been shown to be essential to the cell surface expression
and activity of the receptor, while the latter has been shown to be involved in binding to
accessory proteins (Awata et al. 2001; Hjalm et al. 2001; Ray et al. 1997).
1.5.3.1 Membrane Proximal Region
Investigation of the role of the intracellular tail in expression and activity of the CaR
began with the examination of the aforementioned Alu insertion mutant, which resulted
14
in a truncated receptor with decreased cell surface expression and an inability to
respond to extracellular calcium (Bai et al. 1997). This was followed by a series of
experiments that used a set of CaR mutants that included truncation mutants, 1-865, 1-
874, 1-888 and 1-903, as well as a pair of alanine scan substitutions between Ser875 and
Val883 to further examine the impact of the carboxyl terminal tail on the receptor’s
functionality (Ray et al. 1997). Although all truncation mutants were expressed at the
cell surface, the 1-865 and 1-874 truncation mutants were expressed at a lower level
than the wild-type CaR (Ray et al. 1997). Despite cell surface expression of both the 1-
865 and 1-874 truncation mutants, neither mutant responded to stimulation by
extracellular calcium, while both 1-888 and 1-903 truncation mutants showed biological
activity comparable to wild-type (Ray et al. 1997). Both alanine scan mutants, Ala875-
879 and Ala881-883, were expressed at lower levels on the cell surface when compared
to the wild-type CaR (Ray et al. 1997). The Ala875-879 was unresponsive to
extracellular calcium, while the biological activity of the Ala881-883 mutant was much
lower than the wild-type receptor even when both constructs were expressed at the cell
surface to the same degree as wild-type CaR (Ray et al. 1997). A separate group
examined a set of CaR truncation mutants that included 1-868, 1-886, 1-908 and 1-1024
using an alternate method of measuring CaR activity and found that although all
mutants responded to extracellular calcium, only the 1-868 truncation mutant had a
significantly decreased response compared to wild-type CaR (Gama and Breitwieser
1998). The 1-868 truncation mutant also had an increased rate of desensitisation
compared to the wild-type CaR, while the four other truncation mutants were
desensitised at a rate comparable to wild-type CaR (Gama and Breitwieser 1998). A
third group, using bovine CaR truncation mutants 1-866, 1-895 and 1-929, found that
only the 1-866 truncation mutant was unable to respond to extracellular calcium, as
measured in an assay similar to the one used by Ray et al. (Chang et al. 2001). An
alanine scan between Arg867 and Val895 using the 1-895 truncation mutant indicated
that alanine substitutions throughout the regions 879-883 and 892-895 resulted in a
significant decrease in CaR activity (Chang et al. 2001). Individual alanine substitution
at His880 and Phe882 in full-length bovine CaR also resulted in a reduction in the
biological activity of the receptor (Chang et al. 2001). A large proportion of the H880A
and F882A bovine CaRs expressed in HEK293 cells was retained in the ER, although
they both had glycosylation patterns similar to the wild-type receptor (Chang et al.
2001). It should be noted that both the H880A and F882A mutations occur in a region
of the intracellular tail predicted to form an α-helix. Chang et al. have raised the
15
possibility that an ER retention motif (RXR) beginning at Arg897 might regulate
normal trafficking of the CaR from the ER to the cell surface (Chang et al. 2007). The
work from these three studies highlights the importance of this membrane proximal
region of the CaR’s carboxyl-tail to the overall function of the receptor.
1.5.3.2 Phosphorylation Sites
Prior to the discovery of the CaR, experiments conducted in dissociated bovine
parathyroid cells revealed that protein kinase C (PKC) modulated PTH secretion and the
mobilisation of intracellular calcium (Racke and Nemeth 1993a; Racke and Nemeth
1993b). The regulation of these two processes would later be attributed to the CaR
(Brown et al. 1993). The intracellular tail of the human CaR is predicted to contain three
PKC phosphorylation sites at Thr888, Ser895 and Ser915 (Bai et al. 1998b). Treatment
of HEK293 cells expressing either wild-type CaR or CaR mutated at S895A or S915A,
with the PKC activator, phorbol myristate acetate (PMA), led to an attenuation of the
response to extracellular calcium by the wild-type or mutant CaRs (Bai et al. 1998b).
However, the response to extracellular calcium in HEK293 cells expressing the mutant
CaR construct, T888V, was unaffected by PKC activity, suggesting that PKC
phosphorylation of Thr888 inhibits the biological activity of the CaR (Bai et al. 1998b).
The substitution of Thr888 with hydrophobic residues such as valine, alanine and
tryptophan produced CaRs with an increased responsiveness to its agonists.
Alternatively, CaRs containing mutations at T888D, T888E and T888G were less
responsive than the wild-type CaR to agonist stimulation (Jiang et al. 2002). However,
both sets of mutant CaR constructs exhibited significantly reduced sensitivity to PKC
activity in comparison to the wild-type CaR (Jiang et al. 2002). Treatment of HEK293
cells expressing CaR with the PKC specific inhibitor, GF109203X, negated the
inhibitory effect that activated PKC had on the CaR (Bosel et al. 2003). Experiments
using an antibody specific for the CaR phosphorylated at Thr888 revealed that an
increase in extracellular calcium or acute treatment of the CaR with a calcimimetic
increased phosphorylation of the CaR at Thr888, an effect ablated by treatment with a
calcilytic (Davies et al. 2007). This suggests that the CaR is able to activate PKC, which
in turn phosphorylates the CaR, leading to an inhibition of CaR activity, forming a
negative feedback loop (Davies et al. 2007). It was also observed that after
phosphorylation the Thr888 residue could be dephosphorylated, a process that was
inhibited in the presence of the phosphatase inhibitors calyculin or endothall
thiohydride, suggesting that protein phosphatase 2 is responsible for the
16
dephosphorylation of the CaR (Davies et al. 2007). The antibody specific for the CaR
phosphorylated at Thr888 was able to detect both the mature and immature forms of the
receptor by Western blotting, indicating that both forms are phosphorylated (Davies et
al. 2007). In addition to the PKC phosphorylation sites there are two predicted protein
kinase A (PKA) phosphorylation sites, Ser899 and Ser900, within the intracellular tail
of the CaR (Bosel et al. 2003). Experiments using a PKA specific inhibitor, H-89,
suggested that PKA has only a minor role in the regulation of the CaR (Bosel et al.
2003).
1.6 Calcium-Sensing Receptor Signalling
Just as the major structural elements of the CaR were discussed as either extracellular or
intracellular in the previous section, so too can the receptor’s function be divided by the
plasma membrane into an extracellular component, “sensor”, and an intracellular
component, “transmitter”. The “sensory” aspect of the CaR relates to its ability to detect
changes in the extracellular environment through binding to its agonists, while the
“transmitter” characteristics of the receptor relate to its ability to modulate intracellular
signalling events. These two key facets of the CaR will be outlined below.
1.6.1 Calcium-Sensing Receptor Stimuli
Although extracellular calcium is considered the primary physiological agonist of the
CaR, there are a host of different stimuli to which the receptor is responsive (Hofer and
Brown 2003). The various physiological and pharmacological agonists of the CaR can
be divided into those that can directly induce CaR activation and the allosteric
modulators that sensitise the CaR to its agonists (Brown and MacLeod 2001).
1.6.1.1 Cations
While Ca2+ and Mg2+ may be the only endogenous divalent cations that activate the CaR
there is a growing list of divalent and trivalent cations that are capable of acting as CaR
agonists (Riccardi 2002). In 1990, Nemeth conducted experiments to determine the
CaR’s sensitivity to known cation agonists and found their rank order of potency to be
as follows: La3+ > Gd3+ > Be2+ > Ca2+ = Ba2+ > Sr2+ > Mg2+ (Nemeth and Carafoli
1990). Evidence suggests that not all CaR agonist cations bind to the same region of the
receptor (Hammerland et al. 1999).
17
1.6.1.2 Amino acids
Amino acids were originally shown to act as allosteric modulators of CaR activity in
HEK293 cells stably expressing the CaR (Conigrave et al. 2000). The CaR was
stereoselective for L-amino acids and exhibited greater affinity for large aromatic L-
amino acids (Conigrave et al. 2000). The rank order of potency displayed by the L-
amino acids for the CaR is as follows; L-Phe=L-Trp > L-His > L-Ala > L-Glu > L-Arg
= L-Leu (Conigrave et al. 2000). Although the site of amino acid binding in the CaR is
in the Venus-flytrap domain, it is believed to be distinct from the Ca2+ binding site
(Mun et al. 2005).
1.6.1.3 Pharmacological Agents
Two types of compounds have been designed to act on the CaR, the calcimimetics,
which enhance the sensitivity of the CaR to extracellular calcium, and the calcilytics,
which act as CaR antagonists (Trivedi et al. 2008). The first calcimimetics generated
were the phenylalkylamines, NPS R-467 and NPS R-569, which were shown to be
potent, stereoselective allosteric modulators of the CaR (Nemeth et al. 1998). Currently,
only Cinacalcet-HCl, which is pharmacokinetically more stable than either NPS R-467
and NPS R-569, is commercially available for therapeutic use (Evenepoel 2008). The
calcilytic, NPS 2143, was the first substance, ionic or molecular, identified that was able
to act as a CaR antagonist (Nemeth et al. 2001). Subsequently, structurally different
calcilytics have been designed including Calhex 231 and compound 3 (Brauner-
Osborne et al. 2007). Investigations to determine the binding sites of selected
calcimimetics and calcilytics have revealed that they bind to distinct, but overlapping
regions of the transmembrane domain (Miedlich et al. 2004; Petrel et al. 2004).
1.6.1.4 Polyamines
A number of endogenous polyamines have been experimentally shown to activate the
CaR (Quinn et al. 1997). The concentrations of polyamines used in these experiments to
elicit a CaR-mediated response are higher than their physiological concentrations, but it
should be noted that 0.5 mM of extracellular calcium was routinely used (Riccardi
2002). However, even a modest rise in the concentration of extracellular calcium
dramatically increased the responsiveness of the CaR to polyamines, suggesting that
polyamines might contribute to CaR signalling physiologically (Riccardi 2002). Quinn
et al. measured the efficacies of a group of polyamines and found their order of potency
18
to be as follows: spermine > spermidine >> putrescine, indicating that polyamines with
a higher positive charge were more potent activators of the CaR (Quinn et al. 1997).
1.6.1.5 Polypeptides
Prior to the identification of the CaR, studies examining the effects of polyarginine,
polylysine and protoamine on bovine parathyroid cells revealed that these polypeptides
mimicked the cellular responses observed with extracellular calcium stimulation
(Brown et al. 1991b). Experiments using Chinese hamster ovary cells transiently
expressing the receptor, subsequently confirmed that polypeptides act through the CaR
(Ruat et al. 1996). Amyloid-β peptide, which is excessively produced in the brain of
patients with Alzheimer’s disease, has also been shown to stimulate the CaR and is
proposed to act on the receptor in a fashion similar to spermine, as both molecules have
a similar spacing of positive charges (Brown and MacLeod 2001; Ye et al. 1997a).
1.6.1.6 Aminoglycoside Antibiotics
Like the polypeptides, the polyvalent aminoglycoside antibiotics were originally found
to mimic the effects of extracellular stimulation on cultured bovine parathyroid cells
(Brown et al. 1991a). The aminoglycoside antibiotics were confirmed to act via the CaR
in experiments using HEK293 cells stably expressing the receptor (McLarnon et al.
2002). The order of potency of the aminoglycoside antibiotics tested is as follows:
neomycin > tobramycin > gentamicin > kanamycin, suggesting that their efficacies
positively correlate with the number of attached amino groups (McLarnon et al. 2002).
1.6.1.6 Ionic Strength
Alterations in ionic strength have been shown to influence the sensitivity of the CaR for
its agonists (Brown and MacLeod 2001). It was demonstrated that the CaR expressed in
cultured cells treated with an increase in ionic strength became less sensitive to its
agonists (Quinn et al. 1998). The influence of ionic strength on the CaR’s affinity for
spermine was found to be greater than that of extracellular calcium, suggesting that the
binding efficacies of agonists with a greater number of positive charges would be more
affected by changes in ionic strength (Quinn et al. 1998).
1.6.1.7 pH
Quinn et al. demonstrated that increasing the pH above the physiological range, pH 7.0-
7.8, also increased the agonist sensitivity of the CaR and that a decrease in pH had the
19
reverse effect (Quinn et al. 2004). Part of the variation in CaR agonist affinity by pH
modulation is a result of changes in the ionisation of the charges on the CaR agonists
(Quinn et al. 2004). However, in experiments using HEK293 cells transiently
transfected with any one of the following activating CaR mutants - E191K, F128L,
C129F, Del 543, E127K, and E127A, pH modulation altered CaR agonist sensitivity
(Quinn et al. 2004). These results and the fact that the CaR affinity for extracellular
calcium and magnesium was also influenced by changes in pH suggest that the
alterations in CaR agonist sensitivity by pH is partially the result of molecular changes
to the receptor (Quinn et al. 2004).
1.6.2 Intracellular Signalling Pathways Regulated by the Calcium-Sensing Receptor
The CaR is capable of modulating an extensive and complex array of intracellular
signalling pathways as outlined in Figure 1.2 (Ward 2004). An overview of the better
characterised CaR-mediated pathways will be given below.
1.6.2.1 Phospholipase Signalling
In 1993, studies examining the recently cloned bovine CaR expressed in Xenopus laevis
oocytes identified the phospholipase C (PLC) pathway as being upregulated upon
stimulation of the CaR with extracellular calcium (Brown et al. 1993). CaR activation of
the PLC pathway has been shown to be pertussis toxin sensitive in CaR expressing
Xenopus laevis oocytes and AtT-20 cells but in bovine parathyroid and CaR expressing
HEK293 cells the opposite has been confirmed (Brown et al. 1993; Emanuel et al. 1996;
Kifor et al. 1997b). This suggests that in some cells exhibiting pertussis toxin
insensitive CaR-mediated PLC activity the receptor is coupled to Gq/11 (Kifor et al.
1997b). As indicated in Figure 1.2 an activated G protein subunit interacts with PLC,
leading to the cleavage of phosphatidyl inositol 4,5-bisphosphonate (PIP2) into IP3 and
DAG (Malarkey et al. 1995). IP3 acts as an agonist for the IP3 receptor that upon
activation mobilises calcium from intracellular stores into the cytoplasm, while DAG
can activate PKC (Malarkey et al. 1995; Supattapone et al. 1988).
In addition to PLC, the CaR is also able to activate two other phospholipases,
phospholipase D (PLD) and phospholipase A2 (PLA2) (Kifor et al. 1997a). There are
conflicting reports on how extracellular calcium regulates PLD activity via the CaR.
Kifor et al. showed that downregulation of PKC by pre-treatment with PMA in
parathyroid cells or HEK293 cells stably expressing the CaR abolished PLD activity
20
caused by 2 mM extracellular calcium (Kifor et al. 1997a). In contrast, Huang et al.
demonstrated that PKC down regulated by the same treatment in MDCK cells stably
overexpressing the CaR, did not reduce PLD activity in response to 5 mM extracellular
calcium (Huang et al. 2004). The difference in observed outcomes might be due to the
differences in cell types or levels of extracellular calcium used by the two groups.
Huang et al. went on to show that Rho was involved in CaR-mediated PLD activation
by using the Rho family inhibitor C3 exoenzyme. This led to the abolition of CaR-
mediated PLD activity (Huang et al. 2004). PKC activity is only partially responsible
for CaR-mediated activation of PLA2 (Handlogten et al. 2001). The CaR primarily
regulates PLA2 activity by increasing intracellular calcium levels via Gq activation of
the PLC pathway (Handlogten et al. 2001). The calcium influx causes the PLA2 to
translocate to membranes where it can hydrolyse phospholipids (Purkiss and Boarder
1992). The increase in intracellular calcium also activates calmodulin, which in turn
activates calmodulin-dependent protein kinase (CaMK) (Muthalif et al. 1996). The
activation of these two proteins has been shown to be required for CaR-mediated
activation of PLA2 (Handlogten et al. 2001). However, there is also evidence that CaR-
induced activation of the extracellular signal regulated kinase (ERK) can lead to the
phosphorylation and activation of PLA2 (Kifor et al. 2001). Both PLC and PLD are able
to break down phospholipids to generate phosphatidic acid, which can then be
subsequently hydrolysed by PLA2 to produce free arachidonic acid (Purkiss and
Boarder 1992; Yang et al. 1967).
1.6.2.2 Mitogen Activated Protein Kinase Signalling
The CaR has been implicated in the activation of the following subtypes of mitogen
activated protein kinase (MAPK) signalling cascades: ERK, c-Jun NH2 terminal kinase
(JNK) and p38 MAPK (Ogata et al. 2006).
1.6.2.2.1 Extracellular Signal Regulated Kinase
In 1998 experiments investigating stimulation of rat fibroblasts with either Ca2+ or Gd3+
showed that there was increased activity of the tyrosine kinase, Src, in response to either
cation (McNeil et al. 1998). Rat fibroblasts coexpressing a dominant negative mutant
CaR, R796W, with wild-type CaR exhibited lower Src kinase activity in response to
stimulation with either Ca2+ or Gd3+ than fibroblasts expressing only wild-type CaR,
indicating that this effect was mediated by the CaR (McNeil et al. 1998). As Src kinase
can induce the signalling cascade leading to ERK activation, the effects of Ca2+ or Gd3+
22
stimulation on fibroblasts expressing wild-type CaR, either alone or with the R796W
mutant were examined (McNeil et al. 1998). Not only did these experiments reveal that
ERK activation was mediated through the CaR, but by using a tyrosine kinase inhibitor
selective for Src, herbimycin, McNeil et al. were also able to demonstrate that the CaR
activation of ERK was mediated through the Src kinase (McNeil et al. 1998). A separate
group confirmed that ERK activation by the CaR via the Src kinase was possible in
experiments where HEK293 cells transiently expressing the CaR were stimulated by
extracellular calcium or the calcimimetic, NPS R-467, with or without treatment
involving either of the tyrosine kinase inhibitors, herbimycin and genistein (Kifor et al.
2001). Agonist induced CaR activation of ERK was inhibited in rat ovarian surface
epithelial cells by the transfection of dominant negative mutants of Ras, Raf and MEK,
indicating that the CaR activated ERK via a signalling cascade through the CaR-Src-
Ras-Raf-MEK-ERK pathway as presented in Figure 1.2 (Hobson et al. 2000). However,
Kifor et al. also demonstrated through the use of a PLC specific inhibitor, U-73122, and
a PKC selective inhibitor, GF109203X, that CaR signalling through the PLC pathway
leading to PKC activation could also activate ERK (Kifor et al. 2001). As the effects of
the PLC pathway inhibitors and pertussis toxin treatment on HEK293 cells expressing
the CaR had a synergistic inhibitory effect on ERK activation in response to
extracellular calcium, it can be concluded that the CaR can initiate the PLC pathway
and activate tyrosine kinases to increase ERK activity presumably via coupling to the
Gq/11 and Gi subunits, respectively (Kifor et al. 2001). Phosphatidyl inositol 3 kinase
(PI3K) has been implicated as a component of CaR-mediated ERK activation in CaR
expressing HEK293 cells and ovarian surface epithelial cells in a study that treated both
cell types with the PI3K inhibitors wortmannin and LY294009, resulting in the
inhibition of CaR induced ERK activity (Hobson et al. 2003).
Recently, there have been a number of studies linking the CaR to triple-membrane-
spanning signalling through the EGF receptor (EGFR) (MacLeod et al. 2004; Yano et
al. 2004b). Research into this mode of signalling via the EGFR began when it was
observed that the EGFR undergoes phosphorylation following GPCR activation (Daub
et al. 1996). A chimeric receptor with the extracellular portion of the EGFR and the
platelet derived growth factor receptor’s transmembrane and cytoplasmic domains was
used to show that an EGFR specific extracellular ligand was necessary for EGFR
transactivation by GPCRs (Prenzel et al. 1999). Heparin-binding epidermal growth
factor (HB-EGF) is present on the cell surface as a precursor protein proHB-EGF and
23
can act as a ligand for the EGFR (Dethlefsen et al. 1998). Experiments using inhibitors
to block either HB-EGF specifically or proteolytical processing of proHB-EGF revealed
that HB-EGF was essential for GPCR transactivation of the EGFR (Prenzel et al. 1999).
CaR-mediated ERK activation via triple-membrane-spanning signalling was first
examined in PC-3 cells and subsequently studied in HEK293 cells stably expressing the
CaR, using a combination of inhibitors and neutralising antibodies (MacLeod et al.
2004; Yano et al. 2004b). Treatment of PC-3 and CaR expressing HEK293 cells with
either AG1478, to inhibit the EGFR or GM6001, a broad-spectrum inhibitor of matrix
metalloproteases (MMPs) to inhibit HB-EGF production, reduced the level of CaR
induced stimulation of ERK (MacLeod et al. 2004; Yano et al. 2004b). Antibodies to
HB-EGF and the extracellular domain of EGF receptor also diminished the CaR-
mediated increases in ERK phosphorylation in both cell types (MacLeod et al. 2004;
Yano et al. 2004b). The results of these experiments indicate that stimulation of the CaR
can utilise the key components of triple-membrane-spanning signalling to phosphorylate
ERK (MacLeod et al. 2004; Yano et al. 2004b).
1.6.2.2.2 c-Jun NH2 Terminal Kinase
Studies investigating the role of the CaR in NIH/3T3 cells, a fibroblast cell line, found
that stimulation of the receptor leads to the phosphorylation of JNK, a protein
alternatively known as the stress-activated protein kinase (Hoff et al. 1999). Initiation of
the JNK signalling cascade by the CaR caused morphological changes in the NIH/3T3
cells that were inhibited by the introduction of a dominant-negative kinase that is
upstream of JNK in the pathway (Hoff et al. 1999). Ogata et al. would later show that
CaR agonist stimulation of fibroblasts isolated from rat jaw cysts enhanced
cyclooxygenase-2 expression (Ogata et al. 2006). Western blotting with
phosphospecific antibodies and inhibition experiments with SP600125, a JNK specific
inhibitor, revealed that the CaR-induced expression of cyclooxygenase-2 in fibroblasts
was via JNK signalling (Ogata et al. 2006).
1.6.2.2.3 p38 Mitogen Activated Protein Kinase
Phosphospecific antibodies were used in Western blots to detect increased
phosphorylation of the p38 MAPK in the CaR expressing mouse osteoblastic MC3T3-
E1 cell line in response to treatment with a variety of CaR agonists (Yamaguchi et al.
2000). By measuring the amount of [3H]-thymidine incorporated during DNA synthesis
Yamaguchi et al. was able to demonstrate that CaR induction of the p38 MAPK
24
signalling cascade generated mitogenic responses within the MC3T3-E1 cell line
(Yamaguchi et al. 2000). Using a combination of Western analysis and inhibition
studies, CaR-mediated p38 MAPK signalling has since been detected in rat H-500
Leydig cancer cells where p38 MAPK phosphorylation following CaR stimulation
results in the release of PTH-related protein (PTHrP) and in HK-2G cells, a proximal
tubule human kidney epithelial cell line, in which vitamin D receptor expression levels
are regulated by CaR-mediated p38 signalling (Maiti et al. 2008; Tfelt-Hansen et al.
2003).
1.6.2.3 Inhibition of Cyclic AMP
Prior to the identification of the CaR, it was observed that in bovine parathyroid cells a
range of divalent cations, including Ca2+, Mg2+, Ba2+ and Sr2+, inhibited the dopamine
stimulated accumulation of cyclic adenosine monophosphate (cAMP) in a pertussis
toxin sensitive manner (Chen et al. 1989). Treatment of the cortical thick ascending
limb of rat kidney with antidiuretic hormone leads to the production of cAMP (Ferreira
and Bailly 1998). Stimulation of the endogenously expressed CaR within the cortical
thick ascending limb with either extracellular calcium or neomycin inhibited the
antidiuretic hormone-induced accumulation of cAMP (Ferreira and Bailly 1998).
Another study examined cAMP formation in response to forskolin in HEK293 cells
stably expressing the CaR and noted that stimulation with the cations Ca2+, Mg2+ and
Gd3+ or the calcimimetic NPS R-467 significantly decreased cAMP content in this cell
type (Chang et al. 1998).
1.6.2.4 Rho Signalling
As mentioned above Huang et al. used an inhibitor to demonstrate the involvement of
Rho in CaR-mediated PLD activation (Huang et al. 2004). It was also shown that
extracellular calcium stimulation of MDCK cells stably overexpressing the CaR caused
Rho to translocate to the membrane (Huang et al. 2004). Further evidence of the CaR
acting via the Rho pathway has been reported by other groups examining two separate
CaR-mediated signalling events, the activation of serum response element (SRE)-
mediated gene transcription (Pi et al. 2002) and the production of intracellular Ca2+
oscillations by amino acid stimulation (Rey et al. 2005). The Rho family inhibitor C3
exoenzyme was used in experiments to demonstrate that Rho inhibition leads to the
abolition of CaR-stimulated SRE activity in HEK293 cells stably expressing the CaR
(Pi et al. 2002). When a mutant Rho, that was unaffected by C3 exoenzyme, was also
25
expressed in these cells, CaR-mediated SRE activity was partially restored (Pi et al.
2002). It has also been shown that activation of Rho by the CaR involved the
recruitment of the Rho-guanine nucleotide exchange factor (Rho-GEF) Lbc and filamin
(Pi et al. 2002). Transient calcium oscillations in CaR expressing HEK293 cells caused
by amino acid stimulation were also abrogated by C3 exoenzyme treatment (Rey et al.
2005). Interestingly, the sinusoidal calcium oscillations caused by extracellular calcium
stimulation were unaffected by the addition of C3 exoenzyme (Rey et al. 2005). A
dominant negative filamin peptide was used to block the production of transient calcium
oscillations caused by amino acid stimulation of the CaR. This confirmed the earlier
findings that filamin enhances activation of Rho by the CaR (Rey et al. 2005).
1.7 The Biological Roles of the Calcium-Sensing Receptor
Maintaining systemic calcium homeostasis is regarded as the major physiological role
of the CaR (Brown and MacLeod 2001). Essentially, systemic calcium homeostasis is
the balance within the body between the ingestion and absorption of calcium,
circulating calcium and excreted calcium, which involves the gastrointestinal tract, bone
and kidney (Brown 2000). The CaR expressed in these tissues, as well as in the
parathyroid and thyroid glands, is responsible for regulating the levels of calcium
absorbed or excreted by the body primarily by detecting circulating levels of calcium
within the extracellular fluid (Brown and MacLeod 2001). The CaR responds to a
decrease in the concentration of extracellular calcium by increasing the secretion of
PTH from the parathyroid gland and decreasing the secretion of calcitonin from the
thyroid (Brown 2000). The higher concentration of circulating PTH stimulates bone
resorption, leading to an increased calcium efflux from the skeleton and increased 1,25-
dihydroxyvitamin D3 production from the kidney, which increases calcium absorption
in the gastrointestinal tract (Quarles 2003). As calcitonin inhibits bone resorption and
decreases calcium excretion from the kidneys the CaR-mediated decrease in calcitonin
secretion in response to lower circulating calcium increases the overall level of calcium
retained by the body (Brown 2000).
Following the discovery of the CaR and its role in calcium homeostasis several
inherited disorders of calcium homeostasis were linked to functional abnormalities of
the receptor (Hauache 2001). Primarily, the calcium homeostatic disorders are
associated with mutations of the CaR, which are catalogued on an online database at
http:/data.mch.mcgill.ca/casrdb/, but there are also cases where antibodies to the CaR
26
are believed to interfere with the function of receptor (Thakker 2004). Over 100 CaR
mutations have been identified that are associated with caclium homeostatic disorders
(Pidasheva et al. 2004). First characterised in 1972, familial hypocalciuric
hypercalcaemia (FHH) is an autosomal dominant disorder associated with mild to
moderate hypercalcaemia, inappropriately normal PTH levels and low rates of urinary
calcium excretion that is the result of a loss-of-function CaR mutation on a single allele
(Hendy et al. 2000). There is emerging evidence that the biochemical severity of FHH is
linked to the dominant negative effect CaR mutations have on wild-type CaR activity
(Ward et al. 2006). Inactivating mutations in both copies of the CaR gene result in a
more serious condition known as neonatal severe hyperparathyroidism (NSHPT), which
is characterised by life-threatening severe hypercalcaemia, failure to thrive and
undermineralisation of bone (Tfelt-Hansen and Brown 2005). Without treatment by
parathyroidectomy, NSHPT can lead to multiple fractures, ribcage deformity and
neurodevelopmental disorders (Hendy et al. 2000). Gain-of-function CaR mutations
result in autosomal dominant hypocalcaemia, a generally asymptomatic condition with
patients experiencing mild hypocalcaemia and possibly seizures during childhood
(Hendy et al. 2000). Bartter syndrome type V, with symptoms including hypokalaemic
metabolic alkalosis, hyper-reninaemia, hyperaldosteronism and hypocalcaemia, is also
due to activating CaR mutations (Thakker 2004). Some patients with either autoimmune
hypocalciuric hypercalcaemia or autoimmune acquired hypoparathyroidism have been
shown to have circulating antibodies that recognise the extracellular domain of the CaR,
which are believed to be responsible for their respective conditions (Thakker 2004).
Although the CaR plays a vital role in the maintenance of calcium homeostasis, the
receptor has been detected in a host of tissues unrelated to calcium homeostasis,
including brain, breast and skin (Bikle et al. 1996; Chattopadhyay et al. 1997; Cheng et
al. 1998). Within the different CaR expressing cell types the receptor has been observed
to regulate a multitude of cellular processes including secretion, proliferation,
differentiation, apoptosis and gene expression (Buchan et al. 2001; Komuves et al.
2002; Peiris et al. 2007; Rutten et al. 1999; Tu et al. 2008). Below, the cellular
processes mediated by the CaR will be discussed in relation to several of the tissue
types in which there has been substantial investigation of the receptor’s function.
27
1.7.1 Calcium-Sensing Receptor in the Parathyroid
As mentioned earlier in this chapter, the CaR was originally cloned from parathyroid
cells, where the receptor mediates the inhibition of PTH secretion in response to agonist
stimulation (Brown et al. 1993). A large fraction of the CaR expressed in parathyroid
cells is localised to caveolae, which are regions of the cell membrane containing
signalling molecules and scaffolding proteins (Kifor et al. 1998). As the first cells to be
identified as expressing the CaR, parathyroid cells have been used extensively to
examine the cell signalling properties of the receptor. As outlined above, the CaR
expressed in parathyroid cells was shown to regulate PLC, PLA2, PLD and MAPK
signalling (Kifor et al. 1998; Kifor et al. 2003). However, the link between the induction
of intracellular signals by the CaR and the exocytotic apparatus that secretes PTH from
the cells is still largely unknown (Brown and MacLeod 2001). There is recent data
showing CaR signalling decreases the level of PTH mRNA produced in parathyroid
cells, suggesting an additional means by which the receptor limits the level of
circulating PTH (Carrillo-Lopez et al. 2008). The CaR is also hypothesised to regulate
the proliferation of parathyroid cells based on the evidence of greater parathyroid
hyperplasia in the parathyroid glands associated with decreased CaR function as seen in
patients with NSHPT, or reduced expression as seen in the parathyroid adenomas of
patients with primary hyperparathyroidism (Chen and Goodman 2004; Farnebo et al.
1998). Marked parathyroid cellular hyperplasia has also been observed in CaR knockout
mice (Ho et al. 1995).
1.7.2 Calcium-Sensing Receptor in the Kidney
Following the cloning of the CaR from parathyroid glands, the CaR expression was
detected in the kidney, another organ with a critical role in calcium homeostasis
(Riccardi et al. 1995). A combination of immunohistochemistry and RNA isolation
techniques was used to demonstrate that the CaR is expressed throughout the majority
of the nephron, including the proximal tubule, thick ascending limb, distal tubule and
collecting ducts (Butters et al. 1997; Riccardi et al. 1998). Interestingly, the expression
pattern of the CaR differs between the segments of the nephron (Ba and Friedman
2004). In proximal tubules the CaR is localised to the base of apical brush-border
membranes, in thick ascending limbs it is expressed on basolateral membranes, while in
the collecting ducts it is found at apical plasma membranes (Butters et al. 1997;
Riccardi et al. 1998). As each segment of the nephron has a specialised function it was
postulated that the role of the CaR in each segment would be specific (Brown and
28
Hebert 1997). Inorganic phosphate absorption by proximal tubules is inhibited by PTH,
but can be restored by stimulation of the CaR (Ba and Friedman 2004). In the thick
ascending limb, CaR was found to reduce the activity of the 70-pS apical membrane
potassium channel, which would lead to a reduction in potassium recycling (Wang et al.
1996). Aslanova et al, demonstrated that the intracellular pH within the thick ascending
limb is regulated by the CaR in a chloride-dependent manner (Aslanova et al. 2006).
PTH-dependent absorption of calcium by the cortical ascending limb was inhibited by
stimulation of the CaR, possibly by inhibiting the accumulation of cAMP (Ferreira et al.
1998; Motoyama and Friedman 2002). CaR agonist treatment of collecting duct cells
stably expressing aquaporin 2, inhibited the effects of forskolin-induced trafficking of
aquaporin 2, by reducing cAMP levels, activating the PKC signalling pathway and
increasing the level of actin fibre assembly (Procino et al. 2004).
1.7.3 Calcium-Sensing Receptor in the Gastrointestinal Tract
The gastrointestinal tract consists of a system of organs designed to cope with the
nutrient, electrolyte and fluid absorption requirements of the body, as well as the
secretion of excess electrolytes and fluids (Kirchhoff and Geibel 2006). The CaR has
been identified in a number of the organs that constitute the gastrointestinal tract,
including the oesophagus, stomach, small intestines and colon (Chattopadhyay et al.
1998a; Cheng et al. 1997). Recent experiments using the human oesophageal epithelial
cell line, HET-1A, have provided some insight into the role of the CaR in the
oesophagus, revealing that CaR stimulation led to increased phosphorylation of ERK,
intracellular calcium mobilisation and the secretion of IL-8 (Justinich et al. 2008). In the
stomach, the CaR is expressed in several specialised cells, including mucous epithelial
cells, G cells and parietal cells (Buchan et al. 2001; Busque et al. 2005; Rutten et al.
1999). In human gastric mucous epithelial cells, stimulation of the CaR, which is
primarily expressed at the basolateral membrane, results in increases in both
intracellular calcium levels and the rate of proliferation (Rutten et al. 1999). The CaR
expressed in G cells modulates the secretion of gastrin via the PLC signalling pathway
(Buchan et al. 2001). In response to either multivalent cations or amino acids, the CaR
expressed in parietal cells regulates the activity of a key component of gastric acid
secretion, the H+-K+-ATPase (Busque et al. 2005; Dufner et al. 2005). Research into the
role of the CaR in the intestine has focussed on the colon despite the receptor being
expressed in epithelial cells throughout the intestine (Hebert et al. 2004). Cheng et al.
showed that the colonic CaR in both surface and crypt cells responds to multivalent
29
cations by inducing PLC signalling (Cheng et al. 2002). Activation of the CaR inhibited
the forskolin-stimulated fluid secretion observed in isolated perfused colonic crypt cells,
presumably by inhibiting the cAMP pathway (Cheng et al. 2004). In colonic
myofibroblasts, stimulation of the CaR upregulated the expression and secretion of bone
morphogenetic protein-2 in a PI3K-dependent manner, but decreased the expression of
the bone morphogenetic protein-2 antagonist, Noggin (Peiris et al. 2007). Experiments
examining cultured intestinal cell lines found that the CaR regulated markers of cell
proliferation and differentiation (Chakrabarty et al. 2003; Kallay et al. 1997). The role
of the CaR in the regulation of cell proliferation and differentiation, as well as results of
several studies examining the preventive effects of a high calcium diet against colon
cancer has led to the proposal that the use of therapeutic CaR agonists may reduce the
risk of colon cancer (Kirchhoff and Geibel 2006; Rodland 2004).
1.7.4 Calcium-Sensing Receptor in Bone
Evidence of CaR expression in bone was first obtained in 1997 when a combination of
techniques was used to detect the CaR in the osteoblastic cell line MC3T3-E1
(Yamaguchi et al. 1998). Chang et al would later show that that the CaR is expressed in
osteoblasts of mouse, rat and bovine origin (Chang et al. 1999). It was demonstrated
that stimulation of the CaR expressed in osteoblasts led to an increase in proliferation
(Dvorak et al. 2004). The signalling mechanisms involved in CaR-induced proliferation
observed in osteoblasts is unclear, as one study has reported that JNK signalling was
necessary, while another reported the requirement for ERK, Akt and glycogensynthase
kinase 3β phosphorylation (Chattopadhyay et al. 2004; Dvorak et al. 2004). Following
the detection of the CaR in osteoclasts it was found that stimulation of the receptor
resulted in an inhibition of the bone resorbing activity of the osteoclasts (Kameda et al.
1998). CaR activity has been shown to promote both differentiation and apoptosis
within osteoclasts via the PLC pathway (Mentaverri et al. 2006). Initially the generation
of a CaR knockout mouse was believed to demonstrate a clear functional role of the
receptor in bone as these mice exhibited bone abnormalities and rickets (Garner et al.
2001; Ho et al. 1995). However, when the CaR knockout mice were bred with mice
lacking parathyroid glands a normal bone phenotype was observed, which suggested
that the bone abnormalities detected in the CaR knockout mice resulted from the loss of
CaR expression in the parathyroid glands (Tu et al. 2003). Evidence of alternative
splicing of the CaR supported this notion as the region of the CaR disrupted to generate
the CaR knockout mice could be spliced out without affecting the functionality of the
30
receptor (Oda et al. 2000; Rodriguez et al. 2005). In vivo evidence of the functional
relevance of the CaR in bone has only recently been presented with the generation of
conditional knockout mice that specifically do not express the CaR in bone, which
revealed a profound lack of postnatal growth and skeletal development (Chang et al.
2008). Most of the mice not expressing the CaR in bone did not survive past the first
three weeks after birth and exhibited undermineralisation of the skull, vertebrae and
long bones, as well as fractures of the ribs and long bones (Chang et al. 2008). The
postnatal growth deficiency, undermineralisation and fractures observed in mice deleted
in bone CaR is consistent with the symptoms observed in a patient with NSHPT
involving the effective so-called knockout of the CaR (Ward et al. 2004)
1.7.5 Calcium-Sensing Receptor in the Nervous System
Immunocytochemisty and in situ hybridization studies have revealed a wide distribution
of the CaR throughout the central nervous system with an extremely varied expression
pattern (Ferry et al. 2000; Rogers et al. 1997; Ruat et al. 1995). Not only does the
expression pattern of the CaR within the nervous system overlap with those of two other
Family C GPCRs, the mGluR and GABAB receptors, but it has also been shown in
coimmunoprecipitation experiments that the CaR heterodimerises with these receptors
(Chang et al. 2007; Gama et al. 2001). The highest level of CaR expression is within the
region of the brain known as the subfornical organ which, due to an absence of a blood-
brain barrier, is exposed to systemic fluid (Yano et al. 2004a). There is experimental
evidence indicating that a population of the subfornical organ neurons exhibit a
subthreshold, hyperpolarisation-activated inward current that is potentiated by CaR
stimulation (Washburn et al. 2000a). Furthermore, it has been proposed that the CaR-
mediated current regulates the bursting of action potentials and that subsequent
depolarising afterpotentials of neurones of the subfornical organ can also be modulated
by the CaR (Washburn et al. 2000b). There is also abundant expression of the CaR in
the hippocampus, but there has been little evidence of the role, if any, the receptor plays
in this part of the brain (Yano et al. 2004a). Due to an increase in CaR expression within
the hippocampus, corresponding to the time when long-term potentiation first occurs, it
has been hypothesised that the CaR may be involved in cognitive functions such as
memory and learning (Chattopadhyay et al. 1997). Recently, CaR activity was found to
promote axon growth and branching in developing neurons (Vizard et al. 2008).
Research examining the gonadotropin-releasing hormone neuron cell line, GT1-7,
demonstrated that CaR activation within these cells caused the secretion of cytokines
31
that promoted chemotaxis in astrocytes (Bandyopadhyay et al. 2007). CaR expressed in
the neurons of the hippocampus have been shown to regulate the opening of both
calcium permeable, nonselective cation channels and calcium activated potassium
channels (Vassilev et al. 1997; Ye et al. 1997b). Further studies have revealed that the
CaR-mediated activation of calcium permeable, non-selective cation channels can be
induced by the CaR agonist, amyloid-β peptide, which is excessively produced in
patients with Alzheimer’s disease (Ye et al. 1997a). The expression of the CaR within
the nervous system is not limited to neuronal cells, as the receptor has been detected in
glial cells as well (Yano et al. 2004a). CaR identified in oligodendrocytes and microglia
have also been found to regulate calcium activated potassium channels (Chattopadhyay
et al. 1998b; Chattopadhyay et al. 1999). Chattopadhyay et al. also demonstrated that
agonist stimulation of CaR expressed in astrocytes promoted PTHrP secretion
(Chattopadhyay et al. 2000).
1.7.6 Calcium-Sensing Receptor in Breast
The CaR has been identified in both normal and malignant breast tissue by Northern
analysis and immunohistochemistry (Cheng et al. 1998). In mice, it has been observed
that there is a constant low level of CaR expression in mammary glands throughout
development from prepubertal to adult glands, but the receptor is downregulated during
pregnancy and subsequently upregulated to its highest level of expression during
lactation (VanHouten et al. 2004). It has been demonstrated that PTHrP secretion is
regulated by the CaR in cultured cells derived from the mammary glands (Sanders et al.
2000; VanHouten et al. 2004). However, in normal breast cell lines, CaR stimulation
inhibits PTHrP secretion, while in breast cancer cell lines PTHrP secretion is increased
by CaR stimulation (Sanders et al. 2000; VanHouten et al. 2004). The opposite CaR-
mediated effects on PTHrP secretion have recently been revealed to be a result of the
CaR coupling to Gαi in normal breast cells and Gαs in malignant breast cells
(Mamillapalli et al. 2008). Aside from regulating PTHrP production in normal breast
cells, the CaR has been shown to be important for the transport of calcium into milk
during lactation (Ardeshirpour et al. 2006). The proposed role of the CaR in breast
cancer relates to the metastasis of the breast cancer to bone where the increase in CaR-
mediated PTHrP secretion causes greater bone resorption, which raises the level of
extracellular calcium and further stimulates the CaR leading to further PTHrP secretion,
resulting in a vicious cycle leading to worsening osteolysis (Sanders et al. 2000).
32
1.7.7 Calcium-Sensing Receptor in Epidermal Cells
In 1996, the CaR was identified in cultured human keratinocytes (Bikle et al. 1996).
Results from experiments using mice that were either unable to express full-length CaR
or overexpressed the CaR, suggested that the receptor has a role in the proliferation and
differentiation of keratinocytes (Komuves et al. 2002; Oda et al. 2000; Turksen and
Troy 2003). Recently, knockdown technology was used to decrease the expression
levels of the CaR in human keratinocytes, revealing that aside from its role in
differentiation the CaR is important in cell survival as a decrease in CaR expression in
keratinocytes correlated with an in increase in apoptosis (Tu et al. 2008).
1.8 Interacting Protein Partners of the Calcium-Sensing Receptor
In order to have a better understanding of the mechanisms that govern the CaR’s
functionality, recent work in the CaR field has aimed at identifying proteins that interact
with the receptor (Huang and Miller 2007). As with all GPCRs, the CaR signals through
its interaction with heterotrimeric G proteins, but recently several other proteins have
been identified that interact with the CaR and influence its signalling characteristics
(Huang and Miller 2007). Several identified interacting protein partners of the CaR will
be discussed below.
1.8.1 Filamin
In 2001, two groups performed yeast two-hybrid library screens using the CaR
intracellular tail as bait in order to discover proteins that interact with the CaR and both
studies revealed filamin A as an interaction target for the receptor (Awata et al. 2001;
Hjalm et al. 2001). Following the isolation of a filamin A cDNA fragment from a
human kidney cDNA library, Awata et al. used the yeast two-hybrid system to delineate
a region of filamin A comprised of amino acids 1566-1875 that interacted with the CaR
(Awata et al. 2001). Following the screening of a bovine parathyroid cDNA library
Hjalm et al. performed yeast two-hybrid mapping studies to localise the site of filamin
A to which the CaR binds to residues 1534-1719 (Hjalm et al. 2001). Taken together,
these two studies define filamin A’s minimal CaR binding site as amino acids 1566-
1719. The interaction between the CaR and filamin A was confirmed in mammalian
cells via confocal microscopy and a series of coimmunprecipitation studies (Awata et al.
2001; Hjalm et al. 2001). Zhang and Breitwieser performed a series of
coimmunoprecipitation experiments using truncation and deletion mutants of the CaR
that revealed that the site of interaction for filamin A was between residues 962-981, a
33
domain predicted to form two β-strands in the CaR tail (Zhang and Breitwieser 2005).
Filamin A is a cytoskeletal scaffold protein that has been implicated in the organisation
of signalling molecules (Li et al. 2005). Due to the possible role of filamin A in
intracellular signalling, the effect of disrupting the interaction between the CaR and
filamin A on the ERK pathway was examined using inhibitory peptides (Awata et al.
2001; Hjalm et al. 2001). Awata et al. blocked the binding between the CaR and filamin
A using a myc-tagged peptide corresponding to the filamin A binding site in the CaR
between residues 907-1022 and found that CaR-mediated ERK activity was reduced in
a dose-dependant manner (Awata et al. 2001). Conversely, Hjalm et al. generated a
filamin A fusion construct that contained the CaR interaction domain, amino acids
1534-1719, that was able to significantly disrupt CaR coupling to the ERK pathway
(Hjalm et al. 2001). Peptides corresponding to the CaR binding site in filamin A that
disrupted the interaction between the two proteins were also used to show that this
interaction was important for CaR-mediated Rho signalling (Pi et al. 2002; Rey et al.
2005). Silencing of the filamin A gene in HEK293 cells stably expressing the CaR with
siRNA caused a significant decrease in CaR-mediated JNK activation, indicating the
necessity of the interaction between the CaR and filamin A for the induction of the JNK
signalling cascade by the CaR (Huang et al. 2006a). Experiments that examined the
expression levels of the CaR in M2 cells, a cell line that does not express filamin A,
revealed that CaR expression was almost doubled when filamin A was transfected into
the M2 cells (Zhang and Breitwieser 2005). Inhibition of CaR expression with CaR
antisense cDNA 48 hours after the transfection of CaR with or without filamin A
resulted in a lower level of CaR expression in cells not expressing filamin A, suggesting
that filamin A protects the CaR against degradation (Zhang and Breitwieser 2005).
1.8.2 Potassium Channels
In addition to filamin A, the inwardly rectifying potassium channel Kir4.2 was
identified as a binding partner to the CaR intracellular tail in the yeast two-hybrid
kidney library screen described above (Huang et al. 2007a). The Kir4 family of proteins,
consisting of Kir4.1 and Kir4.2, are channels expressed on the basolateral membrane of
the distal nephron within the kidney and are believed to be involved in the regulation of
membrane potential and recycling potassium for Na,K-ATPases (Lourdel et al. 2002).
The CaR coimmunoprecipitated with both Kir4.1 and Kir4.2 from kidney cortex and
liver, respectively, and confirmed the initial findings of the yeast two-hybrid system
(Huang et al. 2007a). Kir4.2 and the CaR were observed to colocalise at the cell
34
membrane of HEK293 cells stably expressing both proteins, while endogenous CaR and
Kir4.1 colocalised at the basolateral membrane of the distal convoluted tubule in rat
kidney sections (Huang et al. 2007a).
1.8.3 Dorfin
In a third independent yeast two-hybrid library screen that used the CaR intracellular
tail as bait to probe a human kidney cDNA library, an E3 ubiquitin ligase, dorfin, was
identified as an interacting protein partner of the CaR (Huang et al. 2006b). By
interacting with both E2-ubiquitin and a target protein, dorfin is able to transfer the
ubiquitin to the target protein (Niwa et al. 2001; Wojcikiewicz 2004). The yeast two-
hybrid system was used to localise the sites of CaR and dorfin interaction to residues
880-900 in the CaR and amino acids 660-838 of dorfin (Huang et al. 2006b). It should
be noted that residues 660-838 form the carboxyl terminus of dorfin and that removal of
either amino acids between 660-720 or 780-838 disrupted the interaction between dorfin
and the CaR (Huang et al. 2006b). In HEK293 cells transiently expressing the CaR, an
increase in the ubiquitination of the receptor was observed when dorfin was
overexpressed in these cells (Huang et al. 2006b). Further evidence of dorfin regulating
the ubiquitination of the CaR was the reduction in ubiquitination of the receptor
detected in HEK293 cells cotransfected with the CaR and a dominant negative dorfin
construct incapable of catalysing the ubiquitination of substrates (Huang et al. 2006b).
Individual mutation of any of the 16 lysines present either in the intracellular loops or
tail to arginines had no significant impact on the level of CaR ubiquitination (Huang et
al. 2006b). However, when all 16 lysine residues were converted to arginines CaR
ubiquitination was abolished, indicating that the receptor is polyubiquitinated (Huang et
al. 2006b). Increasing the level of dorfin expressed in HEK293 cells transiently
expressing the CaR led to an increased rate of CaR degradation, while the reciprocal
result was observed when a dominant negative dorfin was expressed at increasing levels
(Huang et al. 2006b). Dorfin-mediated degradation of the CaR was shown to occur via
the proteosome when treatment with the proteasomal inhibitor MG132 abolished the
increase in CaR degradation associated with dorfin overexpression (Huang et al.
2006b). Coimmunoprecipitation experiments indicated that the CaR and dorfin are part
of a protein complex that includes the valosin-containing protein, suggesting that the
dorfin-mediated degradation of the CaR proceeds through the ER-associated
degradation (ERAD) pathway (Huang et al. 2006b).
35
1.8.4 Associated Molecule with SH3 Domain of STAM (AMSH)
A more recent screen of a human kidney cDNA yeast two-hybrid library used a CaR-tail
that contained a deletion of the region S895-V1075, which mimicked a naturally
occurring mutation, and identified associated molecule with SH3 domain of STAM
(AMSH) as an interacting partner of the CaR (Herrera-Vigenor et al. 2006). AMSH is
an ubiquitin isopeptodase that is a key regulatory component of endosomal sorting of
the EGFR (McCullough et al. 2004). The AMSH cDNA clone identified in the yeast
two-hybrid library screen corresponded to an AMSH splice variant that was able to bind
to both wild-type CaR and the CaR deletion mutant in a mammalian system following
the cloning of the cDNA fragment into a suitable expression vector (Herrera-Vigenor et
al. 2006). Full-length AMSH was also found to interact with the CaR in HEK293 cells
transfected with both proteins and increasing the level of AMSH expression diminished
the amount of CaR expressed (Herrera-Vigenor et al. 2006). Reduced CaR expression
due to its interaction with AMSH was observed in HEK293 cells transiently expressing
both the CaR and AMSH following stimulation with extracellular calcium (Reyes-
Ibarra et al. 2007).
1.8.3 Receptor-Activity-Modifying Proteins
The receptor-activity-modifying protein (RAMP) family are single-transmembrane
spanning protein that have been shown to affect receptor trafficking, glycosylation,
ligand specificity and second messenger production in several cell types (Morfis et al.
2003). While investigating a new method to examine cell surface expression of the CaR,
Bouschet et al. observed that the receptor was not expressed at the cell surface of COS7
cells (Bouschet et al. 2005). They proposed that the lack of CaR at the cell surface was
due to COS7 cells not expressing any members of the RAMP family and this was tested
by cotransfection of the CaR and the three different RAMPs into COS7 cells (Bouschet
et al. 2005). When the CaR was coexpressed in COS7 cells with either RAMP1 or
RAMP3 the receptor was delivered to the cell surface, but when coexpressed with
RAMP2 there was still no CaR detected at the cell surface (Bouschet et al. 2005).
Colocalisation and coimmunoprecipitation experiments provided evidence that both
RAMP1 and RAMP3 shared the same subcellular location as the CaR and that both
interact with the receptor. As RAMP3 had a greater influence on the cell surface
expression of the CaR than RAMP1, only the effects of RAMP3 on receptor trafficking
and glycosylation, were examined but the results observed are believed to also be valid
for RAMP1 (Bouschet et al. 2005). A high level of CaR was present in the ER and a
36
negligible amount in the Golgi apparatus in the absence of RAMPs, but when
coexpressed with RAMP3 a high level of the receptor was colocalised with RAMP3 in
the Golgi apparatus suggesting that trafficking of the CaR from the ER to Golgi is
reliant on RAMPs (Bouschet et al. 2005). CaR transiently expressed in both COS7 cells
transfected with RAMP3 and in HEK293 cells, which express endogenous RAMP1,
was glycosylated to a greater extent than CaR expressed in COS7 cells without any
RAMPs (Bouschet et al. 2005).
1.8.4 β-Arrestins
β-arrestins are ubiquitously expressed proteins that are involved in the desensitisation
and internalisation of most GPCRs leading to the eventual endocytosis of the receptors
in clathrin-coated pits (DeWire et al. 2007). Measuring luciferase activity in HEK293
cells cotransfected with rat CaR and a SRE-luciferase reporter construct in the presence
and absence of β-arrestins 1 and 2, revealed that both β-arrestin isoforms reduce the
level of CaR-induced luciferase activity (Pi et al. 2005). Interaction between β-arrestin 1
and the CaR was demonstrated in mammalian cells by coimmunoprecipitation
experiments, while colocalisation of the CaR and β-arrestin 2 was shown to occur
following stimulation of the receptor in U2OS cells and was enhanced by the
overexpression of G protein receptor kinase (GRK) 4 (Pi et al. 2005). In yeast two-
hybrid studies, both β-arrestin isoforms were found to interact with a region of the rat
CaR intracellular tail between amino acids 877 and 1079, but neither isoform interacted
with the residues 636-805 of the CaR, which contain the intracellular loops of the
receptor (Pi et al. 2005). In COS7 cells transiently transfected with constructs for β-
arrestin 1 and a fragment of the rat CaR corresponding to residues 877-1079, it was
observed that in cells that were additionally transfected with GRK2 the amount of β-
arrestin 1 coimmunoprecipitated with the CaR fragment was greater than in the absence
of ectopically expressed GRK2 (Pi et al. 2005). Lorenz et al. showed that
overexpression of both β-arrestin 1 and 2 had a negative influence on CaR-mediated
inositol phosphate production in GripTite293 cells, a HEK293 cell line genetically
engineered to have greater adherence. This effect could be abolished by either a PKC
inhibitor or by mutations of all the intracellular PKC phosphorylation sites of the CaR
(Lorenz et al. 2007).
37
1.9 Statement of Aims
As outlined in this chapter the CaR plays a crucial role linking a diverse range of stimuli
to different signalling pathways that in turn lead to a variety of tissue specific cellular
processes. A major determining factor of the biological responses initiated by the CaR
upon stimulation is likely to be the binding of accessory proteins to the intracellular tail
of the receptor. While several of these CaR-tail targets and their functional significance
have already been elucidated from a number of yeast two-hybrid screens using the CaR-
tail as bait, this number is almost certainly not exhaustive and it is anticipated that there
remain many more targets awaiting identification. Moreover, we had the opportunity to
screen a haemopoietic cell line library, not previously used in a yeast two-hybrid screen
with the CaR-tail, which had the potential to reveal many new and exciting targets. This
then forms the underlying hypothesis of this thesis:
That many novel CaR-tail protein targets that potentially influence CaR signalling
and other processes remain to be identified and that at least some of these targets will
be revealed by screening this unique library.
In order to address this hypothesis, other investigators in our laboratory used the LexA
yeast two-hybrid system to screen a mouse pluripotent haemopoietic cell line library
using the CaR-tail as bait. This screen revealed a large number of “potentially
interacting” clones when plated on selective medium. Approximately 130 of these
clones were further confirmed as “potentially interacting” using a Lac Z reporter assay.
The aims of this thesis were then:
AIM 1) To examine 60 of these “potentially interacting” clones to determine that they
are “true positives”. This was performed by plasmid rescue from unique clones,
cotransformation of the purified library plasmid with the CaR-tail plasmid into yeast
and verification of interaction using a Lac Z reporter system. Library plasmids
confirmed as positive underwent sequence analysis and database assessment. A
secondary aim was to determine the unique binding site in the CaR-tail for each of the
confirmed interacting proteins by CaR-tail deletion mapping using the yeast two-hybrid
system.
AIM 2) To examine in more detail two of the partner binding proteins identified above,
(1) Filamin A, a cytoskeletal protein shown previously to interact with the CaR and
influence CaR-mediated cell signalling
38
and
(2) Testin, a LIM domain containing, focal adhesion protein also known to have effects
on the cytoskeleton.
For filamin A, two interacting clones, found to be different to those published
previously, were examined for their ability to directly bind to the CaR-tail using
pulldown techniques and the implications for multiple filamin A binding sites for the
CaR on receptor function discussed.
For testin the aims were to:
(a) Determine the importance of the integrity of the second zinc finger of LIM domain 1
on testin – CaR binding using alanine scan site directed mutagenesis studies.
(b) Examine the ability of testin to interact with the CaR both directly, using pulldown
studies, and in a mammalian system by coimmunoprecipitation studies.
(c) Examine the colocalisation of the CaR and testin in mammalian cells using confocal
microscopy.
(d) Examine the effect of testin on CaR-mediated signalling pathways, specifically the
ERK and Rho signalling pathways using a Western blot-based technique and SRE-
luciferase reporter system, respectively.
(e) Examine the effect of testin on cell morphology and the cytoskeleton, specifically
focal adhesions and actin stress fibre assembly, using testin shRNA knockdown studies
in conjunction with fluorescence microscopy.
39
CChhaapptteerr 22 Materials and Methods
2.1 Materials
2.1.1 Reagents
Item Supplier
Acrylamide (30% Acrylamide/Bis Solution) Bio-Rad Laboratories
Agarose Promega
Ampicillin Sigma-Aldrich
Ammonium persulphate Sigma-Aldrich
Antipain dihydrochloride Sigma-Aldrich
Aprotinin Sigma-Aldrich
Bacto Agar Becton Dickinson
Bacto Peptone Becton Dickinson
Bacto Tryptone Becton Dickinson
Bacto Yeast Extract Becton Dickinson
BCA Protein Assay kit Pierce
Benzamidine (hydrochloride:hydrate) Sigma-Aldrich
β-mercaptoethanol Sigma-Aldrich
BigDye Terminator version 3.1 PerkinElmer
5-Bromo-4-chloro-3indolyl Sigma-Aldrich
B-Dgalactopyranoside (X-gal)
Bovine Serum Albumin New England Biolabs
Bromophenol blue Sigma-Aldrich
Calcium Chloride BDH Chemicals
Coomassie Brilliant Blue R-250 Sigma-Aldrich
Deoxynucleotide triphosphates Promega
Dimethyl sulphoxide BDH Chemicals
Dithiothreitol Sigma-Aldrich
DNA ladder (1 Kb Gibco BRL) Invitrogen
DMEM Thermo Electron Company
DMEM – Calcium Free Invitrogen
Enhanced Chemiluminescence Reagent PerkinElmer
(Western Lightning Plus)
40
Ethylenediaminetetra-acetic Acid AnalaR
Ethanol (Absolute and 95%) Biolab
Ethidium Bromide Calbiochem
Expand High Fidelity PCR System Roche Diagnostics
Fetal calf serum (FCS) Invitrogen
G418 (disulfate salt, cell culture tested) Sigma-Aldrich
Glacial Acetic Acid BDH Laboratory Supplies
Glass beads, acid washed Sigma-Aldrich
D-(+)-Glucose Sigma-Aldrich
DO Supplement -Leu/-Trp/-Ura Clontech
Glutathione Sigma-Aldrich
Glutathione Sepharose 4B Amersham Biosciences
Glycerol Ajax Finechem
Glycine ICN Biomedicals
Goat serum Sigma-Aldrich
HEPES Sigma-Aldrich-USA
Hydrochloric acid (32% (v/v)) Ajax Finechem
Hygromycin B Sigma-Aldrich
Isopropanol Rowe Scientific
Hybond-C super nitrocellulose membrane Amersham
Hyperfilm™ ECL Amersham
Isopropanol β-thiogalactopyranoside Promega
Kanamycin sulphate Sigma-Aldrich
Leupeptin Sigma-Aldrich
Lipofectamine 2000 Reagent Invitrogen
Lithium acetate (dihydrate) Sigma-Aldrich
Luciferase Assay Substrate Promega
Lysozyme Sigma-Aldrich
Magnesium Chloride Sigma-Aldrich
Magnesium Sulphate Sigma-Aldrich
Methanol Ajax Finechem
Opti-MEM Invitrogen
Penicillin-Streptomycin (cell culture tested) Sigma-Aldrich
Pepstatin A Sigma-Aldrich
Phenylmethylsulfonyl Fluoride Roche Diagnostics
41
Polyethylene Glycol 3350 Sigma-Aldrich
Ponceau S George T. Gurr Ltd.
Precision Plus Protein Dual Color Standards Bio-Rad
Prolong Gold Reagent Invitrogen
Protein G sepharose Amersham Biosciences
Protein low molecular weight markers Amersham Biosciences
Potassium Chloride Merck
Potassium Phosphate BDH Chemicals
Qiagen PCR cloning kit Qiagen
Qiagen Maxi Prep kit Qiagen
Qiagen QIAEXII Gel Extraction kit Qiagen
QIAquick PCR purification kit Qiagen
QuikChange Site-Directed Mutagenesis kit Stratagene
Salmon sperm DNA Sigma
Skim milk powder Diploma (Bonland Dairies Pty Ltd)
Sodium Acetate Crown Scientific, WA
Sodium Bicarbonate Ajax Chemicals
Sodium Chloride BDH Chemicals
Sodium Fluoride BDH Chemicals
di-Sodium Hydrogen Orthophosphate BDH Chemicals
Sodium dodecyl sulphate MP Biomedicals, Inc.
NaH2PO4.H2O Merck Pty. Ltd, VIC
Sodium Hydroxide APS Finechem
Sodium Fluoride Ajax Chemicals
Sodium Molybdate BDH AnalaR
Sodium vanadate Sigma-Aldrich
Sodium Dodecyl Sulphate (SDS) MP Biomedicals
N,N,N1,N1-tetramethylethylene (TEMED) Sigma-Aldrich
Tetracycline hydrochloride Sigma-Aldrich
Triton X-100 Roche Diagnostics
Trizma® base Sigma-Aldrich
Trypsin SAFC Biosciences
Tryptophan Sigma-Aldrich
TWEEN 20 Sigma-Aldrich
Urea Sigma
42
Xylene cyanole FF Sigma
Yeast nitrogen base w/o amino acids Becton Dickinson
2.1.2 Plasmids
Item Supplier
pBTM116 Dr Schickwann Tsai, Fred Hutchinson
Cancer Research Centre
pBTM116/CaR-tail Dr Bryan Ward, Sir Charles Gairdner
Hospital
pcDNA3.0/EGFP Karin Kroeger, WAIMR
pcDNA3.1/CaR. FLAG Aaron Magno, Sir
Charles Gairdner Hospital
pET28a/CaR-tail Nuella Cattalini, Sir Charles Gairdner
Hospital
pET-NusA Dr Evan Ingley, WAIMR
pGEX4T.1 Amersham
pSRE-luciferase Prof Jeffrey Pessin, SUNY
pSUPERIOR.retro.neo+gfp Prof Peter Leedman, WAIMR
pVP16 Dr Schickwann Tsai, Fred Hutchinson
Cancer Research Centre
2.1.3 Enzymes
Item Supplier
Alkaline Phosphatase Promega
BamHI Promega
BglII Promega
EcoRI Promega
HindIII Promega
KpnI Promega
NcoI Promega
NotI New England Biolabs
PstI Promega
SalI Promega
T4 DNA ligase Promega
Taq DNA Polymerase Promega
XcmI New England Biolabs
43
XhoI Promega
2.1.4 Cell lines
Item Supplier
HEK293 Assoc Prof Karin Eidne, WAIMR
HEK293-CaR (G418 resistant) Prof Arthur Conigrave, University of
Sydney
HEK293-CaR (Hygromycin resistant ) Dr Donald Ward, Manchester
University
PA317 Prof Peter Leedman, WAIMR
2.1.5 Antibodies
Item Supplier
Goat anti-mouse-Alexa Fluor 546 antibody Invitrogen
Goat anti-mouse-HRP antibody Sigma-Aldrich
Goat anti-rabbit-HRP antibody Promega
Mouse anti-α-Tubulin antibody Sigma-Aldrich
Mouse anti-FLAG M2 monoclonal antibody Sigma-Aldrich
Rabbit anti-ERK antibody Promega
Rabbit anti-GFP antibody Santa Cruz Biotechnology
Rabbit anti-P-ERK antibody Promega
Rabbit anti-P-Y118 paxillin antibody Invitrogen
Rabbit anti-mouse Alexa Fluor-568 antibody Invitrogen
Rabbit anti-mouse HRP antibody Sigma-Aldrich
2.1.6 Equipment
Item Supplier
Centrifuge, Beckman AvantiTM J-301 Beckman Coulter
DNA Thermal Cycler Perkin Elmer
Microcentrifuge IEC Micromax Model
#100,120,220 and 240 IEC
Microscope (Model IMT-2) Olympus
Olympus IX81 inverted microscope Olympus
PTC-100 Programmable Thermal Cycler MJ Research, Inc.
POLARstar OPTIMA plate reader BMG Labtechnologies
44
Varian Spectrophotometer Series 634 Varian Technologies
Scion Imaging Software Scion Corp
Sonifier Cell Disruptor B15 Branson
X-Ray Processor, AGFA CP1000 Scanner AGFA
2.1.7 Commercial Suppliers
Company Address
AGFA Morstel, Belgium
Ajax Chemicals NSW, Australia
Amersham Buckinghamshire, UK
BDH Chemicals Vic, Australia
Becton Dickinson CA, USA
Beckman Coulter USA
Biolab Vic, Australia
Bio-Rad Laboratories CA, USA
BMG Labtechnologies Offenburg, Germany
Branson CT, USA
Calbiochem USA
Clontech USA
Crown Scientific NSW, Australia
Diploma Vic, Australia
George T. Gurr Ltd. London, England
ICN USA
IEC USA
Invitrogen CA, USA
Merck Vic, Austalia
MJ Research Inc. (Bio-Rad) MA, USA
MP Biomedicals, Inc OH, USA
New England Biolabs, Inc MA, USA
Olympus Japan
Perkin Elmer MA, USA
Pierce IL, USA
Promega Wisconsin, USA
Qiagen Hilden, Germany
Roche Diagnostics IN, USA
45
SAFC Biosciences KS, USA
Santa Cruz Biotechnology CA, USA
Scion Corp USA
Sigma-Aldrich MO, USA
Stratagene CA, USA
Thermo Electron Company Vic, Australia
Varian Technologies CA, USA
Whatman® International Ltd Maidstone, England
2.2 Methods
2.2.1 General Methods
2.2.1.1 Tissue Culture Methodology
2.2.1.1.1 Maintenance of Cell Lines
Frozen 1 ml stocks of cells stored in liquid nitrogen were rapidly thawed at 37oC and
added aseptically to prewarmed DMEM (Doublecco’s Modified Eagle Medium)
supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin and 10 μg/mL
streptomycin in a 75 cm2 cell culture flask.
Mammalian cell lines were generally maintained in DMEM supplemented with FCS
and antibiotics in 75 cm2 flasks and incubated at 37°C with 5% CO2. Cells were
passaged at subconfluent levels of approximately 90%. Medium was aspirated from the
flasks containing the cells, which were then washed twice with sterile PBS. After
washing, 2 mL of 1 x trypsin/EDTA solution was added to the cells and incubated for 5
min at 37°C. The trypsin/EDTA was then deactivated with 10 mL DMEM supplemented
with FCS and antibiotics and transferred to a sterile 15 mL falcon tube for
centrifugation at 1, 000 rpm for 1 minute at room temperature. Following this, the
medium was aspirated and the cells were resuspended in 10 mL of fresh medium and 1
mL of the cell suspension transferred to a new flask containing fresh DMEM
supplemented with FCS and antibiotics.
To generate frozen aliquots of cells, following trypsinisation, as described above, the
cell pellets were resuspended in a volume of freezing medium (DMEM with 25% FCS
and 10% DMSO) and aliquoted into cryotubes. The cryotubes were placed in a
polystyrene rack and placed at -70°C overnight before being placed in liquid nitrogen
for long-term storage.
46
2.2.1.1.2 Transfection
Cells were grown to approximately 50-60% confluency in 25 cm2 flasks. Routinely, 5
μg of plasmid DNA was diluted into 500 μL of Opti-MEM and combined with 500 μL
of Opti-MEM containing 15 μL of Lipofectamine2000. This mixture was incubated for
20 min at room temperature. Media was then aspirated from the cells and replaced with
4 ml of DMEM containing 10% FCS but no antibiotics. The 1 ml reactions containing
the DNA and Lipofectamine2000 were then added to the cells and incubated for 4 hr at
37oC with 5% CO2. The medium was again aspirated from each flask and replaced with
5 ml of DMEM supplemented with FCS and antibiotics. Generally, cells were
incubated for 48 hr at 37oC with 5% CO2 prior to cell lysis or other studies.
2.2..1.1.3 Lysis of Cultured Mammalian Cells
Following transfection cells were grown for 48 hr in DMEM with 10% FCS, 100
units/mL penicillin and 100 μg/mL streptomycin before culture medium was removed
from flasks and cell monolayers washed twice with 1 ml of ice-cold PBS, while on ice.
Cells were then lysed in 500 μl of cell lysis buffer (150 mM NaCl, 20 mM Tris pH 6.8,
10 mM EDTA, 1 mM EGTA, 1% Triton X-100,) with 100 mM iodoacetamide and the
protease inhibitors 1 mM PMSF, 0.01 mg/ml aprotinin, antipain, and leupeptin, and 0.1
mg/ml pepstatin A. Lysates were transferred to eppendorf tubes and passed through a 25
gauge needle 10 times on ice, prior to centrifugation at 14 000 rpm for 30 min at 4 °C.
Cleared lysates were transferred to fresh eppendorf tubes for the determination of
protein concentration using the Pierce BCA Protein Assay kit prior to
coimmunoprecipitation.
2.2.1.2 Transformation of Competent Cells
An appropriate aliquot, between 2 and 10 μL of plasmid DNA was added to either 100
or 200 μL of competent bacterial cells, either XL1-Blue or BL21 codon (+) and kept on
ice for 10 min before being heat shocked for 90 sec at 42oC. The competent bacterial
cells were then placed on ice for 2 min before having 800 μL of warmed 2xYT media
added. The reaction was then incubated for 1 hr at 37oC with shaking. Following the
incubation, the cells were spun down at 7,500 rpm for 30 sec in a microcentrifuge at
room temperature and 900 μL of the supernatant was removed and the pellet was
47
resuspended in the remaining 100 μL before being plated out onto LB or 2xYT agar
plates with the appropriate antibiotic.
2.2.1.3 Plasmid DNA Preparation
Small scale production of plasmid DNA was performed using the Wizard® Plus SV
Miniprep DNA Purification System Kit (Promega). A single isolated bacterial colony
was used to inoculate 5 mL of LB with appropriate antibiotic selection and grown
overnight at 37oC with shaking. The next day the culture was centrifuged and DNA
extracted from the pellet according to the manufacturer’s instructions. Purified DNA
was generally eluted into 80 μL of sterile ddH2O except when the DNA was to be used
in the cotransformation of yeast when it was eluted into 50 μL of sterile ddH2O.
Large scale production of plasmid DNA was performed using the Qiagen Maxiprep Kit
according to the manufacturer’s instructions with some minor alterations. A single
isolated bacterial colony was used to inoculate a starter culture of 5 mL of LB with
appropriate antibiotic selection that was grown for approximately 4 hr at 37oC with
shaking. At the end of this incubation the starter culture was transferred to a 3 L flask
containing 500 mL of LB with appropriate antibiotic selection and grown overnight at
37oC with shaking. The following day the cells were pelleted by centrifugation at 4,000
rpm in a Sorvall RC-3 Centrifuge for 20 min at 4°C. The supernatant was removed and
the pellet of cells processed for plasmid DNA extraction in accordance with the kit’s
instructions. The sample was then centrifuged at 4,000 rpm in a Sorvall RC-3
Centrifuge for 30 min at 4°C and the supernatant added to a QIAGEN-tip 500 column,
which had been equilibrated with Buffer QBT.
After the sample had passed through, the column was washed twice with Buffer QC.
Plasmid DNA was eluted from the column with 15 mL of Buffer QF and then
precipitated with 10.5 mL of isopropanol. The DNA was centrifuged at approximately
11,000 rpm for 30 min at 4 °C in an Avanti J-30I Beckman centrifuge using a JA30.50
rotor. The supernatant was removed and the pellet was resuspended in 5 mL of 70%
ethanol and aliquotted into eppendorfs to be centrifuged at 13,200 rpm in a
microcentrifuge at 4oC. The supernatant was removed and the DNA pellet was allowed
to air-dry at room temperature. Once all of the ethanol had evaporated, the DNA in each
eppendorf was redissolved in ddH2O and pooled together into a final volume of 600 μL.
The DNA was quantitated and its quality verified by agarose gel electrophoresis.
48
2.2.1.4 Quantitation of DNA
DNA was quantitated by spectrophotometric analysis using a Varian spectrophotometer.
DNA was diluted with sterile ddH2O and placed in a quartz cuvette where the
absorbance was measured at the ultraviolet wavelength of 260 nm and where 1 OD260
was specified as 50 μg/mL for double stranded DNA and 33 μg/mL for single stranded
oligonucleotides. A reading at the wavelength 280 nm was also taken to measure the
purity of the DNA preparation. An OD260/OD280 value of approximately 1.8 indicated a
pure DNA preparation.
2.2.1.5 Agarose Gel Electrophoresis
DNA was routinely run on a 1% (w/v) agarose gel with agarose dissolved in 1 x TAE
buffer and containing 0.4 μg/mL ethidium bromide for DNA visualisation. A 1 Kb
DNA ladder was run alongside the DNA samples for size determination. Gels were
electrophoresed in a DNA electrophoresis mini-sub DNA tank (Bio-Rad) in 1 x TAE
buffer at 100 volts (V). DNA bands were visualised on a UV transilluminator and
photographed using an IBI Quick Shooter Polaroid camera.
2.2.1.6 Purification of DNA
2.2.1.6.1 Purification of DNA from Agarose Gels
Following electrophoresis on an agarose gel, DNA bands were extracted and purified
using the QIAGEN QIAEX II Gel Extraction Kit according to the manufacturer’s
specifications. Briefly, up to 250 mg of agarose containing the appropriate DNA band
was excised from the gel using a sterile scalpel and placed into eppendorf tubes for
processing. The agarose was first solubilized in 500 μl of QX1 buffer. The DNA was
then absorbed to 20 μl QIAEX II beads and washed once in QXI buffer, then twice with
wash solution containing ethanol before the pellet was dried. DNA was eluted with 20
μl of ddH2O.
2.2.1.6.2 Purification of DNA Using the QIAquick PCR Purification Kit
DNA was purified from restriction enzyme digestions, dephosphorylation reactions or
PCR reactions using the QIAGEN PCR Product Purification kit according to the
manufacturer’s specifications. Briefly, 5 volumes of Buffer PB were added to 1 volume
of the reaction and mixed by inversion. This mixture was then applied to the supplied
spin column and centrifuged at 13,200 rpm for 1 min. DNA bound to the column
49
membrane was then washed in 0.75 ml of Buffer PE and centrifuged at 13,200 rpm for
1 min. Finally, DNA was eluted in 30 μl of ddH20.
2.2.1.7 Ethanol precipitation of DNA
The volume of DNA suspension was made up to 20 μL with ddH2O and 2.5 volumes of
ethanol and 0.1 volumes of 3 mM sodium acetate pH 5.2 were then added and the
sample was incubated for 15 min at room temperature. The sample was then spun for 30
min at 32,000 rpm in a microcentrifuge at room temperature. Following the spin, the
supernatant was removed and the pellet was washed with 70 μL of 75% ethanol. The
sample was then centrifuged at 32,000 rpm in a microcentrifuge for 10 min at room
temperature and the supernatant was again removed. The pellet was then placed into a
desiccator to dry for 30 min.
2.2.1.8 Restriction Enzyme Digestion
Routinely, restriction enzyme digests were performed either as 20 μL or 40 μL reactions
containing 1 x reaction buffer, 1.5 to 5 μg DNA and 10 to 20 units of enzyme.
Reactions were incubated for between 3 to 18 hr at 37oC. Digests requiring two
different restriction enzymes were performed either simultaneously using a mutually
compatible reaction buffer or sequentially with either an ethanol precipitation or agarose
gel extraction step to purify the initial restriction enzyme digestion products as
described in 2.2.1.7 and 2.2.1.6.1 respectively.
2.2.1.9 Dephosphorylation of 5’-Ends
To prevent re-circularisation during ligation, vector DNA digested with a single
restriction enzyme was treated with 20 units of alkaline phosphatase for 1 hr at 37oC to
dephosphorylate 5’ ends. The alkaline phosphatase was inactivated by incubation at
75oC for 10 min. The DNA was then purified from the dephosphorylation reaction
using the QIAGEN PCR Product Purification Kit as described in 2.2.1.6.2.
2.2.1.10 Ligations
DNA ligations were performed using T4 DNA ligase (Promega) in 10 µL reactions
containing 1 x reaction buffer, 3 units of T4 ligase and a vector:insert molar ratio of 1:3.
Control ligations, where insert DNA was excluded, were run alongside all test ligations.
Reactions were incubated at 15ºC overnight and then transformed into XL1-Blue
competent cells as described in 2.2.1.2.
50
Ligation of PCR products into the pDrive cloning vector (Qiagen PCR cloning kit) was
performed according to the manufacturer’s instructions. Four µL (300-400 ng) of DNA
was added to 1 µL (50 ng) pDrive and 5 µL of the 2 x master mix. Reactions were
incubated at 15ºC overnight and then transformed into XL1-Blue competent cells as
described in 2.2.1.2.
2.2.1.11 Reverse Transcriptase-PCR (RT-PCR)
Reverse transcription was performed using the Sensiscript RT-PCR kit (QIAGEN)
according to the manufacturer’s instructions to generate cDNA from mRNA extracted
from selected cell lines that had been diluted to approximately 25 ng/μl in RNAse free
ddH2O. The 20 μL RT-PCR mix contained 50 ng of mRNA (first denatured at 65oC for
5 min), 1x Buffer RT, 500 nM dNTP, 250 ng of random hexamers, 10 U of RNasin and
1 μL of Sensiscript Reverse Transcriptase. The reverse transcription reactions were
incubated at 37oC for 90 min in a Perkin Elmer DNA Thermal Cycler. The cDNA
generated was amplified by PCR as outlined in 2.2.1.7 with 10 μL of cDNA.
2.2.1.12 PCRs Using a Proofreading Enzyme.
For the amplification of DNA fragments that were used for cloning purposes the
Expand High Fidelity PCR system (Roche) was used as described in the manufacturer’s
instructions. The 50 μL PCR reactions contained in 1 x reaction buffer in ddH2O, 7.5 U
of High Fidelity enzyme, 1.5 mM MgCl, 300 μM dNTP, 400 nM of each of the two
appropriate complementary primers (Appendix 1) and either 10 μL of cDNA or 2 ng of
plasmid DNA. Reactions were overlayed with oil and cycled in the Perkin Elmer DNA
Thermal Cycler for 40 cycles as follows:
Phase Temperature Duration Number of Cycles
A: T ep94oC 1 min 40
T ep68oC 1 min
T ep72oC 2 min
B: Te p72oC 10 min 1
C: Te p4oC indefinite 1
51
2.2.1.13 Site-Directed Mutagenesis
Site-directed mutagenesis was performed using the QuickChange site-directed
mutagenesis kit, which is a polymerase chain reaction (PCR) based system. Primers
used in site-directed mutagenesis are outlined in Appendix 1. The 50 μL PCR reaction
contained 5 μL of 10x reaction buffer, 50 ng of template plasmid DNA, 125 ng of each
of the two complementary mutagenesis primers, 1 μL of dNTP mix and 1 μL of Pfu
Turbo DNA polymerase. As the PCR reaction was conducted in a Perkin Elmer DNA
Thermal Cycler it was necessary to overlay the reaction mixtures with mineral oil to
prevent evaporation. The cycling parameters used were as follows:
Phase Temperature Duration Number of Cycles
A: T ep95oC 30 sec Number 1
B: T ep95oC 30 sec Num 16 or 18
T ep55oC 1 min
Te p68oC 2 min per kb of plasmid length
C: Te p4oC indefinite Number1
It should be noted that in Phase B of the PCR reaction the number of cycles is
dependent on whether a single amino acid is being changed (16 cycles) or multiple
amino acids are being mutated (18 cycles). Following the PCR, the reaction mixture
was incubated at 37oC with 1 μL of Dpn I restriction enzyme for 1 hr to remove
methylated parent DNA and then used to transform XL1-Blue competent cells as
outlined in 2.2.1.2
2.2.1.14 DNA Sequencing
DNA sequencing was performed using the Big Dye Terminator Version 3.1 mix. The 10
μL sequencing reactions contained 2 μL of Big Dye Terminator Version 3.1 mix,
approximately 200 ng of plasmid DNA and 25 ng of the appropriate sequencing primer
(see Appendix 1) made up to a final volume of 10 μL with ddH2O. The PCR reactions
were conducted in a MJ Research, Inc., PTC-100 Programmable Thermal Cycler using
the following cycling conditions:
Temperature Duration Number of Cycles
T ep95oC 30 sec Num 25
T ep49oC 15 sec
Te p59oC 4 min
52
Following the PCR the reaction mixture was purified by ethanol precipitation as
outlined in 2.2.1.X. Samples were sent to the Department of Clinical Immunology,
Royal Perth Hospital to be processed using an ABI Prism 3730 48 capillary sequencer.
Sequences generated were compared to published sequences using ClustalW2 at EBI
Tools (http://www.ebi.ac.uk/Tools/clustalw2/index.html) (Chenna et al. 2003) to align
sequences and the Chromas program (version 1.45) to view the chromatograms
generated.
2.2.1.15 Quantification of Protein Concentration Using a BCA Assay Kit
Protein samples were diluted in the appropriate buffer to a final volume of 50 μL. A
standard curve covering a range of protein standards from 500 μg/mL to 15.6 μg/mL
was generated by performing serial two-fold dilutions of bovine serum albumin (BSA)
in the same buffer in a final volume of 50 μL. By adding 50 parts of Reagent A to 1 part
of Reagent B, the BCA reagent was constituted and then 1 mL of it was added to the
prepared protein samples and standards. This solution was then mixed by gentle
inversion and incubated for 30 min at 37oC. After incubation, samples were allowed to
come to room temperature and the protein was quantitated using a Varian
spectrophotometer measuring the absorbance at a wavelength of 562 nm. The protein
sample concentrations were determined using a standard curve relating to BSA
concentration at OD562 reading.
2.2.1.16 Quantification of Protein Concentration Using the Bradford Assay
BSA was serially diluted in the appropriate buffer to create a standard curve including
points between 3 mg/mL and 0.5 mg/mL. To 1 mL of Bradford Reagent, which had
been freshly filtered and was at room temperature, 5 μL of sample or protein standard
was added. After the solution was mixed by gentle inversion it was incubated for 5 min
at room temperature before its absorbance was measured on a Varian spectrophotometer
at a wavelength of 595 nm. The BSA standard curve was used to assess the protein
concentration of samples.
2.2.1.17 Preparation of Gels and Electrophoresis
Gels were prepared and run on a Mini-PROTEAN® II Dual Slab Cell (Bio-Rad). The
separating gel was prepared with 1 x separating buffer (1.5 M Tris pH 8.8, 0.4% (w/v)
SDS), 7.5%, 10% or 15% acrylamide, 0.03% (w/v) APS, 0.1% (v/v) TEMED made up
in ddH2O to a final volume of 7.5 mL. Following the addition of APS and TEMED the
53
gel was immediately poured into the gel cast. The stacking gel was prepared with 1 x
stacking buffer (0.5 M Tris pH 6.8, 0.4% (w/v) SDS), 4% acrylamide, 0.1% APS (w/v)
and 0.1% (v/v) TEMED made up in ddH2O to a final volume of 3.5 mL. Following the
addition of the APS and TEMED the stacking gel was poured immediately into the gel
cast, overlaying the separating gel, and a comb inserted. Both the separating and
stacking gels set within 1 hr at room temperature. Proteins were electrophoresed in
freshly prepared 1 x SDS-polyacrylamide gel electrophoresis (SDS-PAGE) running
buffer (25 mM Tris, 0.1% (w/v) SDS, 192 mM glycine) at 180 V.
2.2.1.18 Western Blotting
After proteins had been separated on a suitable SDS-PAGE percentage gel, they were
transferred to a Hybond-C super nitrocellulose membrane (Amersham) using a Mini
Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to manufacturer’s
instructions. The membrane was placed into the appropriate blocking solution (see
Appendix 2 for solutions and incubation times used for specific antibodies) and
incubated with rotation for 1 hr at room temperature. The blocking solution was then
removed and the membrane incubated with the primary antibody in the appropriate
buffer. Following the incubation with the primary antibody the membrane was washed 3
times in the appropriate buffer and then incubated with a secondary antibody conjugated
to horseradish peroxidise in the appropriate buffer for 1 hr. After incubation with the
secondary antibody the membrane was again washed 3 times and exposed to enhanced
chemiluminescience reagent for 1 min. The membrane was then exposed to Hyperfilm
Film for a range of times from 10 sec to 5 min. Membranes that were to be probed again
using different antibodies were placed in 50 mL of membrane stripping buffer (62.5
mM Tris-HCl ph 6.8, 100 mM β-mercaptoethanol, 2% SDS) for 30 min at 50oC and
then rinsed in ddH2O prior to undergoing the same treatment as above with the different
antibodies.
2.2.1.19 Densitometry
The Western films were scanned using a Cannon Scanjet scanner and saved as grey
scale tif files. Density of bands was measured using Scion Image software with
correction for background.
54
2.2.1.20 Statistical Analysis
Statistical significance was determined by an ANOVA analysis (p<0.05) using SPSS
version 15.0 software.
2.2.2 Identification of Positive Clones from a Yeast Two-Hybrid Library Screen
2.2.2.1 DNA Extraction from Yeast
In the initial screen of the yeast two-hybrid screen of the EMLC.1 mouse pluripotent
haemopoietic cell line library 130 yeast colonies were identified as potentially
containing library proteins that interact with the CaR-tail and were stored as glycerol
stocks. These glycerol stocks were used to inoculate 4 mL of uracil and leucine
deficient media and grown overnight at 30oC with vigorous shaking at 200 rpm. The
next day 1.5 mL of the overnight culture was spun down in a microcentrifuge at 10,000
rpm for 30 sec at room temperature. The supernatant was removed and the pellet
resuspended in 200 μL of Yeast Lysis Buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1
mM EDTA, 1 % SDS). To the resuspended pellet 200 μL of phenol/chloroform-isoamyl
alcohol (1:1 v/v) and approximately 300 mg of acid washed glass beads were added.
This mixture was then vortexed at high speed for 2 min and then spun down in a
microcentrifuge at 14,000 rpm for 10 min at room temperature. The aqueous phase was
transferred to a fresh tube. 8 μL of 10 M ammonium acetate and 300 μL of chilled
ethanol were added prior to being placed at -70oC for a minimum of 1 hr. The solution
was then spun down at 14,000 rpm in a microcentrifuge for 30 min at 4oC. The
supernatant, containing the extracted plasmid, was then removed and stored at -70oC for
future use. The DNA pellet was washed with 75% ethanol, centrifuged and dried in a
dessicator prior to resuspension in 50 μL ddH2O
2.2.2.2 Profiling of Plasmids by Restriction Enzyme Digestion
Plasmid DNA extracted from yeast colonies was used in PCR reactions to amplify the
library inserts using the M13F and VP16-2 primers (see appendix 1 for details). The
PCR mix contained 5 μL of plasmid DNA, 1 x Promega Taq Polymerase Buffer, 2 mM
magnesium chloride, 300 μM dNTP, 125 ng of both the M13F primer and VP16-2
primers and 0.5 μL Taq polymerase. The PCR conditions used were as follows, 1 cycle
of 2 min at 94oC, 35 cycles of denaturation at 94oC for 15 sec, annealing at 60oC for 20
sec and extension at 72oC for 2 min. Amplified DNA was then digested by the
restriction enzyme Hae III at 37oC for a minimum of 3 hr. Both digested and undigested
PCR products were separated on 1% agarose gels. Both the size of the library insert and
55
its HaeIII digestion pattern were examined to determine which clones were unique and
worthy of further investigated.
2.2.2.3 Plasmid Recovery of Library Clones
An aliquot of 7.5 μL of plasmid DNA from each unique library clone was added to 200
μL of HB101 competent cells on ice and incubated for the 30 min and then heat
shocked for 90 sec at 42oC. The HB101 cells were then placed back on ice for 2 min
before having 800 μL of SOC media added and incubated for 1 hr at 37oC with shaking.
Following incubation the cells were washed twice by resuspension in 1 mL of M9
media after microcentrifugation at 7,500 rpm for 30 sec at room temperature. After the
final wash 850 μL of the supernatant was removed and the pellet was resuspended in the
remaining M9 medium before being plated out onto leucine deficient M9 plates that
contain 100 μg/mL of ampicillin. Plates were incubated overnight at 37oC and colonies
picked the next day for DNA extraction using the Promega Wizard Plus SV Miniprep
DNA Purification System according to the manufacturer’s specifications. The purified
plasmid was digested by the restriction enzyme, Not I, to release inserts. Digestion
products were separated on a 1% agarose gel to check the size of the inserts.
2.2.2.4 Cotransformation of Bait and Library Plasmids with Yeast L40
Verification of the interaction between selected library proteins and the CaR-tail in β-
galactosidase colony lift assays required the transformation of yeast with a combination
of constructs. Briefly, L40 yeast were transformed with one of the following
combinations of constructs:
Library clone/pVP16 and CaR-tail/pBTM116 - test
Library clone/pVP16 and ARLE-1/pBTM116 - negative control
Hsp90(520-724)/pVP16 and Cyp40(185-370)/pBTM116 - positive control
Hsp90(520-724)/pVP16, ARLE-1/pBTM116 - negative control
Library clone/pVP16 alone - negative control
The L40 yeast transformed with only the library clone/pVP16 construct acted as a
control to show that the construct could not intrinsically activate the LacZ gene. The
Hsp90 and Cyp40 cotransformation provided a positive control for the system as these
two proteins have previously been shown to interact using the β-galactosidase colony
lift assay (Carrello et al. 1999). ARLE-1 is commonly used as a negative control to test
for non-specific binding and eliminate false positives (Carrello et al. 1999). Prior to
contransformation, L40 yeast were grown on YPAD plates for roughly 3 days at 30oC.
56
After the L40 yeast had grown to an appropriate size colonies were inoculated into 5
mL of YPAD media. The yeast was grown overnight at 30oC with shaking and were
diluted into 50 mL of YPAD media containing 2% glucose to give an optical density
(OD) between 0.3 and 0.4 at an absorbance of 600 nm. The yeast was incubated at 30oC
with shaking until the OD at absorbance 600 nm reached 0.6. The 50 mL of culture was
then spun down at 2500 rpm for 5 min at room temperature in a Sorvall RC-3
Centrifuge, after which the supernatant was removed and the pellet resuspended in 40
mL of TE buffer (10mM Tris-HCl pH 7.5, 1mM EDTA). The resuspended cells were
centrifuged as before and the supernatant was again removed. The pellet, containing the
L40 cells, was then resuspended in 2 mL of TE buffer containing 100 mM lithium
acetate and left at room temperature for 10 min. Approximately 1 μg of each plasmid
DNA and 10 μL of 10 mg/mL salmon sperm DNA (freshly boiled for 10 min to
denature) was added to 100 μL of L40 competent cells. After the addition of 700 μL of
TE buffer containing 40% polyethylene glycol 3350 and 100 mM of lithium acetate the
L40 competent cells were vigorously vortexed for 10 sec and incubated for 30 min at
30oC with shaking. Following the 30 min incubation 88 μL of dimethyl sulphoxide
(DMSO) was added to the cells which were mixed by inversion. The L40 competent
cells were then heat shocked for 7 min at 42oC and put on ice to quickly bring them to
room temperature. Once at room temperature the cells were spun down in a
microcentrifuge at 7500 rpm for 30 sec at room temperature and then the supernatant
was removed. The pellet was then resuspended 1 mL of TE buffer and microcentrifuged
as before and the supernatant once again removed. This was repeated but only 900 μL
of the supernatant was removed with the pellet being resuspended in the remaining 100
μL of TE buffer and spread on appropriate amino acid deficient plates. All transformed
yeast, with the exception of those transformed only with library clone/pVP16, were
grown on uracil, tryptophan and leucine deficient media to select for yeast that
contained both plasmids prior to the β-galactosidase colony lift assay. As yeast
transformed with library clone/pVP16 alone did not contain the pBTM116 vector they
did not have the capacity to produce tryptophan and were grown on medium lacking
only uracil and leucine. The plates were incubated at 30oC for a period of time ranging
between 3 to 6 days before colonies were of a sufficient size to be used in a β-
galactosidase assay.
57
2.2.2.5 β-galactosidase Colony Lift Assays
Once colonies had grown to a sufficient size a piece of Whatman #5 filter paper, which
has been cut to size, is placed onto the plates containing the colonies. Pressure is evenly
applied to the filter paper to ensure that it becomes evenly wetted and that the colonies
stuck to it. The filter is then removed and placed into liquid nitrogen. The filter paper is
initially placed onto the surface of the liquid nitrogen with the colonies facing up for
several sec and then submerged for 10 sec. The filter paper was removed and allowed to
thaw at room temperature before being placed with the colonies facing up onto
Whatman #1 filter paper that has been soaked with 2 mL of Z buffer (60 mM Na2HPO4,
40 mM NaH2PO4.2H20, 10 mM KCl, 1 mM MgSO4.7H20 38.6 mM β-mercaptoethanol,
25 μM X-Gal). The colonies were monitored for colour change representing β-
galactosidase activity over a period of 4 hr.
2.2.3 Protein Interaction Studies
2.2.3.1 Baculoviral Expression and Purification of His-Tagged CaR-Tail
The BacPAK Baculovirus Expression System (Clontech) was used to generate a His-
tagged CaR-tail as per the manufacturer’s instructions. Briefly, Bacfectin was used to
cotransfect His-tagged CaR-tail/BacPAK9 transfer plasmids and linearised BacPAK6
viral DNA into Sf9 insect cells. After 72 hr the primary virus was collected and
subsequently used to infect 300 mL cultures of exponentially growing Sf9 cells. After 3
days cells were divided into 50mL aliquots and collected by centrifugation in an Avanti
J-30I Beckman centrifuge using a JA30.50 rotor at approximately 7,000 rpm for 5 min.
Once the supernatant had been removed the pellet was stored at -80oC. This work was
conducted by Kendall Walker, Laboratory for Molecular Genetics (Neuromuscular
Diseases), Western Australian Institute for Medical Research.
Purification of the His-tagged CaR-Tail began with the resuspension of an aliquotted
pellet of Sf9 insect cells in 2 mL of Buffer 1 (50 mM NaPO4, 300 mM NaCl and 10 mM
imidazole, pH 8.0). The protease inhibitors phenylmethylsulphonylflouride (PMSF) and
benzamidine were immediately added to the resuspended cells at concentrations of 1
mM and 5 mM respectively. Subsequent to the addition of the protease inhibitors the
cells were incubated with 1 mL of 5 mg/mL of lysozyme in Buffer 1 containing 1% of
Triton X-100 on ice for 5 min. Following the incubation the resuspension was subjected
to sonication by pulsing 5 times at 50% duty cycle with a Branson Sonifier Cell
Disruptor B15 on ice and then centrifuged at 32,000 rpm for 1 hr at 4oC in a Sorval RC-
58
90 ultracentrifuge using a Kontron 65.13 rotor. After the centrifugation PMSF and
benzamidine were added to a final concentration of 1 mM and 5 mM, respectively, to
the supernatant. The supernatant was subsequently divided into 2 aliquots. The aliquots
were then incubated for 1 hr at 4oC with rotation in eppendorfs containing
approximately 200 μL of nickel-nitriloacetic acid (Ni-NTA) agarose beads that had
been washed in Buffer 1 3 times. Between the consecutive washings, the Ni-NTA beads
were centrifuged at 1,200 rpm for 1 min at 4oC in a microcentrifuge. The beads were
then washed 8 times in Buffer 2 (50 mM NaPO4, 300 mM NaCl and 20 mM imidazole,
pH 8.0) with 0.2% of Triton X-100. This was followed by a further 8 washes in Buffer 2
without Triton X-100, before being resuspended in 200 μL of renaturation buffer. The
Ni-NTA beads were then divided into 50 μL aliquots to be used in the pulldown assays
outlined in 2.2.3.5.
2.2.3.2 Bacterial Expression and Purification of His-Tagged CaR-Tail
The CaR-tail/pET28a plasmid construct was expressed in BL21 Codon (+) cells. An
isolated colony containing the plasmid was used to inoculate a 100 mL of 2xYT media
containing 100 μg/mL of kanamycin and incubated overnight at 37oC with shaking at
220 rpm. Following the incubation the overnight culture was centrifuged for 10 minutes
in a Sorvall RC-3 Centrifuge at 4,000 rpm. The cell pellet was resuspended in 2 mL of
2xYT and added to 1 L of 2xYT containing 100 ug/mL of kanamycin. The 1 L culture
was incubated for 90 min prior to the addition of isopropanol β-thiogalactopyranoside
(IPTG) to a final concentration of 0.4 mM and further incubated for 1 hr at 37oC. After
the incubation the cells were pelleted at 3,500 rpm for 15 min. The cells were then
resuspended in 25 mL of denaturatiuon buffer (8 M urea, 137 mM NaCl, 2.7 mM KCl,
4.3 mM Na2PO4, 1.4 mM KH2PO4, 20 mM imidazole, 0.2% Triton X-100 and 2.5 mM
β–mercaptoethanol). Cells were lysed on ice by sonication, 5 pulses at 50% duty cycle,
using a Branson Sonifier Cell Disruptor B15. The lysed cells were then ultracentrifuged
for 1 hr at 32,000 rpm in a Sorval RC-90 ultracentrifuge using a Kontron 65.13 rotor.
The supernatant was incubated with pre-washed Ni-NTA agarose beads for 3 hr at 4oC
with rotation. The beads were then washed 5 times with denaturation buffer, leaving a
pellet to which renaturation buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2PO4, 1.4
mM KH2PO4, 20 mM imidazole, 0.2% Triton X-100 and 2.5 mM β–mercaptoethanol)
was added dropwise while the beads remained on ice. The resuspended Ni-NTA beads
were then centrifuged at 3,000 rpm in the Sorvall RC-3 Centrifuge and washed 5 times
in renaturation buffer before being resuspended in 200 μL of renaturation buffer. The
59
Ni-NTA beads were then divided into 50 μL aliquots to be used in the pulldown assays
outlined in 2.2.3.5.
2.2.3.3 Bacterial Expression and Purification of Glutathione S-Transferase (GST)-
Fusion Proteins
The pGEX4T-1 expression plasmid was used to generate both full length proteins and
protein fragments that contained an N-terminal GST-tag. These GST-fusion protein
plasmid constructs were expressed in BL21 Codon (+) cells. An isolated colony
containing a GST fusion protein plasmid was used to inoculate a 100 mL of 2xYT
media containing 100 μg/mL of ampicillin and incubated overnight at 37oC with
shaking at 220 rpm. Following the incubation the overnight culture was centrifuged for
10 minutes in a Sorvall RC-3 Centrifuge at 4,000 rpm. The cell pellet was resuspended
in 2 mL of 2xYT and added to 1 L of 2xYT containing 100 ug/mL of ampicillin. The 1
L culture was incubated for 90 min prior to the addition of IPTG to a final concentration
of 0.4 mM and a further incubation for 1 hr at 37oC. After the incubation the cells were
pelleted at 3,500 rpm for 15 min. The supernatant was removed and the pellet
resuspended in 15 mL of mouse tonicity PBS (MTPBS) to which was added 100 μL of
100 mM PMSF. The cell suspension was stored at -70oC until required.
To begin the purification of the GST-fusion proteins the pellets were thawed and 130
μL of 1 M DTT, 105 μL of 0.5 M EDTA (pH 8.0), 260 μL of 100 mM PMSF and a
complete protease inhibitor tablet (Roche) was added. The bacterial pellet was lysed
with 1 mL of 20 mg/mL of lysozyme on ice for 5 min followed by sonication on ice (3
pulses 50% duty cycle, using a Branson Sonifier Cell Disruptor B15). Insoluble debris
was sedimented by centrifugation at 32,000 rpm for 1 hr at 4oC in a Sorval RC-90
ultracentrifuge using a Kontron 65.13 rotor. The supernatant was then collected and
placed onto a 400 μL 1:1 suspension of glutathione sepharose beads that had been
washed 4 times with MTPBS. The cell lysate was incubated with the beads for 2 hr at
4oC with rotation. Following the incubation the beads were washed 5 times with
MTPBS containing 1 % Triton X-100, 5 mM DTT, 2 mM EDTA and complete protease
inhibitor and subsequently washed 5 more times with MTPBS containing 5 mM DTT, 2
mM EDTA and complete protease inhibitor but no 1% Triton X-100. The beads were
transferred to eppendorfs and protein was eluted from the beads with 500 μL of 10 mM
glutathione in 50 mM Tris pH 8.0 for 15 min at 4oC. Eluates were both purified and
concentrated in Buffer A (10 mM Tris-HCl pH 7.3, 100 mM KCl) using a Centricon
60
microconcentration column. Concentrations of recovered protein were then measured by
using the Bradford Protein Assay.
2.2.3.4 Alternate Purification Method for GST-Testin
An isolated colony containing the plasmid encoding the GST-testin fusion protein was
used to inoculate 50 mL of 2xYT media containing 100 μg/mL of ampicillin and
incubated overnight at 37oC with shaking at 220 rpm. Following overnight incubation, 5
mL of the culture was used to inoculate a fresh 500 mL of 2xYT containing 100 μg/mL
of ampicillin, which was then incubated at 37oC until the OD at absorbance 600 nm
reached 0.6. IPTG, to a final concentration of 0.4 mM, was added and the culture
incubated for 3 hr at 37oC with shaking at 220 rpm prior to being centrifuged for 10
minutes in a Sorvall RC-3 Centrifuge at 4,000 rpm. The supernatant was removed and
the cell pellet was frozen at -70oC. After thawing on ice, the pellet was resuspended in
10 ml of ice-cold STE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl)
containing 0.1 mg/ml lysozyme and 10 mM DTT and placed on ice for 15 min. The
resuspension was sonicated 3 times at 25 % duty cycle with a Branson Sonifier Cell
Disruptor B15 on ice and then centrifuged at 32,000 rpm for 1 hr at 4°C to pellet debris.
The supernatant was then topped up to 20 ml with STE buffer containing of 2 % v/v
Triton X-100. This solution was incubated for 30 min at room temperature with
agitation. A volume of 1 mL of glutathione sepharose 4B beads pre-washed 3 times in
ice-cold PBS was incubated with the supernatant for 1 hr with rotation at room
temperature. The beads were pelleted and then washed three times in PBS. The GST
fusion protein was eluted in 500 μl of 20 mM glutathione in Tris-HCl, pH 8.0 for 15
min at room temperature and then concentrated using a Centricon microconcentration
column.
2.2.3.5 Pulldown Assay with His-Tagged CaR-Tail
Aliquots of Ni-NTA agarose beads (50 μL) with or without bound baculovirally or
bacterially derived His-tagged CaR-tail were resuspended in 500 μL of Buffer A and 25
μg of a GST-fusion protein or GST alone was added. The mixtures were incubated
overnight at 4oC with rotation. The following day, samples were washed 5 times with 1
mL of Buffer A containing 0.2% Triton 100-X and complete protease inhibitor and then
5 times with 1 mL of Buffer A containing only complete protease inhibitor. After the
final wash, bound proteins were eluted from the NI-NTA agarose beads with 50 μL of
Laemmli sample buffer and boiled for 5 min. The NI-NTA agarose beads were spun
61
down in a microcentrifuge and 40 μL of eluted protein was loaded onto a 15% SDS-
PAGE gel to be separated by electrophoresis.
2.2.3.6 Staining and Preservation of Polyacrylamide Gels
The staining of SDS-PAGE gels was performed by incubation of the gel in Coomassie
Brilliant Blue R-250 stain (0.5% (w/v) Coomassie Brilliant Blue R, 10% (v/v) acetic
acid glacial, 30% (v/v) isopropyl alcohol) for 20 minutes at room temperature with
gentle agitation. Gels were then rinsed with Coomassie destain solution (20% (v/v)
methanol, 5% (v/v) acetic acid glacial) to remove excess Coomassie stain before being
incubated with Coomassie destain solution at room temperature with gentle agitation
until protein bands were visible and background staining was minimal.
2.2.3.7 Coimmunoprecipitation
In order to first remove non-specific binding proteins, protein G sepharose beads (40 μl
of a 1:1 slurry) were prepared by washing 3 times in 1 ml of cell lysis buffer and 2 mg
of protein from the extracted cell lysate, was added and the volume made up to 1 ml
with cell lysis buffer containing protease inhibitors. The lysate-bead suspension was
incubated for 1 hr at 4oC with rotation before centrifugation at 14,000 rpm for 2 min at
4oC in a microcentrifuge and the supernatant transferred to a fresh eppendorf tube. The
cleared cell lysate were incubated overnight with rotation at 4oC with 5 μg of either
anti-Flag antibody or anti-GFP antibody for immunoprecipitation of CaR-FLAG or
EGFP-testin respectively. Lysate containing antibody was then incubated with 40 μl of
a 1:1 slurry of pre-washed (as above) protein G sepharose beads for 1 hr at 4 °C. The
bead mixtures was then centrifuged for 1 min at 8,000 rpm at 4οC in a microcentrifuge
and the supernatant discarded. The protein G sepharose beads were washed 6 times
with cell lysis buffer containing protease inhibitors. After the final wash, the
supernatant was discarded and the bound antibody and proteins were eluted into 50 μl
Laemmli sample buffer for 5 min at room temperature. Following centrifugation at
13,200 rpm for 1 min at room temperature the supernatant was loaded onto an SDS-
PAGE gel for electrophoretic separation of protein before transferring to a nitrocellulose
membrane and immunodetection of specific protein.
62
2.2.4 Confocal Microscopy
2.2.4.1 Detection of CaR-FLAG and EGFP-Testin by Confocal Microscopy
Cells transiently expressing CaR-FLAG and EGFP-testin were plated out onto poly-L-
lsine coated coverslips in a 6 well plate 24 hr after transfection. Following a further 24
hr incubation the cells were washed 3 times for 3 min in PBS at room temperature and
then fixed using 4% (v/v) formaldehyde in PBS for 20 min at room temperature. This
was followed by another round of washing in PBS after which the cells were
permeabilized in PBS containing 0.2% (v/v) Triton X-100 at room temperature for 20
min. After permeabilization the cells were again washed in PBS and then blocking
buffer containing 10% (v/v) goat serum and 1% (w/v) BSA in PBS was placed onto the
cells for 1 hr at room temperature. The blocking buffer was removed and the cells were
incubated overnight at room temperature with FLAG antibody diluted 1/250 in PBS
containing 10% (v/v) goat serum and 1% (w/v) BSA. The next day the cells were
washed in PBS and Alexa546-conjugated goat anti-mouse antibody diluted 1/400 in
PBS containing 10% (v/v) goat serum and 1% (w/v) BSA was put onto the cells for 1 hr
at room temperature. After incubation with the secondary antibody the cells were
washed 5 times in PBS for 3 min at room temperature. The coverslips were then
mounted onto slides with antifade mounting media (50 mM Tris-PO4, 50 mM
NaH2PO4.2H2O, 20% (w/v) polyvinyl alcohol, 30% (w/v) glycerol) and sealed with nail
polish. CaR-FLAG was detected at wavelength 568 nm, while EGFP-testin was
detected at 488 nm using a Bio-Rad 1024 UV confocal microscope.
2.2.5 Detection of Signalling Pathway Activity
2.2.5.1 ERK Assay
HEK293 cells stably expressing the CaR were grown in 25 cm2 flasks and upon
reaching approximately 60% confluency were transfected with either EGFP-
testin/pcDNA3 or the vector control, EGFP/pcDNA3. On the day after transfection the
cells were trypsinised and resuspended in 10 mL of DMEM without CaCl2 (Invitrogen)
that had been supplemented with 10% BSA, 100 units/mL penicillin and 100 μg/mL
streptomycin and 0.5 mM CaCl2. The resuspended cells were then plated into 24 well
plates that had been coated with poly-L-lysine and incubated overnight at 37oC with 5%
CO2. 48 hr after transfection the medium was aspirated and replaced with physiological
saline solution. The cells were incubated in physiological saline solution supplemented
with BSA and 0.5mM CaCl2 for 1 hr at 37oC with 5% CO2 before stimulation.
Duplicate wells of cells were stimulated for 5 min with a range of Ca2+ concentrations
63
with or without phenylalanine in physiological saline solution supplemented with BSA
at 37oC with 5% CO2. After the 5 min the cells were placed on ice and washed once in
ice-cold PBS before addition of 100 μL of MAPK lysis buffer (20 mM Tris-HCl ph 7.4,
150 mM NaCl, 25 mM NaF, 2.5 mM EDTA, 35 mM β-glycerophosphate, 1% (v/v)
Triton X-100, 10% (v/v) glycerol) to each well. The 24 well plates were then stored at -
80oC for at least 16 hr. Once the lysates were thawed the duplicates were spun down
and pooled. Protein concentrations measured using the Pierce BCA Protein Assay kit as
described previously in section 2.2.1.15. Following quantification of protein
concentrations, 25 μg of protein was separated on a 12.5% SDS-PAGE gel and
transferred to a Hybond-C super nitrocellulose membrane (Amersham) prior to
detection of ERK and phosphorylated ERK by western analysis as described in section
2.2.1.18.
2.2.5.2 SRE-Luciferase Assay
EGFP-testin/pcDNA3 or the vector control, EGFP/pcDNA3, was cotransfected with the
SRE-luciferase reporter construct into HEK293 cells stably expressing the CaR grown
in 25 cm2 flasks when they reached approximately 60% confluency. On the day after
transfection the cells were trypsinised and resuspended in 10 mL of DMEM
supplemented with FCS and antibiotics prior to being plated into a 24 well plate that
had been coated with poly-L-lysine. The cells were then incubated overnight at 37oC
with 5% CO2. The following day triplicate wells of cells were dosed with a range of
calcium concentrations for 8 hr at 37oC with 5% CO2 in serum-free DMEM
supplemented with antibiotics. At the end of the incubation the cells were placed on ice
and washed once in ice-cold PBS prior to the addition of 180 μL of luciferase lysis
buffer (30 mM Tris-HCl pH 7.8, 2mM EDTA, 10% (v/v) glycerol, 0.1% Triton X-100)
containing 2 mM DTT to the wells. The 24 well plates were then stored at -80oC for at
least 16 hr after which the lysed cells were thawed and protein concentrations of the
lysates were determined using the Bradford Protein Assay, as described in 2.2.1.16. For
each sample 50 μL of lysate was aliquotted into a well of an OptiPlate™ white 96-well
plate. The plate was then placed into a POLARstar OPTIMA plate reader (BMG
Labtechnologies), which dispensed 50 μL of Luciferase Assay Substrate (Pierce) to
each sample and measured the resulting luminescence produced. Luminescence was
normalised to protein concentrations.
64
2.2.6 Generation of the Testin Knockdown HEK293-CaR Stable Cell Line
2.2.6.1 Cloning of Knockdown Target Sequences
Oligonucleotides (Appendix 1) previously shown to knockdown testin in mammalian
cells (Griffith et al. 2005) were purchased from Sigma-Proligo. The sense and antisense
oligonucleotide were first annealed to each other by adding 3 μg of each oligonucleotide
to annealing buffer (50 mM HEPES pH7.4, 100 mM NaCl) in a total volume of 50 μL
that was incubated at 90oC for 4 min, 70oC for 10 min and 37oC for 20 min before being
cooled at 4oC for 15 min. The annealed oligonucleotides were ligated into the
pSUPERIOR.retro.neo+gfp vector which had been linearised by restriction enzyme
digestion using Hind III and Bgl II in the following reaction: 1 μL linearised
pSUPERIOR.retro.neo+gfp, 2 μL annealed oligonucleotides, 0.2 units of Invitrogen T4
DNA ligase and 2 μL of 5 x reaction buffer, made up to a final volume of 10 μL with
sterile ddH2O. The reaction was incubated overnight at room temperature and then
transformed into 200 μL of XL1 competent cells. DNA extracted from transformed
bacterial colonies was tested for the presence of insert by restriction enzyme digestion
with EcoRI and HindIII, which should release an approximately 287 bp fragment
containing the oligonucleotide insert. The insert of the pSUPERIOR construct was
verified by sequencing with the recommended primers (Appendix 1).
2.2.6.2 Generating the Stable Packaging Cell Line
The PA317 viral packaging cell line produces amphotropic virions upon transfection
with the pSUPERIOR.retro.neo+gfp construct containing insert which are suitable for
infecting human cell lines. PA317 cells were maintained in DMEM supplemented with
10% FCS, 100 units/mL penicillin and 10 μg/mL streptomycin. Lipofectamine2000 was
used to transfect 10 μg of the pSUPERIOR.retro.neo+gfp construct containing insert
that had been linearised by restriction enzyme digestion with ScaI into PA317 cells at
approximately 80% confluency in a 75 cm2 flask. A control flask of PA317 cells were
treated in an identical manner but without the addition of vector. The media from both
flasks was replaced 24 hr after transfection with media containing 400 μg/mL G418.
The G418 containing medium was replaced every two days until all the non-transfected
PA317 cells had died.
2.2.6.3 Retroviral Infection of HEK293-CaR Stable Cell Lines
The PA317 viral packaging cells were taken off G418 selection two days prior to
infecting HEK293-CaR stables. The HEK293-CaR stable cells used for the knockdown
65
studies were hygromycin resistant and generously supplied by Dr Donald Ward, from
Manchester University. PA317 cells at approximately 70% confluency were washed
with PBS and incubated with 7 mL of DMEM supplemented with 10% FCS for 24 hr at
37°C with 5% CO2. Following incubation, the medium referred to as ‘viral soup’, was
removed and filtered through a 0.22 μm Millex filter unit into a sterile 15 mL tube. The
filtered viral soup was placed onto HEK293-CaR stable cells at approximately 70%
confluency and the cells incubated for 24 hr at 37°C with 5% CO2. A control flask of
HEK293-CaR stable cells (without the addition of viral soup) grown in 7 mL of DMEM
supplemented with 10% FCS was incubated alongside the test flask. A second viral
soup was generated by again incubating PA317 viral packaging cells with 7 mL of
DMEM supplemented with 10% FCS for 24 hr at 37°C with 5% CO2. The following
day, the second viral soup was removed and processed as before. The first viral soup
was then aspirated from the HEK293-CaR stable cells and the cells were washed with 5
mL of PBS. As before the filtered viral soup was placed onto the cells and incubated for
24 hr at 37°C with 5% CO2. The media on the control HEK293-CaR stable cells was
replaced with 7 mL of DMEM supplemented with 10% FCS. After the second 24 hr
incubation with the second dose of viral soup, the HEK293 cells were passaged into 2
75 cm2 flasks and maintained in DMEM supplemented with 10% FCS, 100 units/mL
penicillin, 10 μg/mL streptomycin, 400 μg/mL G418 and 200 μg/mL hygromycin.
Initially, medium containing the G418 and hygromycin was replaced daily due to the
high confluency of cells but as the number of surviving cells decreased medium was
replaced every two days. As acute stimulation of the CaR with aminoglycoside
antibiotics, such as G418, has been linked to an increase in cell death, after the first
week, the concentration of G418 was reduced to 100 μg/mL, which is routinely used for
selection of G418-resistant HEK293-CaR stable cells. Approximately 2 weeks after the
viral infection all the non-infected HEK293 cells had died. At this point the cells in the
test flask were sorted by flow cytometry, selecting for EGFP-containing cells
2.2.6.4 Enrichment of EGFP-positive Cells Verification of Testin Knockdown
The expression of EGFP by pSUPERIOR.retro.neo+gfp allowed the infected cells to be
identified by cell sorting. Cells were grown to 90% confluency in a 75 cm2 flask,
trypsinised and resuspended in 5 mL of sorting buffer (25 mM HEPES pH 7.0, 2 mM
EDTA, 1% FCS in 1 x PBS). The cells were then sorted using a Fluorescence Activated
Cell Sorter Vantage Cytometer and cells expressing high levels of EGFP were collected
into a small volume of FCS. Collected cells were then placed into a 25 cm2 flask with 5
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mL DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 μg/mL
streptomycin, 100 μg/mL G418 and 200 μg/mL hygromycin and incubated for 2 weeks
at 37oC and 5% CO2. Cells were then sorted a second time, as described above. After
the second round of sorting, which produced over 90% of EGFP positive cells,
knockdown of testin expression was examined by Western analysis as described in
2.2.1.18.
2.2.7 Studies of Morphological and Cytoskeletal Changes.
Cells at 30% to 40% confluency in either 25 cm2 flasks (for morphology studies) or on
fibronectin-coated coverslips in 6 well plates (for cytoskeletal studies) were incubated
in DMEM without supplementation for 3 hr with or without the addition of magnesium.
Magnesium was used in favour of calcium as the CaR agonist because the media used
during treatment would have to be change from DMEM to an alternative HEPES buffer
if calcium was used as calcium chloride precipitates in DMEM at the concentrations
used for stimulation. For the morphological studies, multiple fields of cells were
photographed using a Sony digital camera attached to an Olympus phase contrast
microscope. Actin stress fibres and focal adhesions were detected by
immunofluorescence staining of cells on fibronectin-coated coverslips as outlined
below. HEK293-CaR stable cells were dual stained with both phalloidin-Alexa Fluor-
568, to detect actin stress fibres, and anti-phospho-paxillin-(Y118) antibody followed
by an Alexa Fluor-488 goat anti-mouse secondary antibody, to detect focal adhesions.
As testin knockdown HEK293-CaR stable cells expressed EGFP, they were stained
individually with either phalloidin-Alexa Fluor-568 only or with anti-phospho-paxillin-
(Y118) antibody followed by an Alexa Fluor-568 rabbit anti-mouse secondary antibody.
Following the 3 hr treatment, cells were washed with PBS and then fixed using 4%
(v/v) formaldehyde in PBS for 5 min at 37oC. Cells were then permeabilized in PBS
containing 0.25% (v/v) Triton X-100 at room temperature for 20 min and washed in 100
mM glycine for 10 min before being washed 5 times with TBS for 5 min. Blocking
buffer containing 10% (v/v) goat serum and 1% (w/v) BSA in TBS was placed onto the
cells for 20 min at room temperature with or without the addition of phalloidin-Alexa
Fluor-568. The blocking buffer was removed and the cells incubated for 1 hr at room
temperature with 2.5 μg/mL anti-phospho-paxillin-(Y118) antibody with 10% (v/v) goat
serum and 1% (w/v) BSA in TBS. This was followed by 3 washes with 1% (v/v) BSA
in TBS for 10 min at room temperature. The cells were then stained with either an
Alexa Fluor-488 or Alexa Fluor-568 rabbit anti-mouse secondary antibody with 10%
67
(v/v) goat serum and 1% (w/v) BSA in TBS for 1 hr at room temperature. This was
followed by 3 washes with 1% (w/v) BSA in TBS for 10 min at room temperature. The
coverslips were then mounted onto slides using Prolong Gold reagent and stored at 4oC.
Images of cells were taken using the Olympus IX81 inverted microscope.
68
CChhaapptteerr 33 Identification of Proteins that Interact with the Intracellular Tail of the
Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen
3.1 Introduction
It was originally believed that GPCRs were only capable of interacting with
heterotrimeric G-proteins. However, the limited number of G-protein subunits, 16 Gα, 5
Gβ and 12 Gγ, suggested that this theory was inadequate to account for the ability of
GPCRs to precisely regulate physiological outcomes via specific signalling pathways
(Bockaert and Pin 1999). More recently, it has been shown that GPCRs are capable of
interacting with a vast array of proteins and it is these interactions that govern various
aspects of receptor biology, including subcellular compartmentalisation, trafficking and
degradation, as well as the regulation of signalling events (Bockaert et al. 2004). While
the intracellular loops of GPCRs have been shown to be important in receptor binding
to G proteins, it is the intracellular tail of GPCRs that has proven to be the most
important region for interaction with binding partners (Bockaert et al. 2003).
As summarised in Chapter 1, a number of proteins have been found to interact with the
CaR using a variety of techniques. To identify proteins that interact with the CaR
intracellular tail, several groups have utilised the yeast two-hybrid system to screen both
parathyroid and kidney cDNA libraries using the C-terminal tail of the CaR (Awata et
al. 2001; Herrera-Vigenor et al. 2006; Hjalm et al. 2001; Huang et al. 2006). The yeast
two-hybrid system has been widely used to examine protein-protein interactions in vivo.
In general, the protein of interest is fused to the DNA binding domain of a transcription
factor, forming the “bait”, while proteins generated from the cDNA clones of the library
are fused to the activation domain of the transcription factor, creating the “prey”. If the
protein of interest and a protein generated from a cDNA clone interact, then the
transcription factor is reconstituted as a functional unit and able to induce transcription
of a reporter gene as outlined in Figure 3.1 (Fields and Song 1989). The design of this
system does however mean that only interactions between two proteins can be observed.
Other drawbacks of the yeast two-hybrid system are the possibility of many false
positives or negatives and as a yeast-based system it is not ideal for the detection of
protein interactions that require post-translational modifications (Bockaert et al. 2003).
70
To date, the yeast two-hybrid cDNA libraries screened with the CaR intracellular tail as
“bait” have been generated from tissues that are important to whole body calcium
homeostasis. In order to identify proteins that may interact with the CaR and affect its
function in tissues and processes that are unrelated to calcium homeostasis our
laboratory performed a LexA-based yeast two-hybrid screen of an EMLC.1 mouse
pluripotent haemopoietic cell line library using the intracellular tail of the human CaR,
amino acids 865-1078, as bait. The EMLC.1 cell line library was generated by Dr Evan
Ingley from the Western Australian Institute for Medical Research and has been
successfully used by a number of groups to identify novel interacting protein partners of
a variety of “bait” proteins (Hunter et al. 2005; Ingley et al. 2000; Lim et al. 2002).
Established by Tsai et al. in 1994, the EMLC.1 cell line is a unique model system for
studying the survival, proliferation and differentiation of haemopoietic stem cells (Tsai
et al. 1994). Evidence of CaR expression in haemopoietic stem cells was obtained in
immunocytochemistry studies using a CaR specific antiserum (House et al. 1997). It
was observed in CaR-deficient mice that, despite being able to successfully migrate
from the liver to bone, haemopoietic stem cells failed to become lodged in the endosteal
niche of bone marrow (Adams et al. 2006). In vitro experiments found that the ability of
haemopoietic stem cells derived from CaR-deficient mice to adhere to either fibronectin
or collagen I was significantly lower compared to those from wild-type mice (Adams et
al. 2006). It has been hypothesised that agents that modulate CaR function could be
employed to alter erythropoiesis in a controlled manner (Drueke 2006).
3.2 Results
In the initial screen of the EMLC.1 cell line library performed by Dr Bryan Ward,
approximately 15,000 interacting clones were generated on selective media (-THULL).
A total of 129 of the larger colonies that tested positive for β-galactosidase activity in a
colony lift assay were stored as glycerol stocks. As part of this thesis 60 of the 129
colonies were examined to distinguish between colonies containing true interacting
proteins of the CaR and false positives.
3.2.1 Verification of Clones
The EMLC.1 cell line library had been cloned into the VP16 “prey” vector. Primers
specific for the VP16 vector were used to generate a PCR product from plasmid DNA
extracted from the yeast colonies that corresponded to the inserted cDNA library clone.
The PCR products were subsequently digested with Hae III and by examining both the
72
size of the PCR products and Hae III digestion profiles run on 1% agarose gels (Figure
3.2) unique clones could be identified from the library screen. Plasmids believed to be
unique, based on the profile of the PCR product and Hae III digestion profiles, were
selected for plasmid rescue and tested in the yeast two-hybrid system by examining β-
galactosidase activity. It would later become apparent that selection of unique clones
according to their PCR product and Hae III digestion profiles did not eliminate multiple
copies of clones being selected for plasmid rescue. This was primarily due to many
colonies containing two different clones that may have resulted in PCR products of the
non-interacting clone being produced in favour of the interacting clone. Library clones
were considered as genuine interacting partners of the CaR if yeast colonies
cotransformed with the library clone and CaR-tail exhibited β-galactosidase activity
equivalent to or greater than the positive control with no significant β-galactosidase
activity observed in the negative controls. A colour change in colonies of the positive
control was routinely observed after approximately 2.5-3 hr. Yeast colonies containing
the CaR and an interacting clone that exhibited a colour change at a time point similar to
the positive control were denoted by a “+”. When test colonies displayed β-
galactosidase activity greater than the positive control they were assigned with either
++, for colonies undergoing a colour change after 1-2.5 hr, or +++, for colonies that
turned blue in the first hour. The combination of DNA profiling and verification of the
interactions in β-galactosidase colony lift assays reduced the 60 isolated clones to 14
unique clones that were identified by sequence analysis. The 14 clones represented
seven different proteins that are presented in Table 3.1 alongside their β-galactosidase
activities relative to the positive control. The seven proteins identified in the yeast two-
hybrid screen as being protein binding partners of the CaR were filamin A, filamin B,
testin, 14-3-3 θ, OS-9, Ubc9 and MPc2.
3.2.2 Mapping of Verified Interacting Proteins of the CaR
Overlapping clones discovered in the library screen aided in defining the region of the
identified protein that is required for binding to the CaR. To more precisely define the
regions of the CaR intracellular tail that are important for the interaction of the receptor
with its interacting partners, deletion mapping studies were conducted using the yeast
two-hybrid system. The relative strength of an interaction between the CaR and its
binding partners is expressed as outlined above.
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3.2.2.1 Filamin A
Two clones of filamin A identified in the yeast two-hybrid library screen that do not
overlap are illustrated in Figure 3.3A. The two clones exhibited greater β-galactosidase
activity (++) than the positive control when tested with full-length CaR-tail by the yeast
two-hybrid system. One filamin A clone predominantly corresponded to the filamin
repeat 11, while the second clone was an unusually large clone that was over 2000 bp in
length and encompassed filamin repeats 16-22. Both clones interacted with the same
minimal region of the CaR intracellular tail, amino acids 965-986, as shown in Figure
3.3B. However, both clones interacted more favourably with the CaR-tail(923-1078)
construct (++) than the CaR-tail(965-1078) truncation (+), which suggests that residues
923-965 of the CaR-tail contain elements that enhance binding to filamin A. Therefore
the optimal binding region is considered to encompass residues 923-986 of the CaR.
3.2.2.2 Filamin B
A single clone of filamin B, presented in Figure 3.4A, was also identified in the library
screen. The filamin B clone encompassed filamin repeat 21 and showed β-galactosidase
activity equivalent to that of the positive control. This filamin B clone was found to
interact with the same CaR-tail truncations as the two filamin A clones at an intensity
comparable to that of the positive control in β-galactosidase colony lift assays.
3.2.2.3 Testin
Based on the β-galactosidase colony lift assays, all three clones of testin depicted in
Figure 3.5A that were isolated from the yeast two-hybrid library screen were found to
interact very strongly (+++) with the CaR-tail when compared to the positive control.
The region of overlap between the three clones corresponds to 61 amino acids that are
located within the C-terminal half of the protein. This region corresponded to parts of
LIM domains 1 and 2. The results of yeast two-hybrid mapping for testin clone (148-
357) and testin clone (254-419) are shown in Figure 3.5B and reveal that the only
truncation that interacted with testin was the CaR-tail(865-922) construct. In the β-
galactosidase colony lift assay, the strength of the interaction between testin and the
full-length CaR-tail (+++) was equivalent to that of the CaR-tail(865-922) truncation
(+++).
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3.2.2.4 14-3-3 θ
A full-length clone of 14-3-3 θ was isolated from the screen of the haemopoietic cell
line library, as shown in Figure 3.6A. The results from the yeast two-hybrid mapping
studies indicate that 14-3-3 θ was only able to interact with the CaR-tail(865-922)
construct as presented in Figure 3.6B. The comparative potency of the interaction
between 14-3-3 θ and either the entire CaR-tail or the CaR-tail(865-922) truncation
were equivalent, but much greater (+++) than the strength of the interaction of the
positive control.
3.2.2.5 OS-9
As can be seen in Figure 3.7A, the three OS-9 clones isolated from the yeast two-hybrid
screen all contained the region present in OS-9 isoform 1 that is deleted in isoforms 2
and 3. The largest identified OS-9 clone, spanning residues 430-652, also covers a
region of OS-9 that is deleted in isoforms 3 and 4. The two other clones did not extend
into this deleted region. The minimal site of interaction as determined by the overlap of
the three clones spanned 107 amino acids between amino acids 530 and 636. All three
OS-9 clones were observed to have a much stronger interaction (+++) with the CaR-tail
in the yeast two-hybrid system than the positive control interaction. The results of yeast
two-hybrid mapping studies using OS-9 clone (430-652) and OS-9 clone (530-667)
(conducted by Honours student Bernadette Pederson) narrowed the region of the CaR-
tail responsible for the interaction with OS-9 to amino acids 965-986 (Figure 3.7B).
3.2.2.5 Ubc9
The 124 amino acids forming the overlapping region of the three Ubc9 clones presented
diagrammatically in Figure 3.8A correspond to almost 80% of the protein. The strength
of binding between the Ubc9 clones and the CaR-tail was much greater than that of the
positive control as measured by the β-galactosidase colony lift assay. Testing the
capacity of Ubc9 to bind to the CaR-tail truncations using the full length Ubc9 clone
revealed that residues between 965 and 986 of the CaR are critical for the interaction
between the two proteins, as shown in Figure 3.8B.
3.2.2.6 MPc2
A single 122 amino acid clone of MPc2 that contained a His-rich region was isolated
from the yeast two-hybrid library screen, as seen in Figure 3.9A. While the interaction
between the MPc2 clone and the CaR-tail (++) was not as strong as some of the other
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clones, it was still substantially greater than that, of the Hsp90 and CyP40 positive
control. In Figure 3.9B it can be seen that of the CaR-tail truncations cotransformed
with the MPc2 clone, only the CaR-tail(923-1078) and CaR-tail(965-1078) truncations
were found to interact with MPc2. The strength of the interaction between the MPc2
clone was greater with the CaR-tail truncation 923-1078 (++) than its interaction with
the 965-1078 CaR-tail truncation (+). This suggests that there are elements within the
923-965 region that enhance binding of the CaR to MPc2. Therefore the optimal
binding region is considered to encompass residues 923-986 of the CaR.
3.3 Discussion
In the yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line
library seven proteins that interacted with the intracellular tail of the CaR were revealed.
Although some of the proteins that interact with the CaR-tail share functional
similarities, they represent a diverse collection of proteins. The variety of functions
associated with these CaR binding partners includes those acting as scaffolding proteins
and those involved in the regulation of intracellular signalling, cytoskeletal
organisation, trafficking, degradation, posttranslational modification and transcriptional
repression. The cellular localisation of the proteins that interact with the CaR ranges
from membrane associated to nuclear. In the yeast two-hybrid mapping studies
presented above all seven proteins were shown to require elements contained within
either residues 865-923 or 965-986 of the CaR-tail that were essential for binding.
These two regions of the CaR-tail display the highest level of conservation amongst the
different species (Figure 1.1). This observation is also true of other reported CaR
binding partners for which their site of interaction within the CaR has been mapped.
AMSH interacts with amino acids 865-894 of the CaR, while dorfin binds to CaR
residues 880-900 (Herrera-Vigenor et al. 2006; Huang et al. 2006). An overview of the
properties and functions of the seven interacting protein partners of the CaR-tail
identified in the yeast two-hybrid library screen will be presented below, with possible
links to the CaR emphasised.
3.3.1 Filamins
A number of filamin homologues have been identified, including the three human
isoforms (Stossel et al. 2001). Filamin A was the first member of the filamin family to
be identified when it was isolated from rabbit macrophages in 1975 (Hartwig and
Stossel 1975). Gorlin et al. characterised the human homologue of filamin A 15 years
85
later (Gorlin et al. 1990). Filamin B was identified in a yeast two-hybrid screen of a
human bone marrow library that used the intracellular tail of glycoprotein Ibα as bait,
while filamin C was detected in differentiating human skeletal muscle cells (Takafuta et
al. 1998; van der Ven et al. 2000). There is inconsistency in the nomenclature used to
label the three human isoforms of filamin as they have all been known by a series of
different names (Stossel et al. 2001). Filamin A has been known as actin binding protein
(ABP), ABP280, filamin 1, non-muscle filamin, and α-filamin. Filamin B has been
referred to as filamin homolog 1, filamin 3 and β-filamin. Filamin C has also been
called ABP-L, filamin 2 and γ-filamin (van der Flier and Sonnenberg 2001). The three
human isoforms of filamin range from 2602 to 2705 amino acids in length and share
appoximately 70% sequence homology (Popowicz et al. 2006). The key structural
features common to all vertebrate filamins are an actin-binding domain, 24 repeat
sequences and two hinge regions (Stossel et al. 2001). The N-terminal actin-binding
domain of filamin is similar to those present in other actin filament-binding proteins and
is responsible for the interaction between filamin and the F-actin (van der Flier and
Sonnenberg 2001). Beginning after the actin-binding domain the 24 repeats within the
mammalian filamins consist of approximately 96 amino acids that form antiparallel β-
sheets, which overlap to create an overall rod structure (Popowicz et al. 2006). The two
hinge regions between filamin repeats 15-16 and 23-24 are the least conserved parts of
filamin containing only approximately 50% homology between the three human
isoforms (van der Flier and Sonnenberg 2001). It is the final repeat after the second
hinge that is important for the dimerisation of filamin (Stossel et al. 2001).
Over 30 proteins have been found to interact with the vertebrate filamins, with most
interacting with the C-terminal half containing filamin repeats 15-24 (Feng et al. 2005).
The physiological relevance of many of these interactions has yet to be established but
there are cellular processes that have repeatedly been associated with the interaction
between filamin and its binding partners (Popowicz et al. 2006). Filamin has been
shown to act as a scaffolding protein by providing an anchor for membrane associated
proteins to allow for their precise localisation and recruitment of intracellular signalling
components (Stossel et al. 2001). As an actin binding protein, filamin has also been
shown to provide a link between its interacting protein partners and the cytoskeleton,
which facilitates cellular processes involved in cytoskeletal reoganisation (Popowicz et
al. 2006).
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As mentioned in the introduction, the CaR has already been identified as a binding
partner of filamin A and that this interaction is involved in CaR-mediated signalling
cascades (Awata et al. 2001; Hjalm et al. 2001). However, the isolation of a yeast two-
hybrid library clone corresponding to filamin B is the first evidence that the CaR-tail
interacts with other filamin isoforms. Neither of the filamin A clones identified in this
yeast two-hybrid library screen, corresponding to two distinct sites in filamin, overlap
with the previously reported CaR binding domains. Other identified interacting protein
partners that are able to bind to multiple sites within filamin include PKCα and
Forkhead Box C1 (FOXC1) (Berry et al. 2005; Tigges et al. 2003). The interactions
between the CaR-tail and filamin will be further examined in the following chapter.
3.3.2 Testin
Testin, also known as Tes, was originally identified in 1995, when two isoforms of
testin, presumed to be splice variants, were cloned from a mouse testis germ cell library
(Divecha and Charleston 1995). Further testin homologues have since been identified
that exhibit a high degree of conservation between the species, as can be seen in Figure
3.10. The investigation of a fragile site, FRA7G, on chromosomal band 7q31.2, for
genes with a possible role in carcinogenesis, led to the characterisation of the human
homologue of testin (Tatarelli et al. 2000). Human testin mRNA has been detected in an
extensive range of tissue types, with high levels being detected in the thyroid and
pancreas (Tatarelli et al. 2000). The screening of a panel of human tumour cell lines by
Northern analysis with a testin probe revealed that unlike normal tissue there were many
tumour cell lines that did not express testin, which was subsequently shown to be
predominantly due to CpG methylation of the testin gene (Tatarelli et al. 2000). The
lack of testin expression in tumour cell lines led to the hypothesis that testin may
function as a tumour suppressor and in experiments where testin was exogeneously
expressed in the OVCAR5 and HeLa cancer cell lines, a significant reduction in growth
was observed (Tobias et al. 2001). Sarti et al. would later observe that the testin-
negatitive cancer cell lines, T47D and MES-SA, but not the testin-positive MCF-7
cancer cell line, exhibited a reduction in cell growth following the adenoviral
transduction of testin (Sarti et al. 2005). Adenoviral transduction of testin into T47D
and MES-SA cells also impaired tumourigenicity when these cells were inoculated into
nude mice that were treated with carcinogenic agents (Sarti et al. 2005). Both T47D and
MES-SA cells exhibited higher levels of apoptotic markers following the adenoviral
transduction of testin (Sarti et al. 2005). Further evidence of testin’s role as a tumour
87
suppressor was discovered using testin knockout mice in an established model of gastric
cancer where over 81% of mice without testin developed tumours while only 25% of
mice expressing testin developed tumours (Drusco et al. 2005).
The key structural features of testin shown in Figure 3.5A and 3.10 are a prickle,
espinas, testin (PET) domain and three Lin-11, Isl-1, Mec-3 (LIM) domains, which have
led to testin being classified as a member of the PET/LIM domain family, which is a
subgroup of the LIM domain protein family (Gubb et al. 1999; Tatarelli et al. 2000).
The degree of sequence conservation between the LIM domains of PET/LIM domain
family members, testin, prickle, dyxin and LMO6, is higher than when compared to
other members of the LIM domain containing family, such as paxillin and zyxin (Figure
3.11). LIM domains are proposed to be involved in the coordination of protein-protein
interactions and are comprised of two zinc-fingers that fit the following broad
consensus sequence CX2CX16-23HX2CX2CX2CX16-21CX2-3(C/H/D), where X denotes
any amino acid (Zheng and Zhao 2007). Although all of the LIM domains within the
testin fit the LIM domain consensus sequence, there are several members of the
PET/LIM domain family that have a LIM domain 3 that contains an unusually spaced
zinc-finger (Gubb et al. 1999). As the interaction between testin and the CaR was
discovered in a system that utilises the process of DNA-binding it should be noted that
there is no evidence that LIM domains, despite containing zinc-finger DNA binding
motifs, are capable of binding to DNA (Zheng and Zhao 2007).
Although the CaR is the first receptor that has been identified to interact with testin, a
number of other interacting protein partners of testin has already been identified. Two
groups employing different methods identified the known focal adhesion proteins
mammalian enabled (mena), vasodilator-stimulated phosphoprotein (VASP) and zyxin
as being capable of binding to testin (Coutts et al. 2003; Garvalov et al. 2003). In yeast
two-hybrid library screens using full-length human testin as bait, Coutts et al isolated
clones corresponding to zyxin from a human mammary cDNA library and mena, talin,
actin-like 7A and glutamate-receptor-interacting protein 1 from a mouse testis cDNA
library (Coutts et al. 2003). Clones corresponding to testin were also isolated from both
library screens, demonstrating that testin can interact with itself (Coutts et al. 2003).
Garvalov et al. performed pulldown assays on lysates extracted from HeLa cells using
three bacterially produced GST fusion proteins containing the N-terminal half of testin,
the C-terminal half of testin and full-length testin and probed for known focal adhesion
88
Human 1 MDLENKVKKMGLGHEQGFGAPCLKCKEKCEGFELHFCRKICRNCKCGQEEHDVL 54 Mouse 1 MDLETKMKKMGLGHEQGFGAPCLKCKENCEGFELHFWRKICRNCKCGQEEHDVL 54 Chicken 1 MDLESKVKKMGLGHEQGFGAPCLKCKDKCEGFELHFWRKICRNCKCGQEEHDVL 54 Dog 1 MELEAKVKKMGLGHEQGFGAPCLKCKEKCEGFELHFWRKICRNCKCGQEEHDVL 54 Horse 1 MDLETKVKKMGLGHGQGFGAPCLKCKEKCEGFELHFWRKICRNCKCGQEEHDVL 54
Human 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Mouse 55 LSNEEDRKVGRLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Chicken 55 TSNEEDRKVGKLFEDTKYTTLIAKLKNDGIPMYKRNVMILTNPVPAKKNISINT 108 Dog 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Horse 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNISINT 108
PET Domain
Human 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEEAQHRKKQLAKQLPAHDQDPS 162 Mouse 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Chicken 109 VTYEWAPPVQNQTLARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Dog 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Horse 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162
Human 163 KCHELSPREVKEMEQFVKKYKSEALGVGDVKLPCEMDAQGPKQMNIPGGDRSTP 216 Mouse 163 KCHELSPKEVKEMEQFVKKYKSEALGVGDVKFPSEMNAQGDKVHN-PAGNRHAP 215 Chicken 163 KCHELSPNEVKQMEQFVKKYKNEALGVGDVKLPGELETKATDKNNVNSGDRSTS 216 Dog 163 KCHELSPKEVKEMEQFVKKYKSEALGVGDVKLPREMDAQSTNRMYIPGGDRSTA 216 Horse 163 KCHELSPKEVKEMEQFVKKYKNEALGVGDVKLPREMDAQDPNRMCIPGGDRSTT 216
Zinc Finger
Human 217 AAVGAMEDKSAEHKRTQYSCYCCKLSMKEGDPAIYAERAGYDKLWHPACFVCST 270 Mouse 216 AAV-ASKDKSAESKKTQYSCYCCKHTMNEGEPAIYAERAGYDKLWHPACFICST 268 Chicken 217 AAVGAMEDKSADQKASQYSCYRCKLNMKEGDPAVYAERAGYDKLWHPACFVCCT 270 Dog 217 AAVGAMEDKSAEHKRTQYSCYCCKQSMKEGDPAIYAERAGYDKLWHPACFVCST 270 Horse 217 AAVGAKENKLAENKRTQYSCYCCNLSMKEGDPAIYAERAGYDKLWHPACFVCST 270
Zinc Finger Zinc Finger
Human 271 CHELLVDMIYFWKNEKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324 Mouse 269 CGELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 322 Chicken 271 CSELLVDMIYFWKNGNLYCGRHYCDSEKPRCAGCDELIFSNEYTQAEGQNWHLK 324 Dog 271 CHELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324 Horse 271 CHELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324
Zinc Finger Zinc-Finger
Overlap
Human 325 HFCCFDCDSILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378 Mouse 323 HFCCFDCDHILAGKIYVMVTDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 376 Chicken 325 HFCCFDCDCVLAGEIYVMVNDKPVCRPCYVKKHAAICQGCHNAIDPEVQRVTYN 378 Dog 323 HFCCFDCDNILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378 Horse 323 HFCCFDCDSILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378
Zinc-Finger
Human 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-RMS 421 Mouse 377 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKR-MMS 419 Chicken 379 NFNWHATQECFLCSCCSKCLIGQKFMPVEGMVFCSVECKKKMMS 422 Dog 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-MMS 421 Horse 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-MMS 421 Figure 3.10: A comparison of the amino acid sequence of testin from different mammalian species. Sequences of the human, mouse, chicken, dog and horse testin homologues have been aligned and conserved residues have been highlighted in black ( X ). The PET domain is indicated with a yellow line (―). The zinc-fingers of LIM domain 1, 2 and 3 are marked with blue (―), green (―) and purple (―) lines respectively. The region of overlap between the three clones identified in the yeast two-library screen is denoted with a red line (―).
LIM Domain 1 Zinc Finger 1 Testin 236 CYCCKLSMKEGDPAIYAERAGYDKLWHPACF 266 Prickle 126 CEQCGLKINGGEVAVFASRAGPGVCWHPSCF 156 Dyxin 243 CELCKGAAPPDSPVVYSDRAGYNKQWHPTCF 273 LMO6 186 CEECGKQIGGGDIAVFASRAGLGACWHPQCF 216 Paxillin 324 CGACKKPIAGQ--VVTAM--G--KTWHPEHF 348 Zyxin 384 CGRCHQPLARAQPAVRA0-LGQ--LFHIACF 410 Zinc Finger 2 Testin 267 VCSTCHELLVDMIYFWKNEKLYCGRH 292 Prickle 157 VCFTCNELLVDLIYFYQDGKIHCGRH 182 Dyxin 274 VCAKCSEPLVDLIYFWKDGAPWCGRH 299 LMO6 287 VCTTCQELLVDLIYFYHVGKVYCGRH 242 Paxillin 349 VCTHCQEEIGSRNFFERDGQPYCEKD 374 Zy xin 411 TCHQCAQQLQGQQFYSLEGAPYCEGC 436
LIM Domain 2 Zinc Finger 1 Testin 301 CAGCDELIFSNEYTQAENQNWHLKHF 326 Prickle 191 CSACDEIIFADECTEAEGRHWHMKHF 216 Dyxin 308 CSGCDEIIFAEDYQRVEDLAWHRKHF 333 LMO6 251 CQACDEIIFSPECTEAEGRHWHMDHF 276 Paxillin 383 CYYCNGPILDKVVT-ALDRTWHPEHF 407 Zyxin 444 CNTCGEPITDRMLR-ATGKAYHPHCF 468 Zinc Finger 2 Testin 327 CCFDCDSILAGEIYVMVNDK-PVCKPC 352 Prickle 217 CCLECETVLGGQRYIMKDGR-PFCCGC 242 Dyxin 334 VCEGCEQLLSGRAYIVTKGQ-LLCPTC 359 LMO6 277 CCFECEASLGGQRYVMRQSR-PHCCAC 302 Paxillin 408 FCAQCGAFFGPEGFHEKDGK-AYCRKD 437 Zyxin 469 TCVVCARPLEGTSFIVDQANRPHCVPD 495 LIM Domain 3 Zinc Finger 1 Testin 361 CQGCHNAIDPEVQRVTYNNFSWHASTECFLCS 392 Prickle 251 CETCGEHIGVDHAQMTYDGQHWHATEACFSCA 282 Dyxin -------------------------------- LMO6 311 CDGCGEHIGLDQGQMAYEGQHWHASDRCFCC- 341 Paxillin 442 CGGCARAI-------LENYISALNTLWHPECF 472 Zyxin 504 CSVCSEPIMPEPGRDETVRVVALDKNFHMKCY 535 Zinc Finger 2 Testin 393 CCSKCLIGQKFMPVEG-------MVFCSVEC 416 Prickle 283 QCKASLLGCPFLPKQG-------QIYCSKTC 306 Dyxin ------------------------------- LMO6 342 -–SRCGRALLGRPFLPRRG----LIFCSRAC 366 Paxillin 473 VCRECFTP----FVNGSFFEHDGQPYCEV-H 492 Zyxin 536 KCEDCGKPLSIEADDNGCFPLDGHVLCRK-C 565 Figure 3.11: Comparison of LIM domains. The human amino acid sequence of the LIM domains of four PET/LIM domain containing proteins (testin, prickle, dyxin and LMO6) and two less related LIM domain containing proteins (paxillin and zyxin) were aligned. Conserved amino acids are highlighted in black ( X ).
proteins with a panel of antibodies (Garvalov et al. 2003). Full-length testin was found
to interact with only mena, VASP and actin. However, the N-terminal half of testin
interacted with actin, α-actinin and paxillin, while the C-terminal half bound to mena,
VASP and zyxin. A more recent yeast two-hybrid screen of a rat kidney library
identified testin and αII-spectrin as interacting partners (Rotter et al. 2005). Of all the
interacting protein partners of testin identified, only the functional relevance of the
interaction between testin and zyxin has been demonstrated experimentally. Thus, zyxin
has been shown to recruit testin to focal adhesions (Garvalov et al. 2003).
To further understand how testin functions within the cell, Coutts et al. generated Rat-1
fibroblasts that stably expressed GFP-testin and discovered that these cells exhibited
increased cell spreading on fibronectin in comparison to wild-type Rat-1 fibroblasts
(Coutts et al. 2003). This observation was supported by findings from experiments
where the cell spreading of chicken embryo fibroblasts was enhanced by the
overexpression of testin, but cell motility was found to be reduced (Griffith et al. 2004).
A later study, examining HeLa cells in which testin expression had been knocked down
by RNA interference, revealed that there was a significant reduction in the level of actin
stress fibre assembly in HeLa cells not expressing testin compared to wild-type HeLa
cells (Griffith et al. 2005). This decrease in actin stress fibres coincided with a reduction
in RhoA activity in testin knockdown HeLa cells compared to wild-type HeLa cells
(Griffith et al. 2005). Emerging evidence that testin has a role in the regulation of cell
morphology suggested that, like prickle, another PET containing protein with three LIM
domains, testin might be involved in neural crest migration and axial elongation in the
early development of Xenopus laevis (Dingwell and Smith 2006). Morphilino
oligonucleotides were used to reduce the expression of testin in Xenopus laevis
embryos, which resulted in embryos developing a foreshortened head and shortened
antero-posterior axis (Dingwell and Smith 2006). As the CaR has also been shown to be
localised at actin stress fibres, associated with the actin cytoskeleton and changes in cell
morphology, the interaction between the receptor and testin may influence these
processes (Bouschet et al. 2007; Davies et al. 2006; Rey et al. 2005). Therefore, the
interaction between the CaR and testin was selected for further examination, the results
of which are presented in Chapter 5.
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3.3.3 14-3-3 θ
In 1991, 14-3-3 θ, alternatively known as 14-3-3 τ, was isolated from T cells and
identified as a member of the 14-3-3 family (Nielsen 1991). The 14-3-3 family of
proteins is expressed in a wide range of tissue types and all eukaryotic organisms
examined, displaying a high degree of conservation both between species and isoforms
(Wang and Shakes 1996). 14-3-3 proteins predominantly exist as either homodimers or
heterodimers formed by two 14-3-3 isoforms binding at their N-terminal α-helices
(Yaffe et al. 1997). In vitro studies examining dimerisation equilibria revealed that 70%
of 14-3-3 θ existed as a dimer and that half of the dimerised 14-3-3 θ was as a
heterodimer (Yang et al. 2006). Experimental evidence has linked 14-3-3 proteins to a
variety of cellular processes including signal transduction, cell cycle regulation,
apoptosis, stress response, cytoskeletal organisation and malignant transformation (van
Hemert et al. 2001). The 14-3-3 family primarily acts in these processes through its
interaction with protein binding partners, of which over 300 have been identified
(Coblitz et al. 2006). The interaction between 14-3-3 proteins and their binding partners
allows 14-3-3 to modify the activity of enzymes, regulate subcellular localisation or act
as a scaffolding protein to promote further protein interactions (van Hemert et al. 2001).
Although 14-3-3 proteins have been found to bind to specific phosphorylated motifs
within many of their interacting partners there are several instances where 14-3-3 binds
to non-phosphorylated proteins (Mackintosh 2004). The CaR contains neither of the two
common 14-3-3 consensus binding sequences, RSXpSXP and RXY/FXpSXP, but does
contain an alternate binding motif, RX1-2SX2-3S (where either serine can be
phosphorylated), which has been shown to interact with 14-3-3 θ (Liu et al. 1997; Yaffe
et al. 1997). The RX1-2SX2-3S binding motif in the CaR spans residues 890-895, which
lies in the region of the CaR-tail that was found to bind to 14-3-3 θ in the yeast two-
hybrid system. The putative 14-3-3 binding site in the CaR is adjacent to a putative ER
retention signal, RXR, located between residues 897-899 (Chang et al. 2007; Shikano et
al. 2005), which suggests that 14-3-3 θ may be involved in trafficking the receptor from
the ER. 14-3-3 proteins have been shown to facilitate the surface trafficking of
membrane proteins by one of three proposed mechanisms: scaffolding, clamping and
masking (Shikano et al. 2006). It is the last that is the most likely to occur as a result of
the interaction between the CaR and 14-3-3 θ with the latter binding to the CaR and
sterically masking its ER retention signal (Shikano et al. 2006).
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Interestingly, both the CaR and 14-3-3 θ have been implicated in diarrhoeal processes
(Hebert et al. 2004; Patel et al. 2006). Infantile diarrhoea caused by enteropathogenic
Escherichia coli results in the production of attaching–effacing lesions on the surface of
intestinal epithelial cells (McNamara and Donnenberg 1998). A major feature of the
attaching–effacing lesions is the formation of pedestals, structures made from
polymerised actin, ezrin, talin and myosin (Goosney et al. 2001). When Patel et al.
examined pedestal formation using a cell line model, HeLa cells infected with
enteropathogenic Escherichia coli, 14-3-3 θ was found to have a role in the cellular
organisation of actin and the formation of pedestals (Patel et al. 2006). With the
emerging evidence of the role of the CaR in actin organisation (Davies et al. 2006) it is
possible the interaction between the CaR and 14-3-3 θ may be involved in the formation
of pedestals.
3.3.4 OS-9
Su et al. performed chromosome microdissection to directly isolate transcripts from a
homogeneously staining region of an osteosarcoma cell line, OsA-CL, cDNA library
(Su et al. 1994). The clone designated OS-9 contained the partial sequence of a gene
that was uncharacterised at the time, but was subsequently named OS-9 (Su et al. 1994).
The human OS-9 gene is made up of 15 exons that can be alternatively spliced to
produce four OS-9 isoforms, schematically represented in Figure 3.7A (Kimura et al.
1998; Wang et al. 2007). Northern analysis of human tissue revealed that OS-9 is
expressed in all 16 tissue types tested and it has been proposed that transcription factor
binding-motifs, common to a number of housekeeping genes, found in the 5' upstream
region of OS-9 are responsible for its ubiquitous expression pattern (Kimura et al. 1997;
Su et al. 1996). Characterisation of the OS-9 yeast homologue, Yos9, revealed that it is
a glycosylated protein containing an ER retention motif (Friedmann et al. 2002). Both
mammalian OS-9 and Yos9 were shown to be ER-associated proteins by confocal
microscopy, although there is conjecture as to whether mammalian OS-9 is on the
lumenal or cytoplasmic side of the ER membrane (Bernasconi et al. 2008; Friedmann et
al. 2002; Litovchick et al. 2002).
There is evidence from functional studies examining Yos9 that it plays a role in the ER-
associated degradation (ERAD) pathway (Bhamidipati et al. 2005; Kim et al. 2005;
Szathmary et al. 2005). Yos9, as part of a complex with the 3-hydroxyl-3-
methylglutaryl-coenzyme A reductase degradation ligase, is able to select aberrant
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glycosylated proteins for proteasomal degradation via the ERAD pathway (Gauss et al.
2006). Recently, mammalian OS-9, as part of a ubiquitin ligase complex containing
Hrd1 and SEL1L, has also been shown to be involved in detecting and delivering
terminally misfolded or unassembled glycosylated proteins for ERAD (Christianson et
al. 2008). Bernasconi et al. discovered that in response to acute ER stress there is an
increase in OS-9 transcription, which correlates with an increase in the degradation of
misfolded proteins (Bernasconi et al. 2008) Currently there are no known endogenous
substrates of mammalian OS-9 or Yos9 (Mueller et al. 2008). As the CaR is a
glycosylated protein that is processed in the ER it is possible that OS-9 may participate
in a quality control surveillance mechanism that ensures that only correctly processed
CaR is released from the ER to be expressed at the cell surface.
It should be noted that several other proteins, N-copine, meprin β, hypoxia-inducible
factor 1α (HIF-1α) and transient receptor potential vanilloid 4 (TRPV4), have been
identified in yeast two-hybrid library screens as protein binding partners of OS-9 and
that no common functional outcome has been proposed for their interaction with OS-9.
It has been suggested that N-copine, which interacts with a similar region of OS-9 as the
CaR, recruits OS-9 to the plasma membrane in a calcium dependent manner (Nakayama
et al. 1999). Like the CaR and N-copine, meprin β was also found to interact with the C-
terminal domain of OS-9, although the site of meprin β binding in OS-9 is not as well
defined (Litovchick et al. 2002). It was hypothesised that OS-9 is involved in the
transportation of meprin β from the ER to the Golgi and that this may be a function that
OS-9 performs with other membrane proteins, including the CaR (Litovchick et al.
2002). This notion is supported by evidence that the yeast OS-9 homologue may also
have a role in the ER to Golgi trafficking of glycosylphosphatidylinositol-anchored
proteins (Friedmann et al. 2002). As part of a complex, OS-9 was found to regulate the
ubiquitination and degradation of HIF-1α (Baek et al. 2005) and may mediate the
ubiquitination of the CaR in a similar fashion. Alternatively, the interaction between the
CaR and OS-9 may be protective against degradation, as OS-9 has been shown to
protect the TPRV4 protein from ubiquitination and subsequent degradation (Wang et al.
2007).
The expression of OS-9 in a variety of tumour cell lines and the association in
differentiation-induced myeloid leukemia cells between decreased OS-9 expression and
apoptosis suggests a role in cell viability (Kimura et al. 1998). Vourvouhaki et al.
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demonstrated that murine FDC-P1 cells overexpressing OS-9 had a greater rate of
survival than wild-type FDC-P1 cells when exposed to three different apoptosis-
inducing conditions, IL-3 deprivation, TNFα treatment and staurosporine treatment
(Vourvouhaki et al. 2007). The role of OS-9 in apoptosis may be linked to its interaction
with the CaR as Zhang et al. has demonstrated in rat cardiac myocytes that exposure to
ischaemia/reperfusion conditions also induced apoptosis and that this induction was
associated with an increase in CaR expression (Zhang et al. 2006). Under hypoxic
conditions, like ischaemia, a decrease in OS-9 mRNA has been observed that led to a
decrease in HIF-1α degradation (Baek et al. 2005). If OS-9 regulates the degradation of
CaR as it does HIF-1α, then it is possible that in cells subjected to ischaemia a decrease
in OS-9 expression may result in a lower rate of CaR degradation leading to the
observed increase in CaR expression and a subsequent increase in apoptosis.
3.3.5 Ubc9
The human homologue of Ubc9, also known as ubiquitin-conjugating enzyme E2I
(UBE2I ), was originally cloned in 1996 and found to share 56% amino acid sequence
identity with the yeast homologue (Wang et al. 1996). A year later the mouse
homologue was cloned and its nucleotide sequence was found to be identical to the
human homologue (Tashiro et al. 1997). Due to its sequence similarities with other
ubiquitin-conjugating enzymes, Ubc9 was originally believed to be involved in
ubiquitination until Johnson and Blobbel demonstrated that Ubc9 was an enzyme
involved in the conjugation of the small ubiquitin-related modifier (SUMO) protein to
other proteins (Johnson and Blobel 1997). The process of conjugating SUMO to its
target protein is referred to as SUMOylation and as outlined in Figure 3.12 is very
similar to ubiquitination (Zhao 2007). The initial step of SUMOylation is the ATP-
dependent activation of SUMO by the E1 activating enzyme, which is a heterodimer
made of Aos1 and Uba2 (Dohmen 2004). SUMO is then transferred to the E2
conjugating enzyme, Ubc9, from where it is subsequently attached to a lysine on the
target protein by SUMO specific E3 ligase (Johnson 2004). In the final stage of
SUMOylation, Ubc9, the E3 ligase and the protein being SUMOylated are all part of a
single complex (Johnson 2004). SUMOylation has been shown to regulate the
functionality of target proteins by altering their intracellular localisation and influencing
their interactions with other proteins (Zhao 2007).
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Ubc9 has been reported as a binding partner for a variety of proteins in over 20 yeast
two-hybrid library screens, with only a few being verified as SUMOylation targets,
while many remain untested (Melchior 2000). While the components involved in
SUMOylation, including Ubc9, are more highly expressed in the nucleus, suggesting
that it is primarily a nuclear process, they are also expressed in the cytoplasm (Melchior
2000; Tashiro et al. 1997; Zhao 2007). Therefore it is possible that the CaR may interact
in vivo with Ubc9 and undergo SUMOylation. There are SUMOylation recognition
sites, ψKXE/D (ψ is any large hydrophobic residue) (Rodriguez et al. 2001; Sampson et
al. 2001), within the second and third intracellular loop of the CaR, spanning residues
707-710 and 803-806, respectively. It is also possible that the interaction between the
CaR and Ubc9 can affect the receptor’s function in a SUMOylation independent
manner. Collec et al. recently reported that the stability of the glycoprotein
Lutheran/basal cell adhesion molecules in MDCK cells is regulated by its interaction
with Ubc9 without SUMOylation (Collec et al. 2007). The identification of Ubc9 as an
interacting partner of the CaR in the yeast two-hybrid screen may also be a result of the
sequence and structural similarities between Ubc9 and one of the ubiquitin-conjugating
enzymes, Ubc7 or Ubc8, that are likely to bind to the CaR (Figure 3.13) (Huang et al.
2006; Tong et al. 1997).
3.3.6 MPc2
MPc2 was originally identified as the mouse homologue of the polycomb protein found
in Xenopus, XPc (Alkema et al. 1997). Like MPc2, the human homologue, hPc2, was
also identified by Southern blot hybridisation using a cDNA probe encompassing part
of the coding region of XPc to screen a cDNA library in 1997 (Satijn et al. 1997). As
part of the polycomb group family of proteins both MPc2 and hPc2 interact with other
polycomb proteins to form a multiprotein complex that is involved in transcriptional
repression (Schwartz and Pirrotta 2007). When the SUMOylation of the transcriptional
corepressor, CtBP, was examined in COS-1 cells it was revealed that hPc2 was an E3
ligase that acted in conjunction with the E2 conjugating enzyme, Ubc9 (Kagey et al.
2003). Although both MPc2 and Ubc9 were identified in the yeast two-hybrid screen of
a haemopoietic cell line library it is unlikely that the two proteins act together to
sumoylate the CaR in vivo because MPc2 and its human homologue are nuclear proteins
that are not exposed to the intracellular tail of the CaR.
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CChhaapptteerr 44 Investigating the Interaction Between the Intracellular Tail of the Calcium-
Sensing Receptor and Filamin A
4.1 Introduction
As discussed in the preceding chapter, two filamin A clones were identified in a yeast
two-hybrid screen of a haematopoietic cell line library. The fragments of filamin A that
these clones represented were distinct and did not overlap with the filamin interaction
domain identified in previous studies as the CaR binding site (Awata et al. 2001; Hjalm
et al. 2001), indicating that CaR may interact with multiple regions of filamin A. Both
the yeast two-hybrid system and pulldown assays have been used to identify other
proteins that recognise multiple binding sites within filamin A, including FOXC1,
integrin β7 and PKCα (Berry et al. 2005; Kiema et al. 2006; Tigges et al. 2003).
FOXC1 is a transcription factor that was shown to bind to filamin A at three different
sites, residues 571-866, 867-1154 and 1779-2284 (Berry et al. 2005). Kiema et al.
demonstrated that the intracellular tail of integrin β7 bound to filamin repeats 19 and 21
(Kiema et al. 2006). PKCα was found to interact with two functionally important
regions of filamin A. These correspond to part of the N-terminal domain, which
contains the actin-binding site and also part of the C-terminal domain, which is the site
of filamin dimerisation (Tigges et al. 2003).
In addition to the two filamin A clones identified in the library screen described in
Chapter 3, a filamin B clone was also found to interact with the intracellular tail of the
CaR. As previously mentioned, the two filamin isoforms are structurally similar and
share approximately 70% homology (Popowicz et al. 2006). It was revealed in tissues
coexpressing filamin A and B that the isoforms could form heterodimers and that this
interaction could possibly compensate for the functional defects in one isoform arising
from mutations (Sheen et al. 2002). However, there are clear differences between
filamin A and B, including their level of expression and tissue distribution (Takafuta et
al. 1998). More importantly, filamin A and B have exhibited different functional
outcomes in response to the same stimuli (Glogauer et al. 1998). For example, in M2
cells, which express filamin B, but not filamin A, mechanical force was found to induce
cell death. In A7 cells however, which express both filamin isoforms, there was no
increase in the rate of cell death, suggesting that filamin A, but not filamin B, protects
98
against mechanical stress (Glogauer et al. 1998). Experiments examining the PKCα
phosphorylation of the filamin isoforms revealed that both filamin A and C could be
phosphorylated by this kinase, whereas filamin B could not (Tigges et al. 2003). This
selective phosphorylation of filamin isoforms has been proposed to be the mechanism
responsible for the functional differences observed between filamin A and B (Tigges et
al. 2003). The differences in filamin A and B are particularly relevant to their
interactions with the CaR because of several experiments performed in M2 and A7 cells
that showed that filamin A was involved in CaR signalling and receptor stabilisation
(Awata et al. 2001; Zhang and Breitwieser 2005). No compensatory effects from filamin
B were observed in these experiments (Awata et al. 2001; Zhang and Breitwieser 2005).
In light of the above information, the following interaction studies will focus on the
interactions between the CaR and the multiple binding sites within filamin A.
4.2 Results
4.2.1 Construction of Filamin A GST-Fusion Proteins for Pulldown Studies
To further investigate the possibility that the intracellular tail of the CaR can interact
directly with more than one binding site within filamin A, pulldown assays were
performed. As the data from Chapter 3 and published reports (Awata et al. 2001; Hjalm
et al. 2001) suggested that the CaR-tail may bind to three distinct regions of filamin,
three GST-fusion proteins containing the corresponding filamin A fragments were
created for direct interaction studies. The GST-fusion protein containing the filamin A
sequence from the smaller of the two filamin A cDNA clones identified in the yeast
two-hybrid screen was designated GST-Fil11 as the sequence primarily encoded filamin
repeat 11. The identified filamin B interacting clone predominantly encoded for filamin
repeat 21, which is highly homologous to the corresponding filamin A sequence (Figure
4.1). As results from the yeast two-hybrid mapping studies suggested that filamin A
residues 1714-2380, which form filamin repeats 16-22, contained a CaR-tail interaction
domain, a GST-fusion construct containing the equivalent filamin A sequence of the
filamin B cDNA library clone was made and called GST-Fil21. The final GST-fusion
generated was GST-FilH, which contained the minimum CaR-tail binding domain
identified by Hjalm et al and provided a positive control for these direct interaction
studies (Hjalm et al. 2001).
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Filamin A 2172 SPSGKTHEAEIVEGENHTYCIRFVPAEMGTHTVSV 2206 Filamin B 2135 SPSGRVTEAEIVPMGKNSHCVRFVPQEMGVHTVSV 2169 Filamin A 2207 KYKGQHVPGSPFQFTVGPLGEGGAHKVRAGGPGLE 2241 Filamin B 2170 KYRGQHVTGSPFQFTVGPLGEGGAHKVRAGGPGLE 2204 Filamin A 2242 RAEAGVPAEFSIWTREAGAGGLAIAVEGPSKAEIS 2276 Filamin B 2205 RGEAGVPAEFSIWTREAGAGGLSIAVEGPSKAEIT 2239 Filamin A 2277 FEDRKDGSCGVAYVVQEPGDYEVSVKFNEEHIPDS 2311 Filamin B 2239 FDDHKNGSCGVSYIAQEPGNYEVSIKFNDEHIPES 2274 Filamin A 2312 PF 2313 Filamin B 2275 PY 2276 Figure 4.1: Alignment of the filamin B fragment that binds to the CaR-tail with its filamin A counterpart. Sequences corresponding to the filamin A equivalent (2172-2313) of a filamin B cDNA library clone aligned with filamin B (2135-2276). Conserved residues are highlighted in black ( X ), while conserved substitutions are highlighted in grey ( X ).
4.2.2 Purification of His-tagged CaR-tail from Insect Cells
Previous direct interaction studies have used bacterially produced His-tagged CaR-tail
(His-CaR-tail) purified using a denaturation/renaturation method (Hjalm et al. 2001).
The denaturation/renaturation technique was employed because purification of CaR-tail
extracted from bacteria by other methods does not produce a soluble protein. However,
there is great variability in recovery at the renaturation stage of this purification method,
with only a small percentage of protein being renatured and correctly folded. To counter
these deficiencies an alternate technique was investigated, the baculoviral expression
system. Outlined below is the first use of a baculovirus expression vector system to
generate His-CaR-tail in Sf21 insect cells. Eukaryotic proteins expressed in insect cells
produced using recombinant baculovirus undergo post-translational modifications
similar to those in mammalian cells and are therefore more representative of the
mammalian protein than a bacterially produced counterpart (Jarvis and Finn 1995).
Aliquots taken at different stages of the purification process were separated on a 15%
SDS-PAGE gel and visualised using Coomassie staining (Figure 4.2A). Lanes 1 and 2
contain the lysate from insect cells expressing the His-CaR-tail before and after being
incubated with nickel-chelate agarose beads, respectively. His-CaR-tail was eluted from
nickel-chelate agarose beads with imidazole and the purified receptor fragment was
concentrated by Centricon microconcentration. The purified His- CaR-tail,
approximately 35 kDa in size, is shown in Figure 4.2A Lane 4. The purified protein was
confirmed to correspond to the His-CaR-tail by Western analysis using an anti-His
antibody (Figure 4.2B).
4.2.3 Pulldown Assays Performed Using His-tagged CaR-tail Purified from Insect cells
His-CaR-tail recovered from Sf21 insect cells was immobilised on nickel-chelate
agarose beads before being incubated with one of the three purified GST-filamin
proteins or GST alone, as a negative control. Bound proteins were eluted with sample
buffer and separated on a 15% SDS-PAGE gel before being transferred to a
nitrocellulose membrane for Western analysis with an anti-GST antibody. Both GST-
Fil11 and GST-Fil21 were found to interact with the His-CaR-tail, with the latter
displaying a more efficient interaction (Figure 4.3). Neither GST-FilH nor GST alone
were found to bind to the His-CaR-tail. Neither the three GST-fusion proteins nor GST
alone were able to bind to the nickel-chelate agarose beads in the absence of the His-
CaR-tail. The lack of binding of GST-FilH was suprising as it had previously been
shown to bind to bacterially produced His-CaR-tail (Hjalm et al. 2001).
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4.2.4 Pulldown Assays Performed Using His-tagged CaR-tail Purified from Bacteria
In order to verify that the GST-FilH construct could bind to the CaR-tail as reported
previously, the pulldown assays were repeated using His-CaR-tail purified from bacteria
as described by Hjalm et al. His-CaR-tail expressed in bacteria was purified on nickel-
chelate agarose beads according to the denaturation/renaturation methodology for use in
in vitro interaction studies. The immobilised His-CaR-tail protein was then exposed to
each of the three purified GST-fusion proteins or GST, as the negative control. Bound
proteins were eluted with sample buffer and separated on a 15% SDS-PAGE gel before
being transferred to a nitrocellulose membrane. Western analysis using an anti-GST
antibody revealed that all three GST-fusion proteins, GST-Fil11, GST-FilH and GST-
Fil21, bound to the His-CaR-tail, while GST did not (Figure 4.4). Again the GST-Fil21
protein interacted more efficiently with the His-CaR-tail than the GST-Fil11 protein. In
the absence of His-CaR-tail, neither the GST-fusion proteins nor GST bound to the
nickel-chelate agarose beads.
4.3 Discussion
There is an extensive collection of proteins that regulate a variety of the GPCR
properties through their interaction with the receptor’s intracellular tail. These proteins
include filamin A (Hall and Lefkowitz 2002). Filamin A has been shown to interact
with a variety of proteins, ranging from transmembrane proteins, like the dopamine
receptors and integrins, to components of signalling cascades, such as RhoA and Rac1
(van der Flier and Sonnenberg 2001). The CaR was identified as a binding partner of
filamin A in 2001 and studies have revealed that their interaction is necessary for certain
CaR-mediated signalling pathways and protects the CaR against degradation (Awata et
al. 2001; Hjalm et al. 2001; Huang et al. 2006; Pi et al. 2002; Zhang and Breitwieser
2005).
Interestingly, several of the experiments examining the functional implications of the
CaR and filamin A interaction were performed in M2 cells, which do not express
filamin A and A7 cells, which are M2 cells stably expressing filamin A. Both M2 and
A7 cells express filamin B. CaR-mediated ERK signalling was found by two groups to
occur in A7 cells, but is abolished in M2 cells (Awata et al. 2001; Zhang and
Breitwieser 2005). An increased level of CaR degradation was detected in M2 cells in
comparison to that observed in A7 cells (Zhang and Breitwieser 2005). Increases in
receptor degradation in the absence of filamin have also been observed in experiments
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examining the relative degradation rates of the calcitonin receptor in M2 and A7 cells
(Seck et al. 2003). These findings indicate that, in relation to at least some functions of
the CaR, filamin B is unable to compensate for a lack of filamin A and that the
interaction between the CaR and filamin B observed in the yeast two-hybrid studies
presented in chapter 3 may regulate functions of the CaR distinct from those involving
filamin A. Examining the direct interaction of the identified filamin B clone with the
CaR using in vitro pulldown assays will be the first step towards understanding what
those functions might be. Following confirmation of interaction, further studies could
examine filamin B could perform a similar function to filamin A by acting as a
scaffolding protein bound to a unique set of accessory proteins.
Published reports define filamin A residues 1566-1719 as the site of CaR interaction
(Awata et al. 2001; Hjalm et al. 2001), but direct interaction studies presented above
show that the CaR is also capable of binding to two other distinct regions of filamin A,
residues 1193-1312 and 2172-2313. In pulldown experiments using baculovirus
expressed His-CaR-tail both the GST-Fil11 and GST-Fil21 constructs, which contained
filamin A amino acids 1193-1312 and 2172-2313 respectively, bound to the CaR-tail,
while GST-FilH, which contained filamin A residues 1534-1719, did not (Figure 4.3).
The region of filamin A contained in the GST-FilH fusion protein had previously been
shown by Hjalm et al. to bind to the CaR-tail in direct interaction pulldown studies
using bacterially derived His-CaR-tail (Hjalm et al. 2001). Therefore, the in vitro
interaction studies were repeated using His-CaR-tail purified from bacteria to verify the
reported binding between the GST-FilH construct and the CaR-tail. All three GST-
fusion proteins were found to bind to the bacterially derived His-CaR-tail (Figure 4.4).
The His-CaR-tail purified from baculovirus and that expressed in bacteria and isolated
by denaturation/renaturation might be folded differently with the baculovirus expressed
protein being incapable of binding to the GST-FilH. To determine if the difference in
purification methods account for these observations it will be necessary to repeat the in
vitro pulldown assays using baculovirus expressed His-CaR-tail purified by the
denaturation/renaturation methodology. Alignment of the three identified CaR
interaction domains, filamin A residues 1193-1312, 1534-1719 and 2172-2313, revealed
a high level of homology in the region boxed in Figure 4.5. It should be noted that these
direct interaction studies observe protein binding under artificial conditions that are not
mimicked within the cell. Therefore, from the data presented here, it can only be
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Filamin A 1193-1312 1193 ----------------------------------- 1213 1534-1719 1534 ---PFKVKVLPTHDASKVKASGPGLNTTGVPASLP 1565 2172-2313 2172 ----------------------------------- 2186 1193-1312 1193 --------------LTIEICSEAGLPAEVYIQDHG 1213 1534-1719 1566 VEFTIDAKDAGEGLLAVQITDPEGKPKKTHIQDNH 1600 2172-2313 2172 --------------------SPSGKTHEAEIVEGE 2186 1193-1312 1214 DGTHTITYIPLCPGAYTVTIKYGGQPVPNFPSKLQ 1248 1534-1719 1601 DGTYTVAYVPDVTGRYTILIKYGGDEIPFSPYRVR 1635 2172-2313 2187 NHTYCIRFVPAEMGTHTVSVKYKGQHVPGSPFQFT 2221 1193-1312 1249 VEPAVDTSGVQCYG--PGIEGQGVFREATTEFSVD 1281 1534-1719 1636 AVPTGDASKCTVTVS-IGGHGLGAGIGPTIQIGEE 1661 2172-2313 2222 VGPLGEGGAHKVRAGGPGLERAEAGVPAEFSIWTR 2256 1193-1312 1282 AR---ALTQTGGP----HVKARVANPSGNLTETYV 1309 1534-1719 1662 TV----ITVDTKAAGKGKVTCTVCTPDGSEVDVDV 1692 2172-2313 2257 EAGAGGLAIAVEGPSKAEISFEDRKDGSCGVAYVV 2291 1193-1312 1310 QDR------------------------ 1312 1534-1719 1693 VENEDGTFDIFYTAPQPGKYVICVRFG 1719 2172-2313 2291 QEPGDYEVSVKFNEEHIPDSPF----- 2313 Figure 4.5: A comparison of the amino acid sequence of the identified CaR-binding sites within human filamin A. Sequences corresponding to the three sites of CaR interaction identified in filamin A have been aligned. Conserved residues highlighted in black ( X ) while conserved substitutions are highlighted in grey ( X ). A region of high homology is boxed in red ( ).
concluded that there are at least three possible sites of CaR interaction within filamin A
that contain a highly homologous region of approximately 40 residues.
Although several proteins that bind to multiple sites within filamin A have been
identified (Berry et al. 2005; Kiema et al. 2006; Tigges et al. 2003), there has been little
speculation as to the purpose of the multiple binding sites. In relation to the CaR, there
are some possible advantages that may arise from interacting with filamin A at multiple
sites. The protective effect that the CaR interaction with filamin A provides against
degradation (Zhang and Breitwieser 2005) may be a result of the stabilisation of CaR
dimers assuming that the intracellular tail of one receptor binds to one binding site
within filamin A while the other binds to a second binding site within filamin A,
effectively clamping the two receptors (Figure 4.6A). The multiple CaR binding sites
within filamin A may also assist in its role as a scaffolding protein. The CaR regulates a
host of signalling cascades that contain proteins that bind to the C-terminal end of
filamin A, including Rho A and JNK kinase (JNKK) (Huang et al. 2006; Pi et al. 2002).
By binding to an interaction domain closer to the C-terminus, the receptor is brought
into close proximity to components of CaR-mediated signalling pathways (Figure
4.6B). However, the CaR also regulates the function of large, transmembrane proteins
like the Kir4.2 channel, which binds to the C-terminus of filamin A (Huang et al. 2007;
Wang et al. 2004). The distance between the CaR and Kir4.2 would be determined by
the size of the two proteins and where they are embedded in the membrane. Under these
circumstances the CaR may need to bind to an interaction domain closer to the N-
terminus allowing filamin A to reach and interact with Kir4.2 (Figure 4.6C).
In conclusion, two novel sites of CaR interaction have been identified and verified in
filamin A that may allow it to more efficiently stabilise the CaR dimer and/or act as a
more versatile scaffolding protein by regulating the binding location of its interacting
partners.
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CChhaapptteerr 55 Investigation of the Interaction Between the Intracellular Tail of the
Calcium-Sensing Receptor and Testin and the Implications for Cell
Function
5.1 Introduction
As discussed in Chapter 3, the CaR is the first receptor to be identified as a binding
partner of testin. In this chapter, the nature of the interaction between the CaR and
testin, as well as the functional implications of the interaction will be examined. The
lack of testin expression in particular tumour cell lines (Tatarelli et al. 2000) has already
been discussed, but there are also several reports that CaR expression is decreased in a
number of tumours (Farnebo et al. 1998; Haven et al. 2004; Kifor et al. 1996). It has
been suggested that although the loss of functional CaR is not sufficent to initiate
carcinogenesis, the disruption of CaR function is likely to contribute to the aberrant
physiology of tumours (Rodland 2004). Experiments examining the downstream effects
of CaR activation in cancer cell lines have revealed that the receptor regulates the
expression of several genes associated with malignancy, including E-cadherin, β-
catenin and the c-myc proto-oncogene (Bhagavathula et al. 2007; Kallay et al. 1997).
Increases in CaR-mediated secretion of PTHrP have also been observed in a variety of
cancer cell lines including those derived from the breast, prostate and testis (Sanders et
al. 2000; Tfelt-Hansen 2008). As there is evidence that both testin and the CaR have a
role in tumourigenesis it is possible that their interaction may also be relevant to this
process.
A possible mechanism by which the interaction between the CaR and testin effect
tumourigenesis is through apoptosis. In testin-negative breast and uterine cancer cell
lines the exogenous expression of testin resulted in tumour reduction and increased
apoptosis (Sarti et al. 2005). Several groups have shown that the stimulation of the CaR
can induce apoptosis in a range of cell types including osteoblasts, cardiac myocytes
and HEK293 cells stably expressing the CaR (Mentaverri et al. 2006; Wu et al. 2005;
Zhang et al. 2006). However, the Sindbis Virus induction of apoptosis in AT-3 prostate
cancer cells, fibroblasts and HEK293 cells stably expressing the CaR was prevented by
CaR activation (Lin et al. 1998). The interaction between the CaR and testin may
110
regulate whether CaR stimulation results in either the induction or prevention of
apoptosis.
Another commonality between the CaR and testin is that they have both been shown to
localise at actin stress fibres (Garvalov et al. 2003; Griffith et al. 2004; Hjalm et al.
2001). As mentioned in chapter 3, studies investigating the possible function of testin
have found that it has a role in cytoskeletal processes including actin stress fibre
assembly, cell spreading and cell motility (Coutts et al. 2003; Griffith et al. 2004;
Griffith et al. 2005). In HEK293 cells stably expressing the CaR it was demonstrated
that stimulation of the receptor led to changes in cell morphology and actin stress fibre
reorganisation (Davies et al. 2006). An independent study found that the activation of
the CaR in HEK293 cells stably expressing the receptor induced cell motility and
plasma membrane ruffling, a dynamic process involving the formation and retraction of
cytoplasmic protrusions enriched in filamentous actin (Bouschet et al. 2007).
In light of the evidence of overlap between the biological processes that the CaR and
testin are involved in, it is likely that their interaction, identified in the yeast two-hybrid
system, is physiologically relevant in the mammalian cell. The following chapter will
further examine the CaR and testin relationship and its possible role in cell function.
5.2 Results
5.2.1 Calcium-Sensing Receptor and Testin Interaction Studies
5.2.1.1 Yeast Two-Hybrid Mapping
As outlined in chapter 3, three unique overlapping clones of testin were found to
interact with the intracellular tail of the CaR in a yeast two-hybrid screen of a
haemopoietic cell line library. Analysis of the 61 amino acids present in all three testin
clones, shown in Figure 5.1A, revealed that all three contained a common structural
feature, the second zinc-finger of LIM domain 1. To determine if the zinc-finger motif
was required for binding between the CaR-tail and testin or if the CaR-tail simply
recognised the amino acid sequence of the region per se, the yeast two-hybrid system
was used to investigate binding between the CaR-tail and various mutated testin clone
constructs. Site-directed mutagenesis was performed to substitute an alanine at either
Cys269 or His290 within testin clone 148-357 to interfere with the zinc finger’s
capacity to coordinate metal ions and disrupt its formation. Testin clone 148-357 was
chosen because the overlapping region was contained centrally within this construct.
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Results from β-galactosidase assays displayed in Figure 5.1B indicated that neither
mutant construct was capable of binding to the CaR-tail, while the wild-type testin
construct did interact as observed previously. An alanine scan throughout the loop of
the second zinc-finger was conducted using seven mutant constructs of testin clone 148-
357 to establish the importance of the amino acid sequence to the interaction between
the CaR-tail and testin. Residue Gly270 was omitted from the alanine scan because
there is low conservation of that amino acid amongst species, as seen in Figure 3.10.
Neither a single alanine substitution at residue Met276 nor a tandem alanine substitution
at amino acids Leu288/Tyr289 had an impact on the potency of the interaction between
the CaR-tail and testin clone 148-357. Binding potency between the CaR-tail and testin
clone 148-357 mutants with multiple alanine substitutions at either residues
Ile277/Tyr278/Phe279 or Gly283/Lys284 was less than the strength of the interaction
between the CaR-tail and testin clone 148-357, but equivalent to that of the Hsp90 and
CyP40 positive control. The mutation of amino acids Glu271/Leu272/Leu273,
Val274/Asp275 or Trp280/Lys281/Asn282 to alanines within testin clone 148-357
abolished the interaction between the CaR-tail and testin clone 148-357.
In the previous chapter, residues 865-922 of the CaR were identified as interacting with
testin. To further refine this binding region two more CaR-tail truncation constructs
were created, CaR-tail 865-898 and CaR-tail 899-922, and their ablitiy to interact with
testin investigated in the yeast two-hybrid system. Neither construct was found to
interact with testin clone 148-357 in β-galactosidase assays. This suggests elements
from each of the two constructs are required for testin interaction with the CaR-tail.
5.2.1.2 Cloning of Full-Length Human Testin
To further investigate the interaction between the CaR-tail and testin it was necessary to
obtain a full-length clone of testin. Human testin cDNA was generated by reverse
transcription of RNA extracted by Dr Bryan Ward from the MDA MB231 breast cancer
cell line, which had previously been shown to express testin as detected by Northern
analysis (Tatarelli et al. 2000). The cDNA was subsequently amplified by PCR and
cloned into the pDrive vector and sequenced. Following confirmation that the full-
length human testin clone was error-free it was subcloned into appropriate vectors for
expression in bacteria and mammalian cells.
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5.2.1.3 Direct Interaction Studies
In vitro studies using pulldown assays were proposed to examine the direct interaction
between the CaR-tail and full-length testin. These studies required the purification of
soluble CaR-tail and testin proteins. Bacterially derived, soluble His-tagged CaR-tail
was produced using a denaturation/renaturation method. To express testin as a GST-
fusion protein in bacteria the full-length cDNA of human testin was cloned into the
pGEX4T-1 vector. Expression of GST-testin could be induced by treatment with IPTG
(Figure 5.2A), but no soluble GST-testin could be detected (Figure 5.2B). Soluble GST-
testin could be produced using an alternative purification protocol that included the use
of higher levels of detergent, but this increase in detergent interfered with the release of
the GST-fusion protein from the glutathione beads onto which it had been purified
(Figure 5.2C). An alternative approach to expressing insoluble proteins in a soluble
form has been to create fusion proteins that contain the insoluble protein fused to a
highly soluble protein, such as NusA (Lavallie et al. 1993). A construct containing
NusA fused to human testin was generated, but again no soluble protein was detected
(Figure 5.2D) despite reports of NusA fusion proteins being highly soluble (Harrison
2000). Since we were unable to produce bacterially expressed testin proteins that were
soluble, the approaches to demonstrate direct interaction of testin with the CaR-tail were
not pursued further.
5.2.1.4 Coimmunoprecipitation Studies
Confirmation that the interaction of the CaR and testin occurs in vivo requires the
coexpression of the CaR and testin in a mammalian system. The full-length human
clone of testin was cloned into the mammalian expression vector, pcDNA3/EGFP to
allow the N-terminal EGFP-tagging of testin. The EGFP-testin/pcDNA3 construct was
cotransfected into HEK293 cells with a C-terminal FLAG-tagged CaR/pcDNA3.1
construct used previously by this laboratory (Ward et al. 2004). CaR-FLAG was
immunoprecipitated from the lysates of the cotransfected HEK293 cells with an anti-
FLAG antibody and separated on a 10% SDS-PAGE gel prior to blotting on
nitrocellulose. The Western blot probed with an anti-GFP antibody presented in the
upper panel of Figure 5.3A shows that EGFP-testin was detected in the lane containing
coimmunopreciptates from cells cotransfected with the CaR-FLAG/pcDNA3.1 and
EGFP-testin/pcDNA3 constructs. In the remaining lanes it can be seen that when either
CaR-FLAG or EGFP-testin were expressed alone no EGFP-testin was detected. The
reciprocal experiment is shown in the upper panel of Figure 5.3B where EGFP-testin
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was immunoprecipitated with an anti-GFP antibody from the lysates of the
cotransfected HEK293 cells and run on a 7.5% SDS-PAGE gel prior to western analysis
with an anti-FLAG antibody. Coimmunoprecipitation of CaR-FLAG is observed when
both CaR-FLAG and EGFP-testin are coexpressed in HEK293 cells.
5.2.2 Colocalisation of Testin and the Calcium-Sensing Receptor
Confocal microscopy was employed to investigate the cellular distribution of the CaR
and testin and to determine the degree to which the two proteins colocalise. HEK293
cells cotransfected with the following plasmid combinations: CaR/pcDNA3.1 and
EGFP-testin/pcDNA3, CaR/pcDNA3.1 and EGFP/pcDNA3 empty vector or pcDNA3.1
empty vector and EGFP-testin/pcDNA3, were stained with an anti-FLAG antibody and
a goat anti-mouse secondary antibody conjugated to Alexa Fluor-546 to detect the CaR-
FLAG, while EGFP-testin was detected by the fluorescence emitted from the EGFP tag.
In the left column of images in Figure 5.4 the CaR (red) can be observed at both the cell
surface and within the cytoplasm in HEK293 cells expressing CaR-FLAG (A and D).
EGFP-testin (green) in panels B and H of Figure 5.4, is also present at the cell surface
and in the cytoplasm. The distribution of EGFP (green) throughout the cell, seen in
panel E of Figure 5.4, suggests that the EGFP tag does not interfere with the targeting of
testin to specific subcellular locations. Colocalisation of the CaR-FLAG and EGFP-
testin can be observed either at or near the cell surface in Panel C of Figure 5.4.
5.2.3 The Effects of Testin on Calcium-Sensing Receptor Activated ERK Signalling
As previously mentioned, both the CaR and testin have been shown to be involved in
the process of apoptosis (Sarti et al. 2005; Sun et al. 2006). While it has been
demonstrated that the CaR is able to induce apoptosis via the ERK pathway, the
mechanism by which testin promotes apoptosis is unknown (Sarti et al. 2005; Sun et al.
2006). As testin was found to bind to the membrane proximal region of the CaR
intracellular tail, a region implicated in the receptor’s transduction of the ERK
signalling pathway (Zhang and Breitwieser 2005), the influence of the interaction
between the CaR and testin on ERK signalling was investigated. HEK293 cells stably
expressing the CaR that had been transfected with either EGFP-testin/pcDNA3 or
EGFP/pcDNA3 empty vector were dosed with a range of extracellular calcium
concentrations and the effect on ERK phosphorylation examined by Western blot
analysis using a phosphospecific anti-ERK antibody. Figure 5.5A presents a Western
blot representative of triplicate experiments that show that increasing the concentration
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of extracellular calcium increases the level of phosphorylated ERK produced in
HEK293-CaR cells. However, when comparing HEK293-CaR cells either with or
without EGFP-testin that have been treated with the same amount of extracellular
calcium, there is no significant difference in the level of ERK phosphorylation as
determined by densitometry. This result is depicted graphically in Figure 5.5B.
Although the overexpression of testin does not influence CaR-mediated ERK
phosphorylation in response to extracellular calcium the possibility remained that the
interaction between the CaR and testin may impact on the receptor’s ability to be
allosterically modulated, for example by amino acids (Mun et al. 2004). A
representative Western blot of triplicate experiments is presented in Figure 5.6A and
shows that in the presence of 10 mM phenylalanine there is still a dose dependent
increase in ERK activity in response to increasing concentrations of extracellular
calcium. Densitometric analysis did not reveal any significant difference in the level of
ERK phosphorylation detected in EGFP-testin expressing HEK293-CaR cells compared
with those expressing EGFP empty vector alone, as represented in Figure 5.6B.
5.2.4 The Effects of Testin on Calcium-Sensing Receptor-Mediated Rho Signalling
Transient intracellular calcium oscillations generated by amino acid stimulation of the
CaR were shown to require an intact actin cytoskeleton and functional Rho kinase (Rey
et al. 2005). However, stimulation of the CaR with extracellular calcium produces
sinusoidal intracellular calcium oscillations that are unaffected by disruption to the actin
cytoskeleton, but still require functional Rho kinase (Rey et al. 2005). Activation of the
CaR in HEK293 cells stably expressing the receptor with either extracellular cations or
amino acids also revealed differential signalling in response to the alternate stimuli
(Davies et al. 2006). Treatment of HEK293 cells stably expressing the CaR with either
calcium or magnesium resulted in activation of the Rho kinase that led to actin stress
fibre assembly and morphological changes, while treatment with amino acids failed to
activate processes related to increased Rho activity (Davies et al. 2006). As there is
evidence suggesting a role for both the CaR and testin in changes to the cytoskeletal
structure of cells it was hypothesised that testin may have an effect on CaR-mediated
changes to cell morphology by influencing the receptor’s ability to activate Rho
signalling. Pi et al. has previously demonstrated that treatment of HEK293-CaR cells
with increasing levels of extracellular calcium revealed a dose-dependent increase in
Rho activity as measured using an SRE-luciferase reporter construct (Pi et al. 2002).
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Using the same SRE-luciferase reporter construct, generously supplied by Professor
Jeffrey Pessin from SUNY at Stony Brook, it was found that when HEK293-CaR cells
transfected with EGFP-testin were treated with elevated levels of extracellular calcium,
the Rho activity detected was significantly greater than that observed in HEK293-CaR
cells not transfected with EGFP-testin at the same concentrations of extracellular
calcium, as shown in Figure 5.7A. As evidence that the increase in Rho activity was due
to the overexpression of testin enhancing CaR-mediated Rho signalling and not the
overexpression of testin being solely responsible for the increase in Rho activity,
HEK293 cells, which do not endogenously express the CaR, were transfected with
EGFP-testin or EGFP and exposed to 5 mM of extracellular calcium. There was no
increase in Rho activity above basal observed in HEK293 cells either expressing EGFP-
testin or EGFP upon stimulation with 5 mM extracellular calcium (Figure 5.7B),
indicating that the CaR is required for the overexpression of testin to impact on Rho
signalling.
5.2.5 The Calcium-Sensing Receptor Regulates Changes in Cell Morphology
In order to examine the influence that the interaction between the CaR and testin may
have on the morphology and cytoskeletal organisation of cells, it was necessary to first
replicate the findings of Davies et al, which showed that the CaR had a role in
regulating these processes (Davies et al. 2006). HEK293 cells stably expressing the CaR
were incubated for 3 hr in serum-free DMEM containing a range of extracellular
magnesium concentrations: 0.8 mM, 2.8 mM, 5.8 mM and 8.8 mM. In Figure 5.8 a
graded decrease in the number of cellular extensions formed correlated with an increase
in the extracellular magnesium concentration to which HEK293-CaR cells were
exposed, with virtually no extensions detected at 8.8 mM extracellular magnesium.
Phalloidin-Alexa Fluor-568 was used to detect the formation of actin stress fibres in
HEK293-CaR cells incubated in serum-free DMEM for 3 hr in the presence of
extracellular magnesium at a range of concentrations which included 0.8 mM, 5.8 mM
and 8.8 mM. There are no detectable actin stress fibres in HEK293-CaR cells treated
with 0.8 mM extracellular magnesium, as seen in Figure 5.9A, but in HEK293-CaR
cells incubated with higher concentrations of extracellular magnesium, 5.8 mM and 8.8
mM, there is evidence of actin stress fibre formation, as shown in Figures 5.9C and E,
respectively. These results are consistent with those observed by Davies et al. 2006. As
paxillin phosphorylated Tyr118 is a marker of focal adhesions, the HEK293-CaR cells
described above were also stained with an anti-phospho-paxillin-(Y118) antibody
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followed by an Alexa Fluor-488 rabbit anti-mouse secondary antibody to detect focal
adhesions, which are anchorage points of actin stress fibres (Pellegrin and Mellor 2007).
When HEK293-CaR cells are stimulated with increasing concentrations of extracellular
magnesium, 0.8 mM, 5.8 mM and 8.8 mM, the focal adhesions become more defined at
points at the cell surface, as seen in Figure 5.9B, D and F, respectively.
5.2.6 The Impact of Testin Knockdown on HEK293 Cells Stably Expressing the
Calcium-Sensing Receptor
Having shown that the overexpression of testin accentuates CaR-mediated Rho
signalling in HEK293 cells stably expressing the receptor in section 5.2.5, the effects of
removing testin from HEK293-CaR cells was investigated using shRNA knockdown
technology, specifically the OligoEngine pSUPER RNAi system. The HEK293-CaR
cells used to this point were unsuitable for this process as they were already under G418
selection and the generation of stable testin knockdowns using the OligoEngine
pSUPER RNAi system required the cells to be G418 treated to select for those
expressing the pSUPERIOR.retro.neo+gfp vector. Therefore HEK293-CaR cells that
were under hygromycin selection were used. Incorporation of the
pSUPERIOR.retro.neo+gfp vector into HEK293-CaR cells following viral-mediated
infection resulted in HEK293-CaR cells that also stably expressed EGFP and shRNA-
containing sequences homologous to testin. HEK293-CaR cells that had incorporated
the testin knockdown shRNA were selected by flow cytometry based on their
expression of EGFP. In the initial collection of testin knockdown HEK293-CaR stable
cells it was found that 12.9% of cells sorted contained EGFP. These cells were further
cultured and subjected to a second round of cell sorting to produce a culture in which
98.7% of cells were found to express EGFP. Proteins isolated in lysates from HEK293-
CaR cells and HEK293-CaR stables expressing testin knockdown shRNA were
separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. An anti-testin
antibody was used to detect endogenous testin in both lysates by Western Blot analysis
and showed that efficient knockdown of testin has occurred in cells treated with testin-
targeting shRNA (Figure 5.10).
Results from triplicate experiments examining Rho kinase activity in testin knockdown
HEK293-CaR cells are shown in Figure 5.11. Following an 8 hr incubation with 0.5
mM (basal level) extracellular calcium the level of Rho kinase activity measured in
testin knockdown HEK293-CaR cells using an SRE-luciferase reporter assay was
126
approximately half of that observed in the wild-type HEK293-CaR cell line. However,
upon stimulation of the CaR with 5 mM extracellular calcium there was no significant
difference observed between wild-type and testin knockdown HEK293-CaR cells, as
both exhibited an increase in Rho kinase activity approximately 15-fold greater than the
activity observed in control cells at 0.5 mM extracellular calcium.
Testin knockdown HEK293-CaR cells were examined under serum-starved conditions
to determine if they undergo the same changes in morphology and cytoskeletal structure
as wild-type HEK293-CaR cells in response to the same degree of magnesium
stimulation. Following 3 hr of serum starvation in the presence of 5.8 mM extracellular
magnesium, both wild-type HEK293-CaR cells and testin knockdown HEK293-CaR
cells exhibited similar morphologies as shown in Figure 5.12C and D, respectively.
However, unlike the HEK293-CaR cells grown for 3 hr under serum-free conditions
with an extracellular magnesium concentration of 0.8 mM (Figure 5.12A), testin
knockdown HEK293-CaR cells (Figure 5.12B) showed no signs of stellation, with a
morphology resembling that observed in both serum-starved HEK293-CaR cells and
testin knockdown cells treated with 5.8 mM extracellular magnesium (Figure 5.12C and
D). There were also differences in the cytoskeletal organisation between the wild-type
HEK293-CaR cells and testin knockdown cells, observed following serum starvation for
3 hr at an extracellular magnesium concentration of 0.8 mM. As previously observed,
HEK293-CaR cells showed no signs of actin stress fibre organisation when serum
starved in the presence of 0.8 mM extracellular magnesium, but testin knockdown cells
did exhibit actin stress fibre formation under these conditions, as seen in Figure 5.13A
and B, respectively. Punctate staining, indicating phosphorylated paxillin, was observed
throughout wild-type HEK293-CaR cells treated for 3 hr with 0.8 mM extracellular
magnesium in serum-free media (Figure 5.14A). However, in testin knockdown cells
there is a dramatic decrease in staining corresponding to phospho-paxillin-(Y118) in the
cytoplasm and the staining is concentrated at points at the cell surface representing focal
adhesions, circled in Figure 5.14B. In the presence of 5.8 mM extracellular magnesium
both wild-type HEK293-CaR cells (Figure 5.13C and Figure 5.14C) and testin
knockdown cells (Figure 5.13D and Figure 5.14D) form actin stress fibres and focal
adhesions. The cytoskeletal structure of serum-starved testin knockdown cells in the
presence of 0.8 mM extracellular magnesium (Figure 5.14B) resembles that of
magnesium stimulated HEK293-CaR cells (Figure 5.14D). Table 5.1 summarises the
results of the testin knockdown studies presented in this section.
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5.3 Discussion
5.3.1 The Calcium-Sensing Receptor and Testin Interaction
The results from a yeast two-hybrid library screen presented in Chapter 3 showed that
the CaR interacted with the LIM domain focal adhesion protein testin. This is the first
described instance of testin interacting with a membrane-bound receptor. Studies
conducted in yeast indicated that the interaction between the CaR and testin required the
second zinc finger of the first LIM domain to be intact, with several residues within the
zinc finger being essential for interaction. The site of testin binding was mapped, using
the yeast two-hybrid system, to the membrane proximal region of the CaR-tail. Direct
interaction studies using pulldown assays could not be performed due to the insolubility
of testin fusion proteins, but the interaction between testin and the CaR was confirmed
in coimmunoprecipitation experiments using lysates from mammalian cells
cotransfected with expression plasmids for both proteins. Confocal microscopy studies
revealed that both the CaR and testin colocalised either at or near the cell surface. As
testin bound to a region of the CaR shown to be important for CaR-mediated signalling,
the role of the CaR and testin interaction was examined in relation to two signalling
pathways. The overexpression of testin did not alter the level of CaR-mediated ERK
phosphorylation, but was found to enhance the level of CaR-induced Rho kinase
activity. As the CaR had been recently shown to regulate cell morphology and
cytoskeletal structure via the Rho pathway, the effect of testin knockdown in relation to
these processes was examined. Interestingly, preliminary analysis has shown that the
cell morphology and cytoskeletal structure observed in non-stimulated testin
knockdown HEK293-CaR cells, mimicked the effect seen in CaR agonist stimulated
HEK293-CaR cells.
5.3.2 Sites of Interaction Between the Calcium-Sensing Receptor and Testin Identified
in the Yeast Two-Hybrid System
Testin contains LIM domains, which are protein-protein binding motifs comprised of
two zinc-fingers (Zheng and Zhao 2007). Testin also contains a PET domain, a
characteristic that is shared by the other members of a subfamily of the LIM domain
containing family, including prickle and dyxin (Bekman and Henrique 2002). Yeast
two-hybrid studies presented in Chapter 3 indicated that only the second zinc-finger of
LIM domain 1 was required for binding with the CaR-tail. A comparison between the
amino acid sequences of the LIM domains of the PET/LIM family revealed that the
second zinc-finger of LIM domain 1 displayed the highest degree of conservation
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throughout the family (Figure 3.11). The zinc-finger structure was demonstrated to be
critical for the interaction between the CaR-tail and testin as this interaction was lost
when the zinc-finger was disrupted by mutation of either one of the cysteines or
histidines that coordinate the zinc ion. The results of an alanine scan through the zinc-
finger revealed that binding between the CaR-tail and testin was unaltered upon
substitution of Met276 with alanine, suggesting that this residue is not involved in the
interaction between the CaR and testin. Simultaneous substitution of Leu285 and
Tyr286 also had no influence on binding, indicating that the large hydrophobic residues
located at the end of the second zinc-finger do not participate in CaR recognition.
Alanine scans through the remaining segments of the second zinc finger eliminated
binding between the receptor and testin consistent with a disruption of the structural
integrity of the zinc finger motif or loss of a binding recognition site. Further mapping
studies are necessary to identify specific amino acids essential for CaR-testin
interaction. Since residues within the second zinc finger, corresponding to the putative
CaR interaction domain are highly conserved throughout mammalian species (Figure
3.10) and with other members of the PET/LIM domain family (Figure 3.11), it is
possible that other family members may also interact with the CaR. Further dissection
of the established interaction domain for testin within the membrane proximal region of
the CaR, corresponding to residues 865-922, failed to identify a more specific contact
domain, suggesting that multiple elements might exist within this 57-residue region or
that the individual fragments used in the study disrupted the recognition site.
5.3.3 Calcium-Sensing Receptor and Testin Interaction Studies
Having demonstrated that the CaR and testin interacted within yeast, further studies
were aimed to establish a direct interaction between the two proteins and whether this
occurred in mammalian cells. The insolubility of testin was a problem for the direct
interaction pulldown studies that, despite a number of alternate approaches, could not be
overcome. It was not possible to perform the reciprocal experiment as GST-CaR was
also found to be insoluble (data not shown). Without information from in vitro
pulldown studies it can not be determined if the CaR and testin bind directly or through
an intermediary protein as part of a complex. A combination of coimmunoprecipitation
and colocalisation studies revealed that the interaction between the CaR and testin
occurred in a mammalian system (Figures 5.3 and 5.4). The colocalisation of the CaR
and testin at the cell membrane also meant that the interaction between the two proteins
might regulate the signalling cascades initiated by the CaR in response to extracellular
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stimuli. In order to strengthen the evidence of CaR and testin colocalisation at the cell
membrane, future studies utilising cell surface biotinylation of the receptor could be
performed. Briefly, these experiments would be performed in HEK293 cells
cotransfected with CaR-FLAG and EGFP-testin. Biotinylation would be used to label
all cell surface proteins prior to whole cell lysis. EGFP-testin would then be
immunoprecipitated from the lysates with an anti-GFP antibody and separated on a
SDS-PAGE gel before being transferred to nitrocellulose. Streptavidin would then be
used to detect biotinylated CaR, if it coimmunoprecipitates with EGFP-testin,
demonstrating that the two proteins interact at the cell surface. As other cell surface
proteins to which testin binds may also coimmunoprecipitate, the identity biotinylated
CaR should be confirmed using the FLAG antibody.
5.3.4 The Effects of Testin Binding on Calcium-Sensing Receptor Regulated Signalling
5.3.4.1 Calcium-Sensing Receptor-Mediated ERK Phosphorylation is Unaffected by
Testin Overexpression
There was no obvious candidate CaR-mediated signalling pathway to examine in
relation to the interaction between CaR and testin, as testin has not been previously
associated with a receptor. However, testin was first identified as a tumour suppressor,
an attribute that has recently been associated with the CaR, suggesting possible overlap
between the mechanisms by which the two proteins act (Bhagavathula et al. 2005;
Tatarelli et al. 2000). Sarti et al. showed that the adenoviral transduction of testin into
the T47D and MES-SA cancer cell lines impaired tumourigenicity and increased
apoptosis (Sarti et al. 2005). The CaR has been shown to increase apoptosis in response
to agonist stimulation via the ERK pathway (Sun et al. 2006). The yeast two-hybrid
mapping studies indicated that the membrane proximal region of the CaR-tail contained
the testin interaction domain, a region previously demonstrated to be essential to CaR-
mediated ERK signalling (Zhang and Breitwieser 2005). Experiments using an ERK
assay revealed that overexpression of testin did not alter the level of CaR-mediated
ERK activity in response to either extracellular calcium alone or in the presence of the
amino acid, phenylalanine (Figures 5.5 and 5.6). These findings suggest that testin
neither promotes CaR-induced ERK phosphorylation nor interferes with the interaction
between the CaR and components of the ERK cascade, despite binding to the membrane
proximal region of the CaR-tail critical for CaR-mediated ERK signalling.
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5.3.4.2 Testin Accentuates Calcium-Sensing Receptor-Mediated Rho Kinase Activity
By examining the cellular localisation and binding partners of testin, Coutts et al., found
that the role of this protein in normal tissue was linked to cell spreading and motility
(Coutts et al. 2003). Further studies confirmed the involvement of testin involvement in
a range of cytoskeletal processes including actin stress fibre assembly (Coutts et al.
2003; Griffith et al. 2004; Griffith et al. 2005). Recently, the CaR has also been shown
to have a role in actin stress fibre reorganisation and cell morphology, which was
revealed to be dependent on Rho kinase signalling (Davies et al. 2006). The knockdown
of testin in HeLa cells resulted in a reduction of both actin stress fibre assembly and
Rho A activity, an upstream component of the Rho kinase signalling cascade (Griffith et
al. 2005). Using a SRE-luciferase reporter assay, CaR-mediated Rho kinase activation
was found to be enhanced in HEK293-CaR cells overexpressing testin (Figure 5.7).
shRNA technology was used to knockdown testin in HEK293-CaR cells, which were
then studied to determine the effects of CaR stimulation on Rho kinase activity in the
absence of testin. The basal level of Rho kinase activity detected in testin knockdown
HEK293-CaR cells was significantly lower than that of wild-type HEK293-CaR cells
(Figure 5.11), which is consistent with the decrease in Rho A activity observed in testin
knockdown HeLa cells (Griffith et al. 2005). However, an equivalent level of Rho
kinase activity was detected in testin knockdown HEK293-CaR cells as in HEK293-
CaR cells in response to elevated (5 mM) extracellular calcium (Figure 5.11). Taken
together, these results suggest that, while testin is not essential for CaR-mediated Rho
kinase activation, the interaction of testin with the CaR contributes to increased activity
of this signalling pathway.
5.3.5 The Relationship Between Cell Morphology and the Calcium-Sensing Receptor’s
Interaction with Testin
As previously mentioned, both the CaR and testin have been found to be involved in the
regulation of a series of related processes including cell motility, actin stress fibre
reorganisation and cell morphology (Coutts et al. 2003; Griffith et al. 2004; Griffith et
al. 2005)). In addition to the cytoskeletal processes regulated by the CaR already
described by others (Bouschet et al. 2007; Davies et al. 2006), the current study shows
that stimulation of the CaR in HEK293-CaR cells results in an increase in the formation
of peripheral focal adhesions over that seen in unstimulated cells (0.8 mM Mg2+)
(Figure 5.9B, D and F). Since focal adhesions are anchorage points for actin stress fibre
assembly this would be predicted from the studies of Davies et al, which shows that
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agonist stimulation of HEK293-CaR cells increases actin stress fibre formation (Davies
et al. 2006). However, the studies by Davies et al. and those in this thesis are in contrast
to those of Griffith et al. conducted in HeLa cells where it was shown that peripheral
focal adhesions and actin stress fibre networks were quite extensive even under non-
stimulatory conditions. This difference in basal levels of focal adhesion formation and
actin stress fibre assembly between the two cell lines, HeLa and HEK293-CaR cells
underscores the possibility of cell-type specific differences in cytoskeletal structure
impacting on potential testin modulation of CaR-mediated cytoskeletal changes.
CaR-mediated changes in cell morphology and actin stress fibre assembly require Rho
kinase activation (Davies et al. 2006) and RNAi knockdown of testin at basal agonist
levels reduces Rho A activity with concurrent loss of actin stress fibre assembly
(Griffith et al. 2005). Paradoxically, results presented in Chapter 5 show that, although,
testin overexpression increases Rho kinase activity and testin knockdown reduces it, the
reduction of Rho activity associated with knockdown of testin at basal agonist levels did
not result in the absence of actin stress fibre formation and cell extensions, which might
have been predicted from the studies of Davies et al. with HEK293-CaR cells.
However, these potentially interesting but preliminary results can only be considered
further once certain confirmatory and control studies have been undertaken. Towards
this end, it will be necessary to determine if the increase in stress fibre assembly
observed in the testin knockdown HEK293-CaR cells is specifically CaR-mediated, by
generating and comparing testin knockdown HEK293 cells without the CaR.
Furthermore, the observations made with testin knockdown HEK293-CaR cells with
respect to Rho kinase activation (Figure 5.11), cell morphology (Figure 5.12), actin
stress fibre assembly (Figure 5.13) and focal adhesion formation (Figure 5.14) should
also be evaluated against mock shRNA treated cells, as opposed to the wild-type,
untreated HEK293-CaR cells used in these experiments.
The methodologies used to examine the relationship between Rho signalling and the
CaR-induced cellular alterations differ between the studies presented here and those of
Davies et al in that a downstream effect of Rho kinase activity was examined, while
Davies et al used inhibitors of Rho A and Rho kinase. The drawback of using inhibitors
is that this does not just target CaR-mediated Rho signalling, but inhibits all Rho
signalling throughout the entire cell. However, measuring an outcome of Rho
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signalling, such as SRE-luciferase activity, is limiting because only a single downstream
event is measured and does not account for Rho kinase activity leading to alternate
outcomes. Therefore, despite a decrease in Rho kinase activity being detected via the
SRE-luciferase assay there remains the possibility that CaR-induced Rho signalling has
been diverted to favour another specific response. A proposed mechanism by which
CaR-mediated Rho signalling may be diverted is through interactions between the
intracellular tail of the CaR and specific binding partners, which will be discussed in
further detail in the following chapter. There is an alternative method to measure Rho
activity more directly that is based on the principle that activated Rho exists in a GTP-
bound state, while inactive Rho is bound to GDP (Machesky and Hall, 1996). By using
a GST fusion protein that contains a Rho binding domain motif that specifically binds to
the active, GTP-bound Rho it is possible to perform a pulldown assay using glutathione
beads to detect only active Rho (Ren 1999). Further experiments measuring activated
Rho or inhibiting Rho A and the Rho kinase in CaR-stimulated testin knockdown
HEK293-CaR cells will determine the necessity of the Rho pathway in the observed
morphological and cytoskeletal changes.
In conclusion, testin has been identified as a novel binding partner of the CaR
intracellular tail that was found to enhance CaR-mediated Rho kinase signalling and
may play a role in the receptor’s regulation of cell morphology and cytoskeletal
structure.
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CChhaapptteerr 66 General Discussion
6.1 The Calcium-Sensing Receptor
The CaR was cloned and characterised in an attempt to identify a protein capable of
responding to changes in extracellular calcium and has been shown to be an integral part
of calcium homeostasis (Brown and MacLeod 2001). However, later studies would
reveal that the CaR, unlike most receptors, was able to be activated by a diverse range
of stimuli (Breitwieser et al. 2004). Although extracellular calcium is still considered
the primary physiological agonist of the CaR, the list of additional endogenous stimuli
includes other multivalent cations, amino acids, polyamines, polypetides, as well as
changes in ionic strength and pH (Riccardi 2002). Due to the important physiological
and pathophysiological role of the CaR, a great deal of research has been focused on
examining exogenous CaR agonists that can be used therapeutically to modulate the
receptor’s activity and includes additional multivalent cations and polyamines, as well
as pharmacological agents such as aminoglycoside antibiotics, calcimimetics and
calcilytics (Riccardi 2002). In addition to being able to recognise a multitude of
different agonists, the CaR is also able to initiate a host of different signalling cascades,
including the PLC, MAPK and Rho signalling pathways (Ward 2004). In turn, this array
of intracellular signals is transformed into precise biological outcomes, which include
proliferation, apoptosis, differentiation and gene expression (Brown and MacLeod
2001). Many of the cellular processes regulated by the CaR occur in a tissue-specific
manner and there are even instances where CaR activation has been shown to have
opposing effects on cellular processes in different cells (Huang and Miller 2007). A
simplistic interpretation of the CaR’s translation of extracellular signals into signalling
pathways that result in a variety of biological responses is depicted in Figure 6.1. The
translation of extracellular signals into intracellular signals by the CaR is likely to be
coordinated by several mechanisms working in concert to ensure the correct outcome,
including ligand specific conformational changes of the receptor and receptor
phosphorylation. Another proposed mechanism that may determine the response of the
CaR in a tissue-specific manner may be mediated through the participation of protein
binding partners of the receptor.
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6.2 Interacting Protein Partners of the Calcium-Sensing Receptor
A number of proteins have been found to interact with the CaR, with several being
identified in yeast two-hybrid library screens using the intracellular tail of the CaR as
bait (Awata et al. 2001; Hjalm et al. 2001; Huang and Miller 2007; Huang et al. 2007).
The yeast two-hybrid libraries used in these studies were all generated from tissues
involved in whole body calcium homeostasis. Several years ago, the Ratajczak group
used the CaR-tail as bait to screen a commercially produced brain library to identify
proteins that are involved in processes unrelated to calcium homeostasis. Not a single
interacting protein was isolated from this library screen. This thesis presents results of a
subsequent yeast two-hybrid screen performed by the Ratajczak group of an EMLC.1
mouse pluripotent haemopoietic cell line library using the CaR-tail as bait that
identified several novel CaR binding partners. The seven proteins that were found to
interact with the CaR-tail in this screen, filamin A, filamin B, testin, 14-3-3 θ, OS-9,
Ubc9 and MPc2, are a diverse group of proteins that have been shown to be associated
with a wide variety of cellular functions. Yeast two-hybrid mapping studies revealed
that all seven proteins recognised essential binding elements present within either
residues 865-922 or 965-986 of the CaR-tail. Other reported CaR binding partners with
interaction sites mapped within the CaR-tail, have also been found to bind to either of
these two regions. Within the CaR-tail these two putative contact domains are the most
highly conserved between mammalian CaRs (Figure 1.1). This high level of
conservation suggests that the binding elements recognised in these regions have been
conserved throughout evolution. Additional evidence that these two regions of the CaR-
tail are critical for protein interactions may be obtained in further mapping studies of
other CaR-tail binding partners.
6.3 Interacting Protein Partners of the Calcium-Sensing Receptor Regulate its
Function
While the seven interacting partners of the CaR-tail identified in the yeast two-hyrid
library screen are a diverse group, several have been shown to be associated with
common functional processes. There are several basic mechanisms that determine
whether these accessory proteins can regulate the function of the CaR. In order to
influence the activity of the CaR, the interacting protein partners must obviously be
expressed in the same cell and at the same subcellular location. Gene expression
provides a level of control in cells by determining which binding partners are expressed,
when they are expressed and the level of expression. However, in cells that express both
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the CaR and an interacting protein, the trafficking and subsequent localisation of the
binding partner will dictate whether an interaction between the two proteins can occur.
For instance, the subcellular localisation of testin is dependent on it being in the correct
conformational state to allow binding to zyxin, for its recruitment to specific subcellular
localisations, namely, focal adhesions (Garvalov et al. 2003). Therefore, the interaction
between the CaR and testin is dependent on testin’s recruitment by zyxin to a
subcellular location where the receptor and testin can colocalise. Results presented in
Chapter 5, show that in HEK293-CaR stable cells, stimulation of the CaR with
extracellular magnesium results in the formation of focal adhesions (Figure 5.9). Further
colocalisation studies will need to be performed to establish whether CaR activation
results in greater expression of both the CaR and testin in these newly formed focal
adhesions.
As mentioned, there is some overlap in the biological processes with which some of the
CaR binding partners are associated, which suggests another method by which their
influence on the CaR can be regulated. CaR interacting proteins that promote cellular
processes leading to a common outcome may act together as a complex to regulate CaR
function. The CaR binding partners, filamin A and β-arrestins, were found to form a
complex involved in actin cytoskeletal changes regulated by the activity of other
receptors (Scott et al. 2006). Alternatively, the CaR binding partners may compete for
sites of interaction and therefore the receptor’s function would be regulated by its
affinity for the various accessory proteins. Considering the observed trend for CaR
interacting proteins to bind to only two regions of the intracellular tail, it is very likely
that there is strong competition for these sites.
An outline of CaR processes that are proposed to be regulated by the receptor’s
interaction with accessory proteins is presented below.
6.3.1 The Effect of Interacting protein partners on Calcium-Sensing Receptor
Dimerisation
Dimerisation has been shown to be important for the function of the CaR, which is
predominantly expressed at the cell surface as a homodimer (Bai et al. 1998).
Speculation concerning the roles of multiple CaR binding sites in filamin A that were
identified in Chapter 4, has led to the proposal that if the intracellular tail of each CaR
in a homodimer binds to a distinct site within filamin A, then filamin A may act as a
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clamp to stabilise the CaR homodimer (Figure 4.6A). While there is no direct evidence
to support this theory, the stabilisation of the CaR homodimer in this way could explain
how the interaction between the CaR and filamin A protects the receptor against
degradation perhaps by preventing receptor internalisation (Zhang and Breitwieser
2005).
6.3.2 The Regulation of Calcium-Sensing Receptor Trafficking by Interacting Proteins
Yeast two-hybrid mapping studies presented in Chapter 3 reveal that 14-3-3 θ binds to a
region of the CaR-tail that contains a 14-3-3 consensus binding sequence that lies
adjacent to a potential ER retention motif. The facilitation of the surface trafficking of
membrane proteins by the masking of ER retention motifs has been proposed as a
function of 14-3-3 proteins (Shikano et al. 2006). Once it has been established that the
ER retention signal is specifically able to retain CaR in the ER, the hypothesis that the
masking of the ER retention signal by 14-3-3 θ is required for trafficking of the CaR to
the cell membrane could be tested by examining the trafficking of mutant CaR
constructs containing deletions or mutations of the 14-3-3 binding motif. Alternatively
the movement of CaR from the ER following the knockdown of 14-3-3 θ could be
examined.
OS-9, which was shown to bind to residues 965-987 of the CaR-tail in Chapter 3, has
also been associated with the trafficking of the membrane protein, meprin β from the
ER to the Golgi (Litovchick et al. 2002), whereas Wang et al. has demonstrated that
OS-9 impedes the release of TRPV4 from the ER (Wang et al. 2007). Through its
interaction with OS-9, the CaR may be involved in either of these processes.
6.3.3 The Regulation of Calcium-Sensing Receptor Degradation by Interacting Proteins
OS-9 has also been associated with the degradation of proteins (Baek et al. 2005;
Mueller et al. 2008; Wang et al. 2007). As part of a ubiquitin ligase complex, OS-9 is
involved in the detection and delivery of terminally misfolded or unassembled
glycosylated proteins for degradation by the ERAD pathway (Christianson et al. 2008).
As the CaR is a glycosylated protein that is processed in the ER it is possible that
misfolded CaR is targeted for degradation by OS-9 via the ERAD pathway. Huang et al.
demonstrated that the dorfin-mediated degradation of immature CaR occurred as part of
a protein complex including the valosin-containing protein, suggesting that the observed
degradation was also via the ERAD pathway (Huang et al. 2006b). The binding sites of
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OS-9 and dorfin are distinct allowing for the possibility that they act in unison to target
the CaR for degradation. Coimmunoprecipitation studies could be helpful in revealing
whether OS-9 form part of a complex with the CaR and dorfin suggesting a role for OS-
9 in targeting CaR for ubiquitin mediated degradation. On the other hand, Christianson
et al. demonstrated that OS-9 in conjunction with the Hsp90 paralogue, GRP94, targets
aberrantly folded glycoproteins in the ER lumen via the Hrd1/Sel1 complex for
ubiquitination and proteasomal degradation. The possibility that the CaR represents a
target for OS-9 in this context could also be examined by appropriate
coimmunoprecipitation and subcellular localisation studies.
Experiments examining the oxygen-dependent ubiquitination and degradation of HIF-
1α found that this process was regulated by OS-9 (Baek et al. 2005). In contrast, OS-9
was shown to protect TPRV4 from ubiquitination and degradation (Wang et al. 2007).
Considering the disparity between the observed effects that interaction with OS-9 has
on degradation it is unclear what role, if any, OS-9 plays in CaR degradation. However,
yeast two-hybrid mapping studies have revealed that the site of OS-9 interaction in the
CaR overlaps that of filamin A binding. Since the interaction between the CaR and
filamin A protects the CaR against degradation (Zhang and Breitwieser 2005). It is
possible that filamin A, by competing with OS-9 for binding sites within the CaR-tail, is
able to prevent OS-9 from targeting the receptor for degradation.
6.3.4 Calcium-Sensing Receptor-Mediated Intracellular Signalling is Directed by
Interacting Proteins
As mentioned, the CaR can activate a wide variety of signalling pathways in a cell
specific manner (Ward 2004). For example, in fibroblasts, stimulation of the CaR
initiates the JNK cascade, but this does not occur in the MC3T3-E1 osteoblastic cell
line, (Ogata et al. 2006; Yamaguchi et al. 2000). Recently, a comparison between an
immortalised murine mammary cell line, Comma-D, and mammary epithelial cells
revealed that in the former cAMP production was increased by CaR activation but
inhibited in the latter (Mamillapalli et al. 2008). The difference in CaR signalling was
attributed to a difference in G protein coupling between the CaR in Comma-D cells,
which coupled to Gαs and the CaR in mammary epithelial cells, which coupled to Gαi
(Mamillapalli et al. 2008). This change in G protein coupling was also observed with
the β2 adrenoreceptor, where the change was attributed to the recruitment of PDE4
cAMP phosphodiesterase to the receptor by β-arrestins (Baillie et al. 2003). As β-
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arrestins also interact with the CaR, it is possible that they lead to G protein switching
associated with the CaR. In other members of the GPCR family C, the intracellular
loops have been shown to be crucial for G protein-coupling and Chang et al.
demonstrated that the second and third intracellular loops are important for CaR
signalling (Chang et al. 2000; Pin et al. 1994). Post-translational modifications of these
loops may determine which G proteins bind to the CaR. Both the second and third
intracellular loops of the CaR contain SUMOylation sites that are recognised by a CaR
binding partner identified in Chapter 3, Ubc9. SUMOylation of the tumour necrosis
factor receptors was shown to inhibit their apoptotic signalling (Okura et al. 1996). To
determine if Ubc9 is involved in the selection of G proteins that bind to the CaR, the
possibility of SUMOylation of the CaR must first be investigated.
Scaffold proteins of GPCRs associate with multiple signalling components, linking
them to the receptors. Both filamin and 14-3-3 proteins have both been characterised as
scaffolding proteins (Hall and Lefkowitz 2002). The interaction between the CaR and a
14-3-3 protein was first discovered as part of this study but the interaction between the
CaR and filamin A has already been examined in a number of studies. In relation to
signalling, the interaction with filamin A has been shown to be important for the CaR
activation of the ERK, JNK and Rho pathways (Awata et al. 2001; Hjalm et al. 2001;
Huang et al. 2006a; Pi et al. 2002; Rey et al. 2005). The signalling components JNKK,
from the JNK pathway and Rho A, from the Rho pathway, have been shown to bind to
residues within the filamin A repeats 21-23 and 24, respectively (Marti et al. 1997; Ohta
et al. 2006). Having identified multiple sites of CaR interaction within filamin A it was
proposed that the precise binding site of filamin A to which the CaR may bind could
dictate which signalling components are accessible and therefore, able to be activated.
The role of testin in CaR-mediated signalling was examined in Chapter 5. There was no
observed impact on CaR-induced ERK phosphorylation observed in HEK293-CaR cells
expressing testin. However, the overexpression of testin in HEK293-CaR cells was
found to enhance the induction of Rho signalling by the CaR as measured by the SRE-
luciferase assay. The knockdown of testin in HEK293-CaR cells resulted in a decrease
in Rho kinase activity at basal levels of CaR agonist. However, the Rho activity
detected in both HEK293-CaR cells and testin knockdown HEK293-CaR cells
following stimulation with extracellular calcium was not significantly different. This
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suggests that although testin is not essential for CaR-mediated Rho kinase activity, it
can act to enhance the response.
6.2.5 The Role of the Calcium-Sensing Receptor and its Binding Partners in Cell
Morphology and Organisation of the Cytoskeleton
Studies have demonstrated that the CaR is involved in changes to cell morphology and
rearrangement of the actin cytoskeleton (Bouschet et al. 2007; Davies et al. 2006;
Procino et al. 2004). Stimulation of the CaR resulted in an increase in forskolin-induced
actin stress fibre assemby that was associated with a decrease in cAMP accumulation in
a cell line derived from kidney cortical collecting duct cells (Procino et al. 2004).
Swaney et al. showed in fibroblasts that increases in cAMP accumulation inhibit the
forskolin-mediated formation of focal adhesions and actin stress fibres (Swaney et al.
2006). CaR stimulation by divalent cations and calcimimetics, but not amino acids,
induced cell morphological changes and actin stress fibre assembly that was linked to
Rho kinase activation in HEK293-CaR cells (Davies et al. 2006). In addition to
replicating the findings of Davies et al. in Chapter 5, it was revealed that stimulation of
the CaR in HEK293-CaR cells with extracellular magnesium also induced an increase
in focal adhesion formation (Figure 5.9), as might be predicted since focal adhesions are
anchorage points of actin stress fibres.
Recently, CaR activity was shown to induce plasma membrane ruffling, a process that
involves the formation and retraction of protrusions enriched with filamentous actin,
which required the interaction with a complex containing β-arrestin-1 (Bouschet et al.
2007). Several of the CaR binding partners have also been associated with changes in
cell morphology and cytoskeletal organisation, including filamin A, 14-3-3 proteins and
testin (Birkenfeld et al. 2003; Griffith et al. 2005; Ohta et al. 2006; Vardouli et al.
2005). As a significant portion of the identified CaR interacting proteins are associated
with alterations of cell morphology and cytoskeletal structure, the coordination of their
binding to the CaR-tail may modulate the CaR’s regulation of these processes.
Studies examining the possible relationship between CaR-mediated changes in cell
morphology and cytoskeletal structure and the receptor’s interaction with testin were
conducted using testin knockdown HEK293-CaR cells. Preliminary results have shown
that the cell morphology and cytoskeletal structure observed in non-stimulated testin
knockdown HEK293-CaR cells, mimicked the effect seen in CaR agonist stimulated
146
HEK293-CaR cells. This is potentially an interesting finding but needs to be confirmed
with additional control studies.
6.4 Future Studies
6.4.1 Filamin A
The data presented in Chapter 4 indicates that the CaR can bind to three distinct sites
within filamin A, which contain a highly homologous region of 40 amino acids. Further
mapping studies would be required to more precisely delineate the CaR sites of
interaction within this 40 residue region and/or potentially other regions.
Coimmunprecipitation experiments with a filamin A construct with these three CaR
interacting regions deleted might reveal if this collective loss can be compensated for by
additional regions in filamin A. The hypothesis that filamin A acts as a clamp to
stabilise the CaR homodimer, could be tested by expressing in M2 cells (which do not
express endogenous filamin A) mutant filamin A constructs in which all but one of the
CaR binding sites have been disrupted. It would be predicted that the remaining CaR
binding site should still be sufficient for interaction between a single CaR and filamin A
but without the capacity to bind to two CaRs, the proposed stabilising effect should be
lost. To examine this, Western analysis could be performed to determine if there was
diminished CaR expression in M2 cells cotransfected with the CaR and mutant filamin
A in comparison to the expression of CaR in M2 cells cotransfected with CaR and wild-
type filamin A. The functional importance of the novel CaR-filamin interaction sites on
CaR-mediated processes such as ERK activation could also be examined following the
further delineation of the binding sites in both filamin A, as described above, and the
CaR. If the novel filamin binding domains interact at differential sites within the 923-
986 region of the CaR, then the use of specific filamin-TAT peptides (Hjalm et al.
2001) could be used to block interaction sites and demonstrate the roles of these novel
sites of interaction have in relation to CaR-mediated processes.
6.4.2 Filamin B
In the yeast two-hybrid studies presented in Chapter 3, filamin B was also shown to
bind to the CaR-tail, albeit with a lower binding affinity. Filamin A and B share
approximately 70% homology and are structurally similar proteins (Popowicz et al.
2006). However, several experiments performed in M2 cells that express filamin B but
not filamin A, revealed a role for filamin A in CaR signalling and degradation that was
apparently not compensated for by filamin B (Awata et al. 2001; Zhang and Breitwieser
147
2005). A better understanding of the possible relationship between filamin B and the
CaR requires verification of their interaction by pulldown studies as described in
Chapter 4. Further studies, following the confirmation of the interaction, could examine
the affect of filamin B overexpression and knockdown on various CaR signalling
pathways, including those known to be influenced by filamin A, such as the ERK and
Rho pathways.
6.4.3 Testin
In order to obtain more definitive data from the studies examining CaR-mediated
cellular changes, the methodology will be expanded to include the collection of a
greater number of images of treated and untreated cells. With the increase in number of
cell images it will be possible to use computer software (e.g. Adobe Photoshop) to
perform morphometric analysis on the cells and generate quantitative and statistically
relevant data.
Initial studies performed using testin knockdown HEK293-CaR cells revealed that, in
the presence of basal levels of CaR agonist, these cells exhibited cell morphology and a
cytoskeletal structure that mimicked that observed in stimulated HEK293-CaR cells.
Confirmatory and control studies are necessary to place the significance of these
preliminary results in proper context. To accomplish this, it will be necessary to
determine if the increase in actin stress fibre assembly and focal adhesion formation
observed in the testin knockdown HEK293-CaR cells is specifically CaR-mediated, by
generating and comparing them to testin knockdown CaR-negative HEK293 cells. In
addition, the observed effects of shRA testin knockdown in HEK293-CaR cells must be
evaluated against mock shRNA treated HEK293-CaR cells. To elucidate whether Rho
signalling is necessary for the morphological and cytoskeletal changes observed in
testin knockdown HEK293-CaR cells, experiments inhibiting Rho A and the Rho kinase
must be performed.
The apparent contrasting effects of testin knockdown on actin stress fibre assembly in
HEK293-CaR cells and HeLa cells may well be a cell specific effect, as HEK293-CaR
cells do not contain stress actin fibres at basal levels of CaR agonist but HeLa cells do.
As the knockdown of testin in HeLa cells results in the loss of actin stress fibre
formation it would be interesting to see if CaR agonist stimulation of testin knockdown
HeLa cells overexpressing the CaR would restore actin stress fibre assembly. Also of
148
interest is the cell morphology and cytoskeletal structure present in embryonic
fibroblasts in testin knockout mice compared to embryonic fibroblasts from normal
mice, a resource that has recently been made available. The morphological and
cytokeletal characteristics of the testin negative mouse embryonic fibroblasts could then
be examined in relation to the overexpression and stimulation of the CaR.
Aside from the organisation of the cytoskeleton, other processes where the function of
the CaR and testin overlap include cell motility and cell adhesion. In chicken embryo
fibroblasts the overexpression of testin resulted in a reduction in cell motility (Griffith et
al. 2004). Experiments performed in Boyden’s chambers, have recently revealed that
increasing the concentration of extracellular calcium triggered the migration of MDA
MB 231 cells via the ERK and PLC pathways (Mentaverri et al. 2007). If testin plays a
role in CaR-mediated cell motility, then the overexpression of testin in MDA MB 231
cells should inhibit CaR-induced cell migration. This hypothesis could be tested by
repeating the experiments using the Boyden’s chamber and observing the effects of
testin knockdown and overexpression on CaR-mediated MDA MB 231 cell motility.
Comparative studies with another breast cancer cell line, T47D, which does not express
endogenous testin, could also be highly instructive.
While the effects of testin interaction on CaR-mediated ERK and Rho signalling have
been examined, there are other CaR-induced pathways that may be influenced by the
binding of testin, including the PLC and inhibitory cAMP pathways. Investigating the
impact of the interaction between the CaR and testin on additional signalling pathways
will provide further insight into how the multiple accessory proteins that bind to the
intracellular tail of the CaR regulate the signalling of the receptor.
6.5 Conclusions
Regulation of the CaR’s response to its multitude of stimuli in a cell specific manner is
a key element of the receptor’s role in the body. Like other GPCRs, an important
mechanism for this regulation relate to the accessory proteins that bind to its
intracellular tail. Six novel binding partners of the CaR-tail were identified as part of
this study using the yeast two-hybrid system. The identified CaR interacting proteins
have been associated with a diverse range of functions and additional investigations into
their relationship with the CaR will provide further insight into the functionality of the
receptor. Combining the data from yeast two-hybrid mapping studies presented in this
149
thesis and the known interaction domains of previously identified CaR binding partners,
has revealed two regions of the CaR intracellular tail, 865-922 and 965-986, that appear
to be essential for the interaction of accessory proteins.
Filamin A was the seventh CaR interacting protein identified in the yeast two-hybrid
library screen. Previous studies of the interaction between filamin A and the CaR
suggest that filamin A acts as a scaffolding protein that recruits accessory proteins to the
intracellular tail in order facilitate CaR-mediated signalling. Two additional sites of
CaR interaction within filamin A that may allow it to stabilise the CaR dimer and/or act
as a more versatile scaffolding protein were identified in this study.
The relationship between a novel CaR binding partner, testin, was selected for further
investigation based on the overlap in biological function between the two proteins. The
interaction with testin, while not essential, was found to enhance CaR-mediated Rho
signalling. Preliminary data indicates that testin may be involved in the CaR’s
regulation of changes to cell morphology and cytoskeletal structure.
150
148
((HHaavveenn eett aall.. 22000044;; KKiiffoorr eett aall.. 11999966;; LLaavvaalllliiee eett aall.. 11999933;; LLiinn eett aall.. 11999988;; MMaarrttii eett aall.. 11999977;; MMeennttaavveerrrrii eett aall.. 22000077;; OOggaattaa eett aall.. 22000066;; OOhhttaa eett aall.. 22000066;; OOkkuurraa eett aall.. 11999966;; PPiinn eett aall.. 11999944;;
PPrroocciinnoo eett aall.. 22000044;; SSccootttt eett aall.. 22000066;; SSwwaanneeyy eett aall.. 22000066;; VVaarrddoouullii eett aall.. 22000055)) ((AArrddeesshhiirrppoouurr eett aall.. 22000066;; AAssllaannoovvaa eett aall.. 22000066;; AAwwaattaa eett aall.. 22000011;; BBaa aanndd FFrriieeddmmaann 22000044;; BBaaii 22000044;; BBaaii eett aall.. 11999977;; BBaaii eett aall.. 11999966;; BBaaii eett aall.. 11999988aa;; BBaaii eett aall.. 11999999;; BBaaii eett aall.. 11999988bb;; BBaannddyyooppaaddhhyyaayy eett aall.. 22000077;; BBiikkllee eett aall.. 11999966;; BBoocckkaaeerrtt aanndd PPiinn 11999999;; BBoosseell eett aall.. 22000033;; BBoouusscchheett eett aall.. 22000055;; BBrraauunneerr--OOssbboorrnnee eett aall.. 11999999;; BBrraauunneerr--OOssbboorrnnee eett aall.. 22000077;; BBrroowwnn eett aall.. 11998877;; BBrroowwnn eett aall.. 11999933;; BBrroowwnn aanndd HHeebbeerrtt 11999977;; BBrroowwnn eett aall.. 11999944;; BBrroowwnn 11999999;; BBrroowwnn 22000000;; BBrroowwnn eett aall.. 11999911aa;; BBrroowwnn eett aall.. 11999911bb;; BBrroowwnn aanndd MMaaccLLeeoodd 22000011;; BBuucchhaann eett aall.. 22000011;; BBuutttteerrss eett aall.. 11999977;; CCaarraaffoollii 22000033;; CCaarree eett aall.. 11996666;; CCaarrlliinngg eett aall.. 22000000;; CCaarrrriilllloo--LLooppeezz eett aall.. 22000088;; CChhaakkrraabbaarrttyy eett aall.. 22000033;; CChhaanngg eett aall.. 22000011;; CChhaanngg aanndd SShhoobbaacckk 22000044;; CChhaanngg eett aall.. 22000000;; CChhaanngg eett aall.. 11999988;; CChhaanngg eett aall.. 22000077;; CChhaanngg eett aall.. 11999999;; CChhaattttooppaaddhhyyaayy eett aall.. 11999988aa;; CChhaattttooppaaddhhyyaayy eett aall.. 22000000;; CChhaattttooppaaddhhyyaayy eett aall.. 11999977;; CChhaattttooppaaddhhyyaayy eett aall.. 22000044;; CChhaattttooppaaddhhyyaayy eett aall.. 11999988bb;; CChhaattttooppaaddhhyyaayy eett aall.. 11999999;; CChheenn eett aall.. 11998899;; CChheenn aanndd GGooooddmmaann 22000044;; CChheenngg eett aall.. 11999977;; CChheenngg eett aall.. 11999988;; CChheenngg eett aall.. 22000044;; CChheenngg eett aall.. 22000022;; CChhiikkaattssuu eett aall.. 22000000;; CCiimmaa eett aall.. 11999977;; CCoonniiggrraavvee eett aall.. 22000000;; DDaauubb eett aall.. 11999966;; DDaavviieess eett aall.. 22000077;; DDeetthhlleeffsseenn eett aall.. 11999988;; DDeeWWiirree eett aall.. 22000077;; DDiiaazz eett aall.. 11999977;; DDuuffnneerr eett aall.. 22000055))((DDvvoorraakk eett aall.. 22000044;; EEmmaannuueell eett aall.. 11999966;; EEvveenneeppooeell 22000088;; FFaann eett aall.. 11999977;; FFaann eett aall.. 11999988;; FFaarrnneebboo eett aall.. 11999988;; FFeerrrreeiirraa aanndd BBaaiillllyy 11999988;; FFeerrrreeiirraa eett aall.. 11999988;; FFeerrrryy eett aall.. 22000000;; FFrreeddrriikkssssoonn eett aall.. 22000033;; GGaammaa aanndd BBrreeiittwwiieesseerr 11999988;; GGaammaa eett aall.. 22000011;; GGaarrrreetttt eett aall.. 11999955;; GGoollddssmmiitthh eett aall.. 11999999;; HHaammmmeerrllaanndd eett aall.. 11999999;; HHaannddllooggtteenn eett aall.. 22000011;; HHaauuaacchhee 22000011;; HHaauuaacchhee eett aall.. 22000000;; HHeeaatthh eett aall.. 11999966;; HHeebbeerrtt eett aall.. 22000044;; HHeebbeerrtt eett aall.. 11999966;; HHeennddyy eett aall.. 22000000;; HHeerrrreerraa--VViiggeennoorr eett aall.. 22000066;; HHjjaallmm eett aall.. 22000011;; HHoobbssoonn eett aall.. 22000000;; HHoobbssoonn eett aall.. 22000033;; HHooffeerr aanndd BBrroowwnn 22000033;; HHooffff eett aall.. 11999999;; HHuu eett aall.. 22000000;; HHuu eett aall.. 22000066;; HHuu eett aall.. 22000055;; HHuu eett aall.. 22000022;; HHuu aanndd SSppiieeggeell 22000033;; HHuu aanndd SSppiieeggeell 22000077;; HHuuaanngg aanndd MMiilllleerr 22000077;; HHuuaanngg eett aall.. 22000077aa;; HHuuaanngg eett aall.. 22000044;; HHuuaanngg eett aall.. 22000066aa;; HHuuaanngg eett aall.. 22000066bb;; HHuuaanngg eett aall.. 22000077bb;; JJaanniicciicc eett aall.. 11999955aa;; JJaanniicciicc eett aall.. 11999955bb;; JJiiaanngg eett aall.. 22000022;; JJuussttiinniicchh eett aall.. 22000088))((KKaallllaayy eett aall.. 11999977;; KKaammeeddaa eett aall.. 11999988;; KKiiffoorr aanndd BBrroowwnn 11998888;; KKiiffoorr eett aall.. 11999977;; KKiiffoorr eett aall.. 11999988;; KKiiffoorr eett aall.. 22000033;; KKiiffoorr eett aall.. 22000011;; KKiirrcchhhhooffff aanndd GGeeiibbeell 22000066;; KKoommuuvveess eett aall.. 22000022;; KKuunniisshhiimmaa eett aall.. 22000000;; LLaattrroonniiccoo eett aall.. 11999988;; LLii eett aall.. 22000055;; LLiieennhhaarrddtt eett aall.. 22000000;; LLooppeezz--BBaarrnneeoo aanndd AArrmmssttrroonngg 11998833;; LLoorreennzz eett aall.. 22000077;; LLoorreettzz 22000088;; LLoorreettzz eett aall.. 22000044;; LLoouurrddeell eett aall.. 22000022;; MMaaccLLeeoodd eett aall.. 22000044;; MMaaiittii eett aall.. 22000088;; MMaallaarrkkeeyy eett aall.. 11999955;; MMaammiillllaappaallllii eett aall.. 22000088;; MMccCCuulllloouugghh eett aall.. 22000044;; MMccLLaarrnnoonn eett aall.. 22000022;; MMccNNeeiill eett aall.. 11999988;; MMeennttaavveerrrrii eett aall.. 22000066;; MMiieeddlliicchh eett aall.. 22000044;; MMoorrffiiss eett aall.. 22000033;; MMoottooyyaammaa aanndd FFrriieeddmmaann 22000022;; MMuunn eett aall.. 22000055;; MMuutthhaalliiff eett aall.. 11999966;; NNeemmeetthh eett aall.. 22000011;; NNeemmeetthh eett aall.. 11999988;; NNeemmeetthh aanndd CCaarraaffoollii 11999900;; NNiiwwaa eett aall.. 22000011;; OOddaa eett aall.. 22000000;; OOggaattaa eett aall.. 22000066;; PPaaccee eett aall.. 11999999;; PPaarrmmeennttiieerr eett aall.. 22000022;; PPeeaarrccee eett aall.. 11999966;; PPeeiirriiss eett aall.. 22000077;; PPeettrreell eett aall.. 22000044))((PPii eett aall.. 22000055;; PPii eett aall.. 22000022;; PPiiddaasshheevvaa eett aall.. 22000066;; PPiinn eett aall.. 22000033;; PPoollllaakk eett aall.. 11999933;; PPrreennzzeell eett aall.. 11999999;; PPrroocciinnoo eett aall.. 22000044;; PPuurrkkiissss aanndd BBooaarrddeerr 11999922;; QQuuaarrlleess 22000033;; QQuuiinnnn eett aall.. 22000044;; QQuuiinnnn eett aall.. 11999988;; QQuuiinnnn eett aall.. 11999977;; RRaacckkee aanndd NNeemmeetthh 11999933aa;; RRaacckkee aanndd NNeemmeetthh 11999933bb;; RRaayy eett aall.. 22000077;; RRaayy eett aall.. 11999988;; RRaayy eett aall.. 11999977;; RRaayy eett aall.. 22000044;; RRaayy eett aall.. 11999999;; RReeyy eett aall.. 22000055;; RReeyyeess--CCrruuzz eett aall.. 22000011;; RReeyyeess--IIbbaarrrraa eett aall.. 22000077;; RRiiccccaarrddii 22000022;; RRiiccccaarrddii eett aall.. 11999988;; RRiiccccaarrddii eett aall.. 11999955;; RRooddllaanndd 22000044;; RRooggeerrss eett aall.. 11999977;; RRuuaatt eett aall.. 11999955;; RRuuaatt eett aall.. 11999966;; RRuutttteenn eett aall.. 11999999;; SSaannddeerrss eett aall.. 22000000;; SShheerrwwoooodd eett aall.. 11996666;; SShhoobbaacckk eett aall.. 11998833;; SSiillvvee eett aall.. 22000055;; SSkkeellllyy aanndd FFrraannkklliinn 22000077;; SSuuppaattttaappoonnee eett aall.. 11998888;; TTffeelltt--HHaannsseenn aanndd BBrroowwnn 22000055;; TTffeelltt--HHaannsseenn eett aall.. 22000033;; TThhaakkkkeerr 22000044;; TTrriivveeddii eett aall.. 22000088;; TTuu eett aall.. 22000088;; TTuurrkksseenn aanndd TTrrooyy 22000033;; VVaannHHoouutteenn eett aall.. 22000044;; VVaassssiilleevv eett aall.. 11999977;; VViizzaarrdd eett aall.. 22000088))((AAddaammss eett aall.. 22000066;; AAllkkeemmaa eett aall.. 11999977;; BBaaeekk eett aall.. 22000055;; BBeerrnnaassccoonnii eett aall.. 22000088;; BBeerrrryy eett aall.. 22000055;; BBhhaammiiddiippaattii eett aall.. 22000055;; BBoocckkaaeerrtt eett aall.. 22000033;; BBoocckkaaeerrtt eett aall.. 22000044;; BBoouusscchheett eett aall.. 22000077;; CChhrriissttiiaannssoonn eett aall.. 22000088;; CCoobblliittzz eett aall.. 22000066;; CCoolllleecc eett aall.. 22000077;; CCoouuttttss eett aall.. 22000033;; DDaavviieess eett aall.. 22000066;; DDiinnggwweellll aanndd SSmmiitthh 22000066;; DDiivveecchhaa aanndd
149
CChhaarrlleessttoonn 11999955;; DDoohhmmeenn 22000044;; DDrruueekkee 22000066;; DDrruussccoo eett aall.. 22000055;; FFeenngg eett aall.. 22000055;; FFiieellddss aanndd SSoonngg 11998899;; FFrriieeddmmaannnn eett aall.. 22000022;; WWaanngg eett aall.. 11999966aa;; WWaarrdd eett aall.. 22000044;; WWaarrdd eett aall.. 22000066;; WWaarrdd 22000044;; WWaarrdd eett aall.. 11999988;; WWaasshhbbuurrnn eett aall.. 22000000aa;; WWaasshhbbuurrnn eett aall.. 22000000bb;; WWeessss 11999977;; WWoojjcciikkiieewwiicczz 22000044;; YYaammaagguucchhii eett aall.. 11999988;; YYaammaagguucchhii eett aall.. 22000000;; YYaanngg eett aall.. 11996677;; YYaannoo eett aall.. 22000044aa;; YYaannoo eett aall.. 22000044bb;; YYee eett aall.. 11999977aa;; YYee eett aall.. 11999977bb;; YYuu eett aall.. 22000044;; ZZhhaanngg aanndd BBrreeiittwwiieesseerr 22000055;; ZZhhaanngg eett aall.. 22000022;; ZZhhaanngg eett aall.. 22000011;; ZZhhaaoo eett aall.. 11999999))((CCaarrrreelllloo eett aall.. 11999999;; GGaarrvvaalloovv eett aall.. 22000033;; GGaauussss eett aall.. 22000066;; GGoorrlliinn eett aall.. 11999900;; GGrriiffffiitthh eett aall.. 22000044;; GGrriiffffiitthh eett aall.. 22000055;; GGuubbbb eett aall.. 11999999;; HHaarrttwwiigg aanndd SSttoosssseell 11997755;; KKaaggeeyy eett aall.. 22000033;; KKiimm eett aall.. 22000055;; KKiimmuurraa eett aall.. 11999977;; KKiimmuurraa eett aall.. 11999988;; LLiittoovvcchhiicckk eett aall.. 22000022;; LLiiuu eett aall.. 11999977;; MMaacckkiinnttoosshh 22000044;; MMccNNaammaarraa aanndd DDoonnnneennbbeerrgg 11999988;; MMeellcchhiioorr 22000000;; MMuueelllleerr eett aall.. 22000088;; NNaakkaayyaammaa eett aall.. 11999999;; NNiieellsseenn 11999911;; PPaatteell eett aall.. 22000066;; PPooppoowwiicczz eett aall.. 22000066;; RRooddrriigguueezz eett aall.. 22000011;; RRootttteerr eett aall.. 22000055;; SSaammppssoonn eett aall.. 22000011;; SSaarrttii eett aall.. 22000055;; SSaattiijjnn eett aall.. 11999977;; SScchhwwaarrttzz aanndd PPiirrrroottttaa 22000077;; SShhiikkaannoo eett aall.. 22000055;; SShhiikkaannoo eett aall.. 22000066;; SSttoosssseell eett aall.. 22000011;; SSuu eett aall.. 11999966;; SSuu eett aall.. 11999944;; SSzzaatthhmmaarryy eett aall.. 22000055;; TTaakkaaffuuttaa eett aall.. 11999988;; TTaasshhiirroo eett aall.. 11999977;; TTaattaarreellllii eett aall.. 22000000;; TToobbiiaass eett aall.. 22000011;; TToonngg eett aall.. 11999977;; vvaann ddeerr FFlliieerr aanndd SSoonnnneennbbeerrgg 22000011;; vvaann ddeerr VVeenn eett aall.. 22000000;; vvaann HHeemmeerrtt eett aall.. 22000011;; VVoouurrvvoouuhhaakkii eett aall.. 22000077;; WWaanngg aanndd SShhaakkeess 11999966;; WWaanngg eett aall.. 11999966bb;; WWaanngg eett aall.. 22000077;; YYaaffffee eett aall.. 11999977;; YYaanngg eett aall.. 22000066;; ZZhhaaoo 22000077;; ZZhheenngg aanndd ZZhhaaoo 22000077))((GGoooossnneeyy eett aall.. 22000011;; HHoouussee eett aall.. 11999977;; HHuunntteerr eett aall.. 22000055;; IInngglleeyy eett aall.. 22000000;; JJoohhnnssoonn 22000044;; JJoohhnnssoonn aanndd BBlloobbeell 11999977;; LLiimm eett aall.. 22000022;; TTssaaii eett aall.. 11999944;; vvaann HHeemmeerrtt eett aall.. 22000011;; WWaanngg eett aall.. 11999966bb;; ZZhhaanngg eett aall.. 22000066)) ((BBaaiilllliiee eett aall.. 22000033;; BBeekkmmaann aanndd HHeennrriiqquuee 22000022;; BBhhaaggaavvaatthhuullaa eett aall.. 22000077;; BBhhaaggaavvaatthhuullaa eett aall.. 22000055;; BBiirrkkeennffeelldd eett aall.. 22000033;; BBrreeiittwwiieesseerr eett aall.. 22000044;; CCoouuttttss eett aall.. 22000033;; GGllooggaauueerr eett aall.. 11999988;; HHaallll aanndd LLeeffkkoowwiittzz 22000022;; KKiieemmaa eett aall.. 22000066;; MMaacchheesskkyy aanndd HHaallll 11999966;; MMuunn eett aall.. 22000044;; PPeelllleeggrriinn aanndd MMeelllloorr 22000077;; SSeecckk eett aall.. 22000033;; SShheeeenn eett aall.. 22000022;; SSuunn eett aall.. 22000066;; TTaakkaaffuuttaa eett aall.. 11999988;; TTffeelltt--HHaannsseenn 22000088;; TTiiggggeess eett aall.. 22000033;; WWaanngg eett aall.. 22000044;; WWuu eett aall.. 22000055))
((RReenn eett aall.. 11999999))
CChhaapptteerr 77 References
Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, Kos CH, Pollak MR, Brown EM, Scadden DT. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439(7076):599-603.
Alkema MJ, Jacobs J, Voncken JW, Jenkins NA, Copeland NG, Satijn DPE, Otte AP, Berns A, vanLohuizen M. 1997. MPc2, a new murine homolog of the Drosophila polycomb protein is a member of the mouse polycomb transcriptional repressor complex. Journal of Molecular Biology 273(5):993-1003.
Ardeshirpour L, Dann P, Pollak M, Wysolmerski J, VanHouten J. 2006. The calcium-sensing receptor regulates PTHrP production and calcium transport in the lactating mammary gland. Bone 38(6):787-793.
Aslanova UF, Morimoto T, Farajov EI, Kumagai N, Nishino M, Sugawara N, Ohsaga A, Maruyama Y, Tsuchiya S, Takahashi S and others. 2006. Chloride-dependent intracellular pH regulation via extracellular calcium-sensing receptor in the medullary thick ascending limb of the mouse kidney. Tohoku Journal of Experimental Medicine 210(4):291-300.
Awata H, Huang C, Handlogten M, Miller R. 2001. Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein. Journal of Biological Chemistry 276(37):34871-34879.
Ba J, Friedman P. 2004. Calcium-sensing receptor regulation of renal mineral ion transport. Cell Calcium 35(3):229-237.
Baek JH, Mahon PC, Oh J, Kelly B, Krishnamachary B, Pearson M, Chan DA, Giaccia AJ, Semenza GL. 2005. OS-9 interacts with hypoxia-inducible factor 1 alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1 alpha. Molecular Cell 17(4):503-512.
Bai M. 2004. Structure-function relationship of the extracellular calcium-sensing receptor. Cell Calcium 35(3):197-207.
Bai M, Janicic N, Trivedi S, Quinn SJ, Cole DEC, Brown EM, Hendy GN. 1997. Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Journal of Clinical Investigation 99(8):1917-1925.
Bai M, Quinn S, Trivedi S, Kifor O, Pearce S, Pollak M, Krapcho K, Hebert S, Brown E. 1996. Expression and characterization of inactivating and activating mutations in the human Ca-0(2+)-sensing receptor. Journal of Biological Chemistry 271(32):19537-19545.
Bai M, Trivedi S, Brown E. 1998a. Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. Journal of Biological Chemistry 273(36):23605-23610.
Bai M, Trivedi S, Kifor O, Quinn SJ, Brown EM. 1999. Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function. Proc Natl Acad Sci U S A 96(6):2834-9.
Bai M, Trivedi S, Lane C, Yang Y, Quinn S, Brown E. 1998b. Protein kinase C phosphorylation of threonine at position 888 in Ca-0(2+)-sensing receptor (CaR) inhibits coupling to Ca2+ store release. Journal of Biological Chemistry 273(33):21267-21275.
150
Baillie GS, Sood A, McPhee I, Gall I, Perry SJ, Lefkowitz RJ, Houslay MD. 2003. beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from G(s) to G(i). Proceedings of the National Academy of Sciences of the United States of America 100(3):940-945.
Bandyopadhyay S, Jeong KH, Hansen JT, Vassilev PM, Brown EM, Chattopadhyay N. 2007. Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons. Journal of Neuroscience Research 85(4):882-895.
Bekman E, Henrique D. 2002. Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene. Mechanisms of Development 119:S77-S81.
Bernasconi R, Pertel T, Luban J, Molinari M. 2008. A dual task for the Xbp1-responsive OS-9 variants in the mammalian endoplasmic reticulum - Inhibiting secretion of misfolded protein conformers and enhancing their disposal. Journal of Biological Chemistry 283(24):16446-16454.
Berry FB, O'Neill MA, Coca-Prados M, Walter MA. 2005. FOXC1 transcriptional regulatory activity is impaired by PBX1 in a filamin a-mediated manner. Molecular and Cellular Biology 25(4):1415-1424.
Bhagavathula N, Hanosh AW, Nerusu KC, Appelman H, Chakrabarty S, Varani J. 2007. Regulation of E-cadherin and beta-catenin by Ca2+ in colon carcinoma is dependent on calcium-sensing receptor expression and function. International Journal of Cancer 121(7):1455-1462.
Bhagavathula N, Kelley EA, Reddy M, Nerusu KC, Leonard C, Fay K, Chakrabarty S, Varani J. 2005. Upregulation of calcium-sensing receptor and mitogen-activated protein kinase signalling in the regulation of growth and differentiation in colon carcinoma. British Journal of Cancer 93(12):1364-1371.
Bhamidipati A, Denic V, Quan EM, Weissman JS. 2005. Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Molecular Cell 19(6):741-751.
Bikle DD, Ratnam A, Mauro T, Harris J, Pillai S. 1996. Changes in calcium responsiveness and handling during keratinocyte differentiation - Potential role of the calcium receptor. Journal of Clinical Investigation 97(4):1085-1093.
Birkenfeld J, Betz H, Roth D. 2003. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta. Biochemical Journal 369:45-54.
Bockaert J, Marin P, Dumuis A, Fagni L. 2003. The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks. FEBS Letters 546(1):65-72.
Bockaert J, Pin JP. 1999. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO Journal 18(7):1723-1729.
Bockaert J, Roussignol G, Becamel C, Gavarini S, Joubert L, Dumuis A, Fagni L, Marin P. 2004. GPCR-interacting proteins (GIPs): nature and functions. Biochemical Society Transactions 32:851-855.
Bosel J, John M, Freichel M, Blind E. 2003. Signaling of the human calcium-sensing receptor expressed in HEK293-cells is modulated by protein kinases A and C. Experimental and Clinical Endocrinology and Diabetes 111(1):21-26.
Bouschet T, Martin S, Henley JM. 2005. Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane. Journal of Cell Science 118(20):4709-4720.
Bouschet T, Martin S, Kanamarlapudi V, Mundell S, Henley JM. 2007. The calcium-sensing receptor changes cell shape via a beta-arrestin-1-ARNO-ARF6-ELMO protein network. Journal of Cell Science 120(15):2489-2497.
151
Brauner-Osborne H, Jensen A, Sheppard P, O'Hara P, Krogsgaard-Larsen P. 1999. The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. Journal of Biological Chemistry 274(26):18382-18386.
Brauner-Osborne H, Wellendorph P, Jensen AA. 2007. Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors. Current Drug Targets 8(1):169-184.
Breitwieser G, Miedlich S, Zhang M. 2004. Calcium sensing receptors as integrators of multiple metabolic signals. Cell Calcium 35(3):209-216.
Brown E, Enyedi P, Leboff M, Rotberg J, Preston J, Chen C. 1987. High Extracellular Ca-2+ and Mg-2+ Stimulate Accumulation of Inositol Phosphates in Bovine Parathyroid Cells. FEBS Letters 218(1):113-118.
Brown E, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger M, Lytton J, Hebert S. 1993. Cloning and Characterization of an Extracellular Ca2+-Sensing Receptor from Bovine Parathyroid. Nature 366(6455):575-580.
Brown E, Hebert S. 1997. Calcium-receptor-regulated parathyroid and renal function. Bone 20(4):303-309.
Brown E, Pollak M, Hebert S. 1994. Cloning and Characterization of Extracellular Ca2+-Sensing Receptors From Parathyroid and Kidney - Molecular Physiology and Pathophysiology of Ca2+-Sensing. Endocrinologist 4(6):419-426.
Brown EM. 1999. Physiology and pathophysiology of the extracellular calcium-sensing receptor. American Journal of Medicine 106(2):238-253.
Brown EM. 2000. The extracellular Ca2+-sensing receptor: central mediator of systemic calcium homeostasis. Annual Revue of Nutrition 20:507-33.
Brown EM, Butters R, Katz C, Kifor O. 1991a. Neomycin Mimics the Effects of High Extracellular Calcium Concentrations on Parathyroid Function in Dispersed Bovine Parathyroid Cells. Endocrinology 128(6):3047-3054.
Brown EM, Katz C, Butters R, Kifor O. 1991b. Polyarginine, Polylysine, and Protamine Mimic the Effects of High Extracellular Calcium Concentrations on Dispersed Bovine Parathyroid Cells. Journal of Bone and Mineral Research 6(11):1217-1225.
Brown EM, MacLeod RJ. 2001. Extracellular calcium sensing and extracellular calcium signaling. Physiology Revue 81(1):239-297.
Buchan A, Squires P, Ring M, Meloche R. 2001. Mechanism of action of the calcium-sensing receptor in human antral gastrin cells. Gastroenterology 120(5):1128-1139.
Butters RR, Chattopadhyay N, Nielsen P, Smith CP, Mithal A, Kifor O, Bai M, Quinn S, Goldsmith P, Hurwitz S and others. 1997. Cloning and characterization of a calcium-sensing receptor from the hypercalcemic New Zealand white rabbit reveals unaltered responsiveness to extracellular calcium. Journal of Bone and Mineral Research 12(4):568-579.
Carafoli E. 2003. The calcium-signalling saga: tap water and protein crystals. Nature Reviews Molecular Cell Biology 4(4):326-332.
Care AD, Sherwood LM, Potts JT, Aurbach GD. 1966. Perfusion of Isolated Parathyroid Gland of Goat and Sheep. Nature 209(5018):55-&.
Carling T, Szabo E, Bai M, Ridefelt P, Westin G, Gustavsson P, Trivedi S, Hellman P, Brown EM, Dahl N and others. 2000. Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. Journal of Clinical Endocrinology and Metabolism 85(5):2042-7.
Carrello A, Ingley E, Minchin RF, Tsai S, Ratajczak T. 1999. The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90. Journal of Biological Chemistry 274(5):2682-2689.
152
Carrillo-Lopez N, Alvarez-Hernandez D, Gonzalez-Suarez I, Roman-Garcia P, Valdivielso JM, Fernandez-Martin JL, Cannata-Andia JB. 2008. Simultaneous changes in the calcium-sensing receptor and the vitamin D receptor under the influence of calcium and calcitriol. Nephrology Dialysis Transplantation 23(11):3479-3484.
Chakrabarty S, Radjendirane V, Appelman H, Varani J. 2003. Extracellular calcium and calcium sensing receptor function in human colon carcinomas: Promotion of E-cadherin expression and suppression of beta-catenin/TCF activation. Cancer Research 63(1):67-71.
Chang W, Pratt S, Chen TH, Bourguignon L, Shoback D. 2001. Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation. Journal of Biological Chemistry 276(47):44129-36.
Chang W, Shoback D. 2004. Extracellular Ca2+-sensing receptors - an overview. Cell Calcium 35(3):183-196.
Chang WH, Chen TH, Pratt S, Shoback D. 2000. Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C. Journal of Biological Chemistry 275(26):19955-19963.
Chang WH, Pratt S, Chen TH, Nemeth E, Huang ZM, Shoback D. 1998. Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera. Journal of Bone and Mineral Research 13(4):570-580.
Chang WH, Tu CL, Cheng ZQ, Rodriguez L, Chen TH, Gassmann M, Bettler B, Margeta M, Jan LY, Shoback D. 2007. Complex formation with the type B gamma-aminobutyric acid receptor affects the expression and signal transduction of the extracellular calcium-sensing receptor - Studies with hek-293 cells and neurons. Journal of Biological Chemistry 282(34):25030-25040.
Chang YH, Tu CL, Chen TH, Komuves L, Oda Y, Pratt SA, Miller S, Shoback D. 1999. Expression and signal transduction of calcium-sensing receptors in cartilage and bone. Endocrinology 140(12):5883-5893.
Chattopadhyay N, Cheng I, Rogers K, Riccardi D, Hall A, Diaz R, Hebert SC, Soybel DI, Brown EM. 1998a. Identification and localization of extracellular Ca2+-sensing receptor in rat intestine. American Journal of Physiology-Gastrointestinal and Liver Physiology 37(1):G122-G130.
Chattopadhyay N, Evliyaoglu C, Heese O, Carroll R, Sanders J, Black P, Brown EM. 2000. Regulation of secretion of PTHrP by Ca2+-sensing receptor in human astrocytes, astrocytomas, and meningiomas. American Journal of Physiology-Cell Physiology 279(3):C691-C699.
Chattopadhyay N, Legradi G, Bai M, Kifor O, Ye C, Vassilev P, Brown E, Lechan R. 1997. Calcium-sensing receptor in the rat hippocampus: A developmental study. Developmental Brain Research 100(1):13-21.
Chattopadhyay N, Yano S, Tfelt-Hansen J, Rooney P, Kanuparthi D, Bandyopadhyay S, Ren X, Terwilliger E, Brown E. 2004. Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts. Endocrinology 145(7):3451-3462.
Chattopadhyay N, Ye CP, Yamaguchi T, Kifor O, Vassilev PM, Nishimura R, Brown EM. 1998b. Extracellular calcium-sensing receptor in rat oligodendrocytes: Expression and potential role in regulation of cellular proliferation and an outward K+ channel. Glia 24(4):449-458.
Chattopadhyay N, Ye CP, Yamaguchi T, Nakai M, Kifor O, Vassilev PM, Nishimura RN, Brown EM. 1999. The extracellular calcium-sensing receptor is expressed
153
in rat microglia and modulates an outward K+ channel. Journal of Neurochemistry 72(5):1915-1922.
Chen CJ, Barnett JV, Congo DA, Brown EM. 1989. Divalent-Cations Suppress 3',5'-Adenosine-Monophosphate Accumulation by Stimulating a Pertussis Toxin-Sensitive Guanine Nucleotide-Binding Protein in Cultured Bovine Parathyroid Cells. Endocrinology 124(1):233-239.
Chen RA, Goodman WG. 2004. Role of the calcium-sensing receptor in parathyroid gland physiology. American Journal of Physiology-Renal Physiology 286(6):F1005-F1011.
Cheng I, Kifor O, Chattopadhyay N, Butters RR, Cima RR, Hebert SC, Brown EM, Soybel DI. 1997. Expression of an extracellular Ca2+-sensing receptor in rat gastrointestinal tract. Gastroenterology 112(4):A1139-A1139.
Cheng I, Klingensmith ME, Chattopadhyay N, Kifor O, Butters RR, Soybel DI, Brown EM. 1998. Identification and localization of the extracellular calcium-sensing receptor in human breast. Journal of Clinical Endocrinology and Metabolism 83(2):703-7.
Cheng SX, Geibel JP, Hebert SC. 2004. Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor. Gastroenterology 126(1):148-158.
Cheng SX, Okuda M, Hall AE, Geibel JP, Hebert SC. 2002. Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion. American Journal of Physiology-Gastrointestinal and Liver Physiology 283(1):G240-G250.
Chikatsu N, Fukumoto S, Takeuchi Y, Suzawa M, Obara T, Matsumoto T, Fujita T. 2000. Cloning and characterization of two promoters for the human calcium-sensing receptor (CaSR) and changes of CaSR expression in parathyroid adenomas. Journal of Biological Chemistry 275(11):7553-7557.
Christianson JC, Shaler TA, Tyler RE, Kopito RR. 2008. OS-9 and GRP94 deliver mutant alpha 1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD. Nature Cell Biology 10(3):272-U13.
Cima RR, Cheng I, Klingensmith ME, Chattopadhyay N, Kifor O, Hebert SC, Brown EM, Soybel DI. 1997. Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa. American Journal of Physiology-Gastrointestinal and Liver Physiology 36(5):G1051-G1060.
Coblitz B, Wu M, Shikano S, Li M. 2006. C-terminal binding: An expanded repertoire and function of 14-3-3 proteins. Febs Letters 580(6):1531-1535.
Collec E, El Nemer W, Gauthier E, Gane P, Lecomte MC, Dhermy D, Cartron JP, Colin Y, Van Kim CL, Rahuel C. 2007. Ubc9 interacts with Lu/BCAM adhesion glycoproteins and regulates their stability at the membrane of polarized MDCK cells. Biochemical Journal 402:311-319.
Conigrave AD, Quinn SJ, Brown EM. 2000. L-amino acid sensing by the extracellular Ca2+-sensing receptor. Proceedings of the National Acadademy of Sciences of the United States of America 97(9):4814-9.
Coutts AS, MacKenzie E, Griffith E, Black DM. 2003. TES is a novel focal adhesion protein with a role in cell spreading. Journal of Cell Science 116(5):897-906.
Daub H, Weiss FU, Wallasch C, Ullrich A. 1996. Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379(6565):557-560.
Davies S, Gibbons C, Vizard T, Ward D. 2006. Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids. American Journal of Physiology-Cell Physiology 290(6):C1543-C1551.
154
Davies SL, Ozawa A, McCormick WD, Dvorak MM, Ward DT. 2007. Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. Journal of Biological Chemistry 282(20):15048-15056.
Dethlefsen SM, Raab G, Moses MA, Adam RM, Klagsbrun M, Freeman MR. 1998. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-Like growth factor independently of protein kinase C. Journal of Cellular Biochemistry 69(2):143-153.
DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK. 2007. beta-arrestins and cell signaling. Annual Review of Physiology 69:483-510.
Diaz R, Hurwitz S, Chattopadhyay N, Pines M, Yang YH, Kifor O, Einat MS, Butters R, Hebert SC, Brown EM. 1997. Cloning, expression, and tissue localization of the calcium-sensing receptor in chicken (Gallus domesticus). American Journal of Physiology-Regulatory Integrative and Comparative Physiology 42(3):R1008-R1016.
Dingwell KS, Smith JC. 2006. Tes regulates neural crest migration and axial elongation in Xenopus. Developmental Biology 293(1):252-267.
Divecha N, Charleston B. 1995. Cloning and Characterization of 2 New Cdnas Encoding Murine Triple Lim Domains. Gene 156(2):283-286.
Dohmen RJ. 2004. SUMO protein modification. Biochimica Et Biophysica Acta-Molecular Cell Research 1695(1-3):113-131.
Drueke TB. 2006. Haematopoietic stem cells - role of calcium-sensing receptor in bone marrow homing. Nephrology Dialysis Transplantation 21(8):2072-2074.
Drusco A, Zanesi N, Roldo C, Trapasso F, Farber JL, Fong LY, Croce CM. 2005. Knockout mice reveal a tumor suppressor function for Testin. Proceedings of the National Academy of Sciences of the United States of America 102(31):10947-10951.
Dufner MM, Kirchhoff P, Remy C, Hafner P, Muller MK, Cheng SX, Tang LQ, Hebert SC, Geibel JP, Wagner CA. 2005. The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands. American Journal of Physiology-Gastrointestinal and Liver Physiology 289(6):G1084-G1090.
Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, Riccardi D. 2004. Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proceedings of the National Academy of Sciences of the United States of America 101(14):5140-5145.
Emanuel RL, Adler GK, Kifor O, Quinn SJ, Fuller F, Krapcho K, Brown EM. 1996. Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line. Molecular Endocrinology 10(5):555-565.
Evenepoel P. 2008. Calcimimetics in chronic kidney disease: evidence, opportunities and challenges. Kidney International 74(3):265-275.
Fan G, Goldsmith PK, Collins R, Dunn CK, Krapcho KJ, Rogers KV, Spiegel AM. 1997. N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface. Endocrinology 138(5):1916-22.
Fan GF, Ray K, Zhao XM, Goldsmith PK, Spiegel AM. 1998. Mutational analysis of the cysteines in the extracellular domain of the human Ca2+ receptor: effects on cell surface expression, dimerization and signal transduction. FEBS Letters 436(3):353-356.
Farnebo F, Hoog A, Sandelin K, Larsson C, Farnebo LO. 1998. Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas. Surgery 124(6):1094-1098.
155
Feng SJ, Lu X, Kroll MH. 2005. Filamin binding to glycoprotein Ib alpha signals talin-directed alpha IIb beta 3 activation. Blood 106(11):115a-115a.
Ferreira MCD, Bailly C. 1998. Extracellular Ca2+ decreases chloride reabsorption in rat CTAL by inhibiting cAMP pathway. American Journal of Physiology-Renal Physiology 44(2):F198-F203.
Ferreira MCD, Helies-Toussaint C, Imbert-Teboul M, Bailly C, Verbavatz JM, Bellanger AC, Chabardes D. 1998. Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney - Inhibition of hormone-dependent camp accumulation by extracellular Ca2+. Journal of Biological Chemistry 273(24):15192-15202.
Ferry S, Traiffort E, Stinnakre J, Ruat M. 2000. Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes. European Journal of Neuroscience 12(3):872-884.
Fields S, Song O-k. 1989. A novel genetic system to detect protein–protein interactions. Nature 340(6230):245-246.
Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB. 2003. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Molecular Pharmacology 63(6):1256-1272.
Friedmann E, Salzberg Y, Weinberger A, Shaltiel S, Gerst JE. 2002. YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins. Journal of Biological Chemistry 277(38):35274-35281.
Gama L, Breitwieser GE. 1998. A carboxyl-terminal domain controls the cooperativity for extracellular Ca2+ activation of the human calcium sensing receptor. A study with receptor-green fluorescent protein fusions. Journal of Biological Chemistry 273(45):29712-8.
Gama L, Wilt S, Breitwieser G. 2001. Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons. Journal of Biological Chemistry 276(42):39053-39059.
Garrett JE, Capuano IV, Hammerland LG, Hung BCP, Brown EM, Hebert SC, Nemeth EF, Fuller F. 1995. Molecular-Cloning and Functional Expression of Human Parathyroid Calcium Receptor Cdnas. Journal of Biological Chemistry 270(21):12919-12925.
Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Kocher T, Piddini E, Griffiths G, Way M. 2003. The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions. Journal of Cell Biology 161(1):33-39.
Gauss R, Jarosch E, Sommer T, Hirsch C. 2006. A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery. Nature Cell Biology 8(8):849-U102.
Glogauer M, Arora P, Chou D, Janmey PA, Downey GP, McCulloch CAG. 1998. The role of actin-binding protein 280 in integrin-dependent mechanoprotection. Journal of Biological Chemistry 273(3):1689-1698.
Goldsmith PK, Fan GF, Ray K, Shiloach J, McPhie P, Rogers KV, Spiegel AM. 1999. Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor. Journal of Biological Chemistry 274(16):11303-9.
Goosney DL, DeVinney R, Finlay BB. 2001. Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals. Infection and Immunity 69(5):3315-3322.
156
Gorlin JB, Yamin R, Egan S, Stewart M, Stossel TP, Kwiatkowski DJ, Hartwig JH. 1990. Human Endothelial Actin-Binding Protein (Abp-280, Nonmuscle Filamin) - a Molecular Leaf Spring. Journal of Cell Biology 111(3):1089-1105.
Griffith E, Coutts AS, Black DM. 2004. Characterisation of chicken TES and its role in cell spreading and motility. Cell Motility and the Cytoskeleton 57(3):133-142.
Griffith E, Coutts AS, Black DM. 2005. RNAi knockdown of the focal adhesion protein TIES reveals its role in actin stress fibre organisation. Cell Motility and the Cytoskeleton 60(3):140-152.
Gubb D, Green C, Huen D, Coulson D, Johnson G, Tree D, Collier S, Roote J. 1999. The balance between isoforms of the Prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs. Genes & Development 13(17):2315-2327.
Hall R, Lefkowitz R. 2002. Regulation of G protein-coupled receptor signaling by scaffold proteins. CIRCULATION RESEARCH 91(8):672-680.
Hammerland LG, Krapcho KJ, Garrett JE, Alasti N, Hung BCP, Simin RT, Levinthal C, Nemeth EF, Fuller FH. 1999. Domains determining ligand specificity for Ca2+ receptors. Molecular Pharmacology 55(4):642-648.
Handlogten ME, Huang CF, Shiraishi N, Awata H, Miller RT. 2001. The Ca2+-sensing receptor activates cytosolic phospholipase A(2) via a G(q)alpha-dependent ERK-independent pathway. Journal of Biological Chemistry 276(17):13941-13948.
Hartwig JH, Stossel TP. 1975. Isolation and Properties of Actin, Myosin, and a New Actin-Binding Protein in Rabbit Alveolar Macrophages. Journal of Biological Chemistry 250(14):5696-5705.
Hauache OM. 2001. Extracellular calcium-sensing receptor: structural and functional features and association with diseases. Braz J Med Biol Res 34(5):577-84.
Hauache OM, Hu JX, Ray K, Xie RY, Jacobson KA, Spiegel AM. 2000. Effects of a calcimimetic compound and naturally activating mutations on the human Ca2+ receptor and on Ca2+ receptor/metabotropic glutamate chimeric receptors. Endocrinology 141(11):4156-4163.
Haven CJ, van Puijenbroek M, Karperien M, Fleuren GJ, Morreau H. 2004. Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma. Journal of Pathology 202(1):86-94.
Heath H, Odelberg S, Jackson CE, Teh BT, Hayward N, Larsson C, Buist NRM, Krapcho KJ, Hung BC, Capuano IV and others. 1996. Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. Journal of Clinical Endocrinology and Metabolism 81(4):1312-1317.
Hebert S, Cheng S, Geibel J. 2004. Functions and roles of the extracellular Ca2+-sensing receptor in the gastrointestinal tract. Cell Calcium 35(3):239-247.
Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, Bouvier M. 1996. A peptide derived from a beta(2)-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. Journal of Biological Chemistry 271(27):16384-16392.
Hendy GN, D'Souza-Li L, Yang B, Canaff L, Cole DE. 2000. Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 16(4):281-96.
Herrera-Vigenor F, Hernandez-Garcia R, Valadez-Sanchez M, Vazquez-Prado J, Reyes-Cruz G. 2006. AMSH regulates calcium-sensing receptor signaling through direct interactions. Biochemical and Biophysical Research Communications 347(4):924-930.
157
Hjalm G, MacLeod RJ, Kifor O, Chattopadhyay N, Brown EM. 2001. Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase. Journal of Biological Chemistry 276(37):34880-7.
Hobson SA, McNeil SE, Lee F, Rodland KD. 2000. Signal transduction mechanisms linking increased extracellular calcium to proliferation in ovarian surface epithelial cells. Experimental Cell Research 258(1):1-11.
Hobson SA, Wright J, Lee F, McNeil SE, Bilderback T, Rodland KD. 2003. Activation of the MAP kinase cascade by exogenous calcium-sensing receptor. Molecular and Cellular Endocrinology 200(1-2):189-198.
Hofer AM, Brown EM. 2003. Extracellular calcium sensing and signalling. Nature Reviews Molecular Cell Biology 4(7):530-538.
Hoff AO, Cote GJ, Fritsche HA, Jr., Qiu H, Schultz PN, Gagel RF. 1999. Calcium-induced activation of a mutant G-protein-coupled receptor causes in vitro transformation of NIH/3T3 cells. Neoplasia (New York) 1(6):485-491.
House MG, Kohlmeier L, Chattopadhyay N, Kifor O, Yamaguchi T, Leboff MS, Glowacki J, Brown EM. 1997. Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. Journal of Bone and Mineral Research 12(12):1959-1970.
Hu J, Hauache O, Spiegel AM. 2000. Human Ca2+ receptor cysteine-rich domain. Analysis of function of mutant and chimeric receptors. J Biol Chem 275(21):16382-9.
Hu J, Jiang J, Costanzi S, Thomas C, Yang W, Feyen J, Jacobson K, Spiegel A. 2006. A missense mutation in the seven-transmembrane domain of the human Ca2+ receptor converts a negative allosteric modulator into a positive allosteric modulator. Journal of Biological Chemistry 281(30):21558-21565.
Hu J, McLarnon S, Mora S, Jiang J, Thomas C, Jacobson K, Spiegel A. 2005. A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+. Journal of Biological Chemistry
280(6):5113-5120. Hu J, Reyes-Cruz G, Chen W, Jacobson K, Spiegel A. 2002. Identification of acidic
residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator. Journal of Biological Chemistry 277(48):46622-46631.
Hu J, Spiegel A. 2003. Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function. TRENDS IN ENDOCRINOLOGY AND METABOLISM 14(6):282-288.
Hu JX, Spiegel AM. 2007. Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators. Journal of Cellular and Molecular Medicine 11(5):908-922.
Huang C, Miller RT. 2007. The calcium-sensing receptor and its interacting proteins. Journal of Cellular and Molecular Medicine 11(5):923-934.
Huang C, Sindic A, Hill CE, Hujer KM, Chan KW, Sassen M, Wu Z, Kurachi Y, Nielsen S, Romero MF and others. 2007a. Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function. American Journal of Physiology-Renal Physiology 292(3):F1073-1081.
Huang CF, Hujer KM, Wu ZZ, Miller RT. 2004. The Ca2+-sensing receptor couples to G alpha(12/13) to activate phospholipase D in Madin-Darby canine kidney cells. American Journal of Physiology-Cell Physiology 286(1):C22-C30.
158
Huang CF, Wu ZZ, Hujer KM, Miller RT. 2006a. Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling. FEBS Letters 580(7):1795-1800.
Huang Y, Niwa J, Sobue G, Breitwieser G. 2006b. Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin. Journal of Biological Chemistry 281(17):11610-11617.
Huang Y, Zhou YB, Yang W, Butters R, Lee HW, Li SY, Castiblanco A, Brown EM, Yang JJ. 2007b. Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor. Journal of Biological Chemistry 282(26):19000-19010.
Hunter M, Angelicheva D, Tournev I, Ingley E, Chan DC, Watts GF, Kremensky I, Kalaydjieva L. 2005. NDRG1 interacts with APO A-I and A-II and is a functional candidate for the HDL-C QTL on 8q24. Biochemical and Biophysical Research Communications 332(4):982-992.
Ingley E, Sarna MK, Beaumont JG, Tilbrook PA, Tsai S, Takemoto Y, Williams JH, Klinken SP. 2000. HS1 interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells. Journal of Biological Chemistry 275(11):7887-7893.
Janicic N, Pausova Z, Cole DEC, Hendy GN. 1995a. Insertion of an Alu Sequence in the Ca2+-Sensing Receptor Gene in Familial Hypocalciuric Hypercalcemia and Neonatal Severe Hyperparathyroidism. American Journal of Human Genetics 56(4):880-886.
Janicic N, Soliman E, Pausova Z, Seldin MF, Szpirer C, Hendy GN. 1995b. Mapping of the Calcium-Sensing Receptor Gene (Casr) to Human Chromosome-3q13.3-21 by Fluorescence in-Situ Hybridization, and Localization to Rat Chromosome-11 and Mouse Chromosome-16. Journal of Bone and Mineral Research 10:S377-S377.
Jiang Y, Zhang Z, Kifor O, Lane C, Quinn S, Bai M. 2002. Protein kinase C (PKC) phosphorylation of the Ca2+-sensing receptor (CaR) modulates functional interaction of g proteins with the CaR cytoplasmic tail. Journal of Biological Chemistry 277(52):50543-50549.
Johnson ES. 2004. Protein modification by SUMO. Annual Review of Biochemistry 73:355-382.
Johnson ES, Blobel G. 1997. Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. Journal of Biological Chemistry 272(43):26799-26802.
Justinich CJ, Mak N, Pacheco I, Mulder D, Wells RW, Blennerhassett MG, MacLeod RJ. 2008. The extracellular calcium-sensing receptor (CaSR) on human esophagus and evidence of expression of the CaSR on the esophageal epithelial cell line (HET-1A). American Journal of Physiology-Gastrointestinal and Liver Physiology 294(1):G120-G129.
Kagey MH, Melhuish TA, Wotton D. 2003. The polycomb protein Pc2 is a SUMO E3. Cell 113(1):127-137.
Kallay E, Kifor O, Chattopadhyay N, Brown EM, Bischof MG, Peterlik M, Cross HS. 1997. Calcium-dependent c-myc proto-oncogene expression and proliferation of CACO-2 cells: A role for a luminal extracellular calcium-sensing receptor. Biochemical and Biophysical Research Communications 232(1):80-83.
Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M, Izumi N, Kawashima H, Ozawa H, Ikeda K and others. 1998. Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochemical and Biophysical Research Communications 245(2):419-422.
159
Kiema T, Lad Y, Jiang PJ, Oxley CL, Baldassarre M, Wegener KL, Campbell ID, Ylanne J, Calderwood DA. 2006. The molecular basis of filamin binding to integrins and competition with talin. Molecular Cell 21(3):337-347.
Kifor O, Brown EM. 1988. Relationship between Diacylglycerol Levels and Extracellular Ca-2+ in Dispersed Bovine Parathyroid Cells. Endocrinology 123(6):2723-2729.
Kifor O, Diaz R, Butters R, Brown EM. 1997. The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. Journal of Bone and Mineral Research 12(5):715-25.
Kifor O, Diaz R, Butters R, Kifor I, Brown EM. 1998. The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. Journal of Biological Chemistry 273(34):21708-21713.
Kifor O, Kifor I, Moore FD, Butters RR, Brown EM. 2003. m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR. Journal of Biological Chemistry 278(33):31167-31176.
Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, Brown EM. 2001. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. American Journal of Physiology-Renal Physiology 280(2):F291-302.
Kifor O, Moore FD, Wang P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM. 1996. Reduced immunostaining for the extracellular Ca sensing receptor in primary and uremic secondary hyperparathyroidism. Journal of Clinical Endocrinology and Metabolism 81(4):1598-1606.
Kim W, Spear ED, Ng DTW. 2005. Yos9p detects and targets misfolded glycoproteins for ER-associated degradation. Molecular Cell 19(6):753-764.
Kimura Y, Nakazawa M, Tsuchiya N, Asakawa S, Shimizu N, Yamada M. 1997. Genomic organization of the OS-9 gene amplified in human sarcomas. Journal of Biochemistry 122(6):1190-1195.
Kimura Y, Nakazawa M, Yamada M. 1998. Cloning and characterization of three isoforms of OS-9 cDNA and expression of the OS-9 gene in various human tumor cell lines. Journal of Biochemistry 123(5):876-882.
Kirchhoff P, Geibel J. 2006. Role of calcium and other trace elements in the gastrointestinal physiology. World Journal of Gastroenterology 12(20):3229-3236.
Komuves L, Oda Y, Tu CL, Chang WH, Ho-Pao CL, Mauro T, Bikle DD. 2002. Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation. Journal of Cellular Physiology 192(1):45-54.
Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, Nakanishi S, Jingami H, Morikawa K. 2000. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407(6807):971-977.
Latronico AC, Abell AN, Arnhold IJP, Liu X, Lins TSS, Brito VN, Billerbeck AE, Segaloff DL, Mendonca BB. 1998. A unique constitutively activating mutation in third transmembrane helix of luteinizing hormone receptor causes sporadic male gonadotropin-independent precocious puberty. Journal of Clinical Endocrinology and Metabolism 83(7):2435-2440.
Lavallie ER, Diblasio EA, Kovacic S, Grant KL, Schendel PF, Mccoy JM. 1993. A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the Escherichia-Coli Cytoplasm. Bio-Technology 11(2):187-193.
160
Li S, Huang S, Peng S. 2005. Overexpression of G protein-coupled receptors in cancer cells: Involvement in tumor progression. International Journal of Oncology 27(5):1329-1339.
Lienhardt A, Garabedian M, Bai M, Sinding C, Zhang Z, Lagarde J, Boulesteix J, Rigaud M, Brown E, Kottler M. 2000. A large homozygous or heterozygous in-frame deletion within the calcium-sensing receptor's carboxylterminal cytoplasmic tail that causes autosomal dominant hypocalcemia. Journal of Clinical Endocrinology and Metabolism 85(4):1695-1702.
Lim R, Winteringham LN, Williams JH, McCulloch RK, Ingley E, Tiao JYH, Lalonde JP, Tsai SW, Tilbrook PA, Sun Y and others. 2002. MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1. Journal of Biological Chemistry 277(43):40997-41008.
Lin K, Chattopadhyay N, Bai M, Alvarez R, Dang C, Baraban J, Brown E, Ratan R. 1998. Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249(2):325-331.
Litovchick L, Friedmann E, Shaltiel S. 2002. A selective interaction between OS-9 and the carboxyl-terminal tail of meprin beta. Journal of Biological Chemistry 277(37):34413-34423.
Liu YC, Liu YH, Elly C, Yoshida H, Lipkowitz S, Altman A. 1997. Serine phosphorylation of Cbl induced by phorbol ester enhances its association with 14-3-3 proteins in T cells via a novel serine-rich 14-3-3-binding motif. Journal of Biological Chemistry 272(15):9979-9985.
Lopez-Barneo J, Armstrong CM. 1983. Depolarizing Response of Rat Parathyroid Cells to Divalent-Cations. Journal of General Physiology 82(2):269-294.
Lorenz S, Frenzel R, Paschke R, Breitwieser GE, Miedlich SU. 2007. Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: Role of G protein-coupled receptor kinases, protein kinase C and beta-arrestins. Endocrinology 148(5):2398-2404.
Loretz CA. 2008. Extracellular calcium-sensing receptors in fishes. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology 149(3):225-245.
Loretz CA, Pollina C, Hyodo S, Takei Y, Chang W, Shoback D. 2004. cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus). Journal of Biological Chemistry 279(51):53288-97.
Lourdel S, Paulais M, Cluzeaud F, Bens M, Tanemoto M, Kurachi Y, Vandewalle A, Teulon J. 2002. An inward rectifier K+ channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4-Kir5.1 heteromeric channels. Journal of Physiology-London 538(2):391-404.
Machesky LM, Hall A. 1996. Rho: A connection between membrane receptor signalling and the cytoskeleton. Trends in Cell Biology 6(8):304-310.
Mackintosh C. 2004. Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes. Biochemical Journal 381:329-342.
MacLeod RJ, Yano S, Chattopadhyay N, Brown EM. 2004. Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism. Biochemical and Biophysical Research Communications 320(2):455-60.
Maiti A, Hait NC, Beckman MJ. 2008. Extracellular calcium-sensing receptor activation induces vitamin D receptor levels in proximal kidney HK-2G cells by
161
a mechanism that requires phosphorylation of p38 alpha MAPK. Journal of Biological Chemistry 283(1):175-183.
Malarkey K, Belham CM, Paul A, Graham A, Mclees A, Scott PH, Plevin R. 1995. The Regulation of Tyrosine Kinase Signaling Pathways by Growth-Factor and G-Protein-Coupled Receptors. Biochemical Journal 309:361-375.
Mamillapalli R, VanHouten J, Zawalich W, Wysolmerski J. 2008. Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells. Journal of Biological Chemistry 283(36):24435-24447.
Marti A, Luo ZJ, Cunningham C, Ohta Y, Hartwig J, Stossel TP, Kyriakis JM, Avruch J. 1997. Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis factor-alpha activation of SAPK in melanoma cells. Journal of Biological Chemistry 272(5):2620-2628.
McCullough J, Clague MJ, Urbe S. 2004. AMSH is an endosome-associated ubiquitin isopeptidase. Journal of Cell Biology 166(4):487-492.
McLarnon SJ, Holden D, Ward DT, Jones MN, Elliott AC, Riccardi D. 2002. Aminoglycoside antibiotics induce pH-sensitive activation of the calcium-sensing receptor. Biochemical and Biophysical Research Communications 297(1):71-77.
McNamara BP, Donnenberg MS. 1998. A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway. Fems Microbiology Letters 166(1):71-78.
McNeil SE, Hobson SA, Nipper V, Rodland KD. 1998. Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. Journal of Biological Chemistry 273(2):1114-20.
Melchior F. 2000. SUMO - Nonclassical ubiquitin. Annual Review of Cell and Developmental Biology 16:591-+.
Mentaverri R, Abdoune R, Lion J, Brazier M, Kamel S. 2007. High extracellular calcium directly stimulates MDA-MB 231 human breast cancer cell by a mechanism involving the calcium sensing receptor. Journal of Bone and Mineral Research 22:S292-S292.
Mentaverri R, Yano S, Chattopadhyay N, Petit L, Kifor O, Kamel S, Terwilliger EF, Brazier M, Brown EM. 2006. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis. FASEB Journal 20(14):2562-+.
Miedlich SU, Gama L, Seuwen K, Wolf RM, Breitwieser GE. 2004. Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site. Journal of Biological Chemistry 279(8):7254-7263.
Morfis M, Christopoulos A, Sexton PM. 2003. RAMPs: 5 years on, where to now? Trends in Pharmacological Sciences 24(11):596-601.
Motoyama HI, Friedman PA. 2002. Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs. American Journal of Physiology-Renal Physiology 283(3):F399-F406.
Mueller B, Klemm EJ, Spooner E, Claessen JH, Ploegh HL. 2008. SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins. Proceedings of the National Academy of Sciences of the United States of America 105(34):12325-12330.
Mun H, Culverston E, Franks A, Collyer C, Clifton-Bligh R, Conigrave A. 2005. A double mutation in the extracellular Ca2+-sensing receptor's Venus flytrap
162
domain that selectively disables L-amino acid sensing. Journal of Biological Chemistry 280(32):29067-29072.
Mun HC, Franks AH, Culverston EL, Krapcho K, Nemeth EF, Conigrave AD. 2004. The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing. Journal of Biological Chemistry 279(50):51739-44.
Muthalif MM, Benter IF, Uddin MR, Malik KU. 1996. Calcium/calmodulin-dependent protein kinase II alpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A(2) in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells. Journal of Biological Chemistry 271(47):30149-30157.
Nakayama T, Yaoi T, Kuwajima G, Yoshie O, Sakata T. 1999. Ca2+-dependent interaction of N-copine, a member of the two C2 domain protein family, with OS-9, the product of a gene frequently amplified in osteosarcoma. Febs Letters 453(1-2):77-80.
Nemeth E, Delmar E, Heaton W, Miller M, Lambert L, Conklin R, Gowen M, Gleason J, Bhatnagar P, Fox J. 2001. Calcilytic compounds: Potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone. Journal of Pharmacology and Experimental Therapeutics 299(1):323-331.
Nemeth E, Steffey M, Hammerland L, Hung B, Van Wagenen B, DelMar E, Balandrin M. 1998. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proceedings of the National Acadademy of Sciences of the United States of America 95(7):4040-4045.
Nemeth EF, Carafoli E. 1990. The Role of Extracellular Calcium in the Regulation of Intracellular Calcium and Cell-Function - Introduction. Cell Calcium 11(5):319-321.
Nielsen PJ. 1991. Primary Structure of a Human Protein-Kinase Regulator Protein. Biochimica Et Biophysica Acta 1088(3):425-428.
Niwa J, Ishigaki S, Doyu M, Suzuki T, Tanaka K, Sobue G. 2001. A novel centrosomal RING-finger protein, Dorfin, mediates ubiquitin ligase activity. Biochemical and Biophysical Research Communications 281(3):706-713.
Oda Y, Tu CL, Chang WH, Crumrine D, Komuves L, Mauro T, Elias PM, Bikle DD. 2000. The calcium sensing receptor and its alternatively spliced form in murine epidermal differentiation. Journal of Biological Chemistry 275(2):1183-1190.
Ogata S, Kubota Y, Satoh S, Ito S, Takeuchi H, Ashizuka M, Shirasuna K. 2006. Ca2+ a stimulates COX-2 expression through calcium-sensing receptor in fibroblasts. Biochemical and Biophysical Research Communications 351(4):808-814.
Ohta Y, Hartwig JH, Stossel TP. 2006. FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling. Nature Cell Biology 8(8):803-U35.
Okura T, Gong LM, Kamitani T, Wada T, Okura I, Wei CF, Chang HM, Yeh ETH. 1996. Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. Journal of Immunology 157(10):4277-4281.
Pace AJ, Gama L, Breitwieser GE. 1999. Dimerization of the calcium-sensing receptor occurs within the extracellular domain and is eliminated by Cys -> Ser mutations at Cys(101) and Cys(236). Journal of Biological Chemistry 274(17):11629-11634.
Parmentier ML, Prezeau L, Bockaert J, Pin JP. 2002. A model for the functioning of family 3 GPCRs. Trends in Pharmacological Sciences 23(6):268-274.
Patel A, Cummings N, Batchelor M, Hill PJ, Dubois T, Mellits KH, Frankel G, Connerton I. 2006. Host protein interactions with enteropathogenic Escherichia
163
coli (EPEC): 14-3-3 tau binds Tir and has a role in EPEC-induced actin polymerization. Cellular Microbiology 8(1):55-71.
Pearce S, Bai M, Quinn S, Kifor O, Brown E, Thakker R. 1996. Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. Journal of Clinical Investigation 98(8):1860-1866.
Peiris D, Pacheco I, Spencer C, MacLeod RJ. 2007. The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts. American Journal of Physiology-Gastrointestinal and Liver Physiology 292(3):G753-G766.
Pellegrin S, Mellor H. 2007. Actin stress fibres. Journal of Cell Science 120(20):3491-3499.
Petrel C, Kessler A, Dauban P, Dodd R, Rognan D, Ruat M. 2004. Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain. Journal of Biological Chemistry 279(18):18990-18997.
Pi M, Oakley R, Gesty-Palmer D, Cruickshank R, Spurney R, Luttrell L, Quarles L. 2005. beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization. Molecular Endocrinology 19(4):1078-1087.
Pi M, Spurney RF, Tu Q, Hinson T, Quarles LD. 2002. Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor. Endocrinology 143(10):3830-8.
Pidasheva S, Grant M, Canaff L, Ercan O, Kumar U, Hendy G. 2006. Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly. Human Molecular Genetics 15(14):2200-2209.
Pin J, Galvez T, Prezeau L. 2003. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacology and Therapeutics 98(3):325-354.
Pin JP, Joly C, Heinemann SF, Bockaert J. 1994. Domains Involved in the Specificity of G-Protein Activation in Phospholipase C-Coupled Metabotropic Glutamate Receptors. Embo Journal 13(2):342-348.
Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, Seidman JG. 1993. Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75(7):1297-303.
Popowicz GM, Schleicher M, Noegel AA, Holak TA. 2006. Filamins: promiscuous organizers of the cytoskeleton. Trends in Biochemical Sciences 31(7):411-419.
Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. 1999. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402(6764):884-888.
Procino G, Carmosino M, Tamma G, Gouraud S, Laera A, Riccardi D, Svelto M, Valenti G. 2004. Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells. Kidney International 66(6):2245-2255.
Purkiss JR, Boarder MR. 1992. Stimulation of Phosphatidate Synthesis in Endothelial-Cells in Response to P2-Receptor Activation - Evidence for Phospholipase-C and Phospholipase-D Involvement, Phosphatidate and Diacylglycerol Interconversion and the Role of Protein-Kinase-C. Biochemical Journal 287:31-36.
Quarles LD. 2003. Extracellular calcium-sensing receptors in the parathyroid gland, kidney, and other tissues. Current Opinion in Nephrology and Hypertension 12(4):349-355.
164
Quinn S, Bai M, Brown E. 2004. pH sensing by the calcium-sensing receptor. Journal of Biological Chemistry 279(36):37241-37249.
Quinn SJ, Kifor O, Trivedi S, Diaz R, Vassilev P, Brown E. 1998. Sodium and ionic strength sensing by the calcium receptor. Journal of Biological Chemistry 273(31):19579-19586.
Quinn SJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, Brown E. 1997. The Ca2+-sensing receptor: A target for polyamines. American Journal of Physiology-Cell Physiology 42(4):C1315-C1323.
Racke FK, Nemeth EF. 1993a. Cytosolic Calcium Homeostasis in Bovine Parathyroid Cells and Its Modulation by Protein-Kinase-C. Journal of Physiology-London 468:141-162.
Racke FK, Nemeth EF. 1993b. Protein-Kinase-C Modulates Hormone-Secretion Regulated by Extracellular Polycations in Bovine Parathyroid Cells. Journal of Physiology-London 468:163-176.
Ray K, Adipietro KA, Chen C, Northup JK. 2007. Elucidation of the role of peptide linker in calcium-sensing receptor activation process. Journal of Biological Chemistry 282(8):5310-5317.
Ray K, Clapp P, Goldsmith P, Spiegel A. 1998. Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. Journal of Biological Chemistry 273(51):34558-34567.
Ray K, Fan GF, Goldsmith PK, Spiegel AM. 1997. The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction. Journal of Biological Chemistry 272(50):31355-61.
Ray K, Ghosh SP, Northup JK. 2004. The role of cysteines and charged amino acids in extracellular loops of the human Ca(2+) receptor in cell surface expression and receptor activation processes. Endocrinology 145(8):3892-903.
Ray K, Hauschild BC, Steinbach PJ, Goldsmith PK, Hauache O, Spiegel AM. 1999. Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca2+ receptor critical for dimerization - Implications for function of monomeric Ca2+ receptor. Journal of Biological Chemistry 274(39):27642-27650.
Ren XD, Kiosses WB, Schwartz MA. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. Embo Journal 18(3):578-585.
Rey O, Young SH, Yuan JZ, Slice L, Rozengurt E. 2005. Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G(12), Rho, Filamin-A, and the actin cytoskeleton. Journal of Biological Chemistry 280(24):22875-22882.
Reyes-Cruz G, Hu J, Goldsmith PK, Steinbach PJ, Spiegel AM. 2001. Human Ca(2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants. Journal of Biological Chemistry 276(34):32145-51.
Reyes-Ibarra AP, Garcia-Regalado A, Ramirez-Rangel I, Esparza-Silva AL, Valadez-Sanchez M, Vazquez-Prado J, Reyes-Cruz G. 2007. Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism. Molecular Endocrinology 21(6):1394-1407.
Riccardi D. 2002. Cell surface, ion-sensing receptors. Experimental Physiology 87(4):403-411.
Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, Hebert SC. 1998. Localization of the extracellular Ca2+ polyvalent cation-sensing protein in rat kidney. American Journal of Physiology-Renal Physiology 43(3):F611-F622.
165
Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC. 1995. Cloning and Functional Expression of a Rat-Kidney Extracellular Calcium Polyvalent Cation-Sensing Receptor. Proceedings of the National Academy of Sciences of the United States of America 92(1):131-135.
Rodland K. 2004. The role of the calcium-sensing receptor in cancer. Cell Calcium 35(3):291-295.
Rodriguez MS, Dargemont C, Hay RT. 2001. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. Journal of Biological Chemistry 276(16):12654-12659.
Rogers K, Dunn C, Hebert S, Brown E. 1997. Localization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization. Brain Research 744(1):47-56.
Rotter B, Bournier O, Nicolas G, Dhermy D, Lecomte MC. 2005. alpha II-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts. Biochemical Journal 388:631-638.
Ruat M, Molliver M, Snowman A, Snyder S. 1995. Calcium Sensing Receptor - Molecular cloning in rat and localization to nerve-terminals. Proceedings of the National Acadademy of Sciences of the United States of America 92(8):3161-3165.
Ruat M, Snowman AM, Hester LD, Snyder SH. 1996. Cloned and expressed rat Ca2+-sensing receptor - Differential cooperative responses to calcium and magnesium. Journal of Biological Chemistry 271(11):5972-5975.
Rutten MJ, Bacon KD, Marlink KL, Stoney M, Meichsner CL, Lee FP, Hobson SA, Rodland KD, Sheppard BC, Trunkey DD and others. 1999. Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells. American Journal of Physiology-Gastrointestinal and Liver Physiology 277(3):G662-G670.
Sampson DA, Wang M, Matunis MJ. 2001. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SURIO-1 modification. Journal of Biological Chemistry 276(24):21664-21669.
Sanders JL, Chattopadhyay N, Kifor O, Yamaguchi T, Butters RR, Brown EM. 2000. Extracellular calcium-sensing receptor expression and its potential role in regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology 141(12):4357-64.
Sarti M, Sevignani C, Calin GA, Aqeilan R, Shimizu M, Pentimalli F, Picchio MC, Godwin A, Rosenberg A, Drusco A and others. 2005. Adenoviral transduction of TESTIN gene into breast and uterine cancer cell lines promotes apoptosis and tumor reduction in vivo. Clinical Cancer Research 11(2):806-813.
Satijn DPE, Olson DJ, vanderVlag J, Hamer KM, Lambrechts C, Masselink H, Gunster MJ, Sewalt RGAB, vanDriel R, Otte AP. 1997. Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis. Molecular and Cellular Biology 17(10):6076-6086.
Schwartz YB, Pirrotta V. 2007. Polycomb silencing mechanisms and the management of genomic programmes. Nature Reviews Genetics 8(1):9-22.
Scott M, Pierotti V, Storez H, Lindberg E, Thuret A, Muntaner O, Labbe-Jullie C, Pitcher J, Marullo S. 2006. Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins. Molecular and Cellular Biology 26(9):3432-3445.
Seck T, Baron R, Horne WC. 2003. Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling. Journal of Biological Chemistry 278(12):10408-10416.
166
Sheen VL, Feng YY, Graham D, Takafuta T, Shapiro SS, Walsh CA. 2002. Filamin A and Filamin B are co-expressed within neurons during periods of neuronal migration and can physically interact. Human Molecular Genetics 11(23):2845-2854.
Sherwood LM, Potts JT, Care AD, Mayer GP, Aurbach GD. 1966. Evaluation by Radioimmunoassay of Factors Controlling Secretion of Parathyroid Hormone. Nature 209(5018):52-&.
Shikano S, Coblitz B, Sun HY, Li M. 2005. Genetic isolation of transport signals directing cell surface expression. Nature Cell Biology 7(10):985-U85.
Shikano S, Coblitz B, Wu M, Li M. 2006. 14-3-3 proteins: regulation of endoplasmic reticulum localization and surface expression of membrane proteins. Trends in Cell Biology 16(7):370-375.
Shoback D, Thatcher J, Leombruno R, Brown E. 1983. Effects of Extracellular Ca++ and Mg++ on Cytosolic Ca++ and Pth Release in Dispersed Bovine Parathyroid Cells. Endocrinology 113(1):424-426.
Silve C, Petrel C, Leroy C, Bruel H, Mallet E, Rognan D, Ruat M. 2005. Delineating a Ca2+ binding pocket within the Venus flytrap module of the human calcium-sensing receptor. Journal of Biological Chemistry 280(45):37917-37923.
Skelly BJ, Franklin RJM. 2007. Mutations in genes causing human familial isolated hyperparathyroidism do not account for hyperparathyroidism in Keeshond dogs. Veterinary Journal 174(3):652-654.
Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, Shapiro SS. 2001. Filamins as integrators of cell mechanics and signalling. Nature Reviews Molecular Cell Biology 2(2):138-145.
Su YA, Hutter CM, Trent JM, Meltzer PS. 1996. Complete sequence analysis of a gene (OS-9) ubiquitously expressed in human tissues and amplified in sarcomas. Molecular Carcinogenesis 15(4):270-275.
Su YA, Trent JM, Guan XY, Meltzer PS. 1994. Direct Isolation of Genes Encoded within a Homogeneously Staining Region by Chromosome Microdissection. Proceedings of the National Academy of Sciences of the United States of America 91(19):9121-9125.
Sun YH, Liu MN, Li H, Shi S, Zhao YJ, Wang R, Xu CQ. 2006. Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis. Biochemical and Biophysical Research Communications 350(4):942-948.
Supattapone S, Worley PF, Baraban JM, Snyder SH. 1988. Solubilization, Purification, and Characterization of an Inositol Trisphosphate Receptor. Journal of Biological Chemistry 263(3):1530-1534.
Swaney JS, Patel HH, Yokoyama U, Head BP, Roth DM, Insel PA. 2006. Focal adhesions in (myo) fibroblasts scaffold adenylyl cyclase with phosphorylated caveolin. Journal of Biological Chemistry 281(25):17173-17179.
Szathmary R, Bielmann R, Nita-Lazar M, Burda P, Jakob CA. 2005. Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Molecular Cell 19(6):765-775.
Takafuta T, Kim JH, Shapiro SS. 1998. ABP-280 (alpha-filamin), beta-filamin and gamma-filamin are all expressed in alternatively spliced forms in a tissue-specific manner. Blood 92(10):347a-347a.
Tashiro K, Pando MP, Kanegae Y, Wamsley PM, Inoue S, Verma IM. 1997. Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of I kappa B alpha. Proceedings of the National Academy of Sciences of the United States of America 94(15):7862-7867.
Tatarelli C, Linnenbach A, Mimori K, Croce CM. 2000. Characterization of the human TESTIN gene localized in the FRA7G region at 7q31.2. Genomics 68(1):1-12.
167
Tfelt-Hansen J. 2008. The role of calcium-sensing receptor and signalling pathways in the pathophysiology in two in vitro models of malignant hypercalcemia: The rat rice H-500 leydig testis cancer and prostate cancer (PC-3) cells. Expression and regulation of pituitary tumor transforming gene in Leydig testis cancer and astrocyte and astrocytoma cells. Danish Medical Bulletin 55(1):17-46.
Tfelt-Hansen J, Brown E. 2005. The calcium-sensing receptor in normal physiology and pathophysiology: A review. Critical Reviews in Clinical Laboratory Sciences 42(1):35-70.
Tfelt-Hansen J, MacLeod RJ, Chattopadhyay N, Yano S, Quinn S, Ren X, Terwilliger EF, Schwarz P, Brown EM. 2003. Calcium-sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells. American Journal of Physiology-Endocrinology and Metabolism 285(2):E329-37.
Thakker R. 2004. Diseases associated with the extracellular calcium-sensing receptor. Cell Calcium 35(3):275-282.
Tigges U, Koch B, Wissing J, Jockusch BM, Ziegler WH. 2003. The F-actin cross-linking and focal adhesion protein filamin A is a ligand and in vivo substrate for protein kinase C alpha. Journal of Biological Chemistry 278(26):23561-23569.
Tobias ES, Hurlstone AFL, MacKenzie E, McFarlane R, Black DM. 2001. The TES gene at 7q31,1 is methylated in tumours and encodes a novel growth-suppressing LIM domain protein. Oncogene 20(22):2844-2853.
Tong H, Hateboer G, Perrakis A, Bernards R, Sixma TK. 1997. Crystal structure of murine/human Ubc9 provides insight into the variability of the ubiquitin-conjugating system. Journal of Biological Chemistry 272(34):21381-21387.
Trivedi R, Mithal A, Chattopadhyay N. 2008. Recent updates on the calcium-sensing receptor as a drug target. Current Medicinal Chemistry 15(2):178-186.
Tsai S, Bartelmez S, Sitnicka E, Collins S. 1994. Lymphohematopoietic Progenitors Immortalized by a Retroviral Vector Harboring a Dominant-Negative Retinoic Acid Receptor Can Recapitulate Lymphoid, Myeloid, and Erythroid Development. Genes & Development 8(23):2831-2841.
Tu CL, Chang WH, Xie ZJ, Bikle DD. 2008. Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes. Journal of Biological Chemistry 283(6):3519-3528.
Turksen K, Troy TC. 2003. Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo. Mechanisms of Development 120(6):733-744.
van der Flier A, Sonnenberg A. 2001. Structural and functional aspects of filamins. Biochimica Et Biophysica Acta-Molecular Cell Research 1538(2-3):99-117.
van der Ven PFM, Wiesner S, Salmikangas P, Auerbach D, Himmel M, Kempa S, Hayess K, Pacholsky D, Taivainen A, Schroder R and others. 2000. Indications for a novel muscular dystrophy pathway: gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin. Journal of Cell Biology 151(2):235-247.
van Hemert MJ, de Steensma HY, van Heusden GPH. 2001. 14-3-3 proteins: key regulators of cell division, signalling and apoptosis. Bioessays 23(10):936-946.
VanHouten J, Dann P, McGeoch G, Brown EM, Krapcho K, Neville M, Wysolmerski JJ. 2004. The calcium-sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport. J Clin Invest 113(4):598-608.
Vardouli L, Moustakas A, Stournaras C. 2005. LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by
168
transforming growth factor-beta. Journal of Biological Chemistry 280(12):11448-11457.
Vassilev PM, HoPao CL, Kanazirska MPV, Ye CP, Hong K, Seidman CE, Seidman JG, Brown EM. 1997. Ca-o-sensing receptor (CaR)-mediated activation of K+ channels is blunted in CaR gene-deficient mouse neurons. Neuroreport 8(6):1411-1416.
Vizard TN, O'Keeffe GW, Gutierrez H, Kos CH, Riccardi D, Davies AM. 2008. Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor. Nature Neuroscience 11(3):285-291.
Vourvouhaki E, Carvalho C, Aguiar P. 2007. Model for Osteosarcoma-9 as a potent factor in cell survival and resistance to apoptosis. Physical Review E 76(1):-.
Wang WF, Shakes DC. 1996. Molecular evolution of the 14-3-3 protein family. Journal of Molecular Evolution 43(4):384-398.
Wang WH, Lu M, Hebert SC. 1996a. Cytochrome P-450 metabolites mediate extracellular Ca2+-induced inhibition of apical K+ channels in the TAL. American Journal of Physiology-Cell Physiology 40(1):C103-C111.
Wang WM, Sluys LJ, DeBorst R. 1996b. Interaction between material length scale and imperfection size for localisation phenomena in viscoplastic media. European Journal of Mechanics a-Solids 15(3):447-464.
Wang Y, Fu X, Gaiser S, Kottgen M, Kramer-Zucker A, Walz G, Wegierski T. 2007. OS-9 regulates the transit and polyubiquitination of TRPV4 in the endoplasmic reticulum. Journal of Biological Chemistry 282(50):36561-36570.
Wang ZR, Eldstrom JR, Jantzi J, Moore ED, Fedida D. 2004. Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization. American Journal of Physiology-Heart and Circulatory Physiology 286(2):H749-H759.
Ward B, Magno A, Davis E, Hanyaloglu A, Stuckey B, Burrows M, Eidne K, Charles A, Ratajczak T. 2004. Functional deletion of the calcium-sensing receptor in a case of neonatal severe hyperparathyroidism. Journal of Clinical Endocrinology and Metabolism 89(8):3721-3730.
Ward BK, Cameron FJ, Magno AL, McDonnell CM, Stuckey BGA, Ratajczak T. 2006. A novel homozygous deletion in the calcium-sensing receptor ligand-binding domain associated with neonatal severe hyperparathyroidism. Journal of Pediatric Endocrinology & Metabolism 19(1):93-100.
Ward D. 2004. Calcium receptor-mediated intracellular signalling. Cell Calcium 35(3):217-228.
Ward D, Brown E, Harris H. 1998. Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. Journal of Biological Chemistry 273(23):14476-14483.
Washburn DLS, Anderson JW, Ferguson AV. 2000a. The calcium receptor modulates the hyperpolarization-activated current in subfornical organ neurons. Neuroreport 11(14):3231-3235.
Washburn DLS, Anderson JW, Ferguson AV. 2000b. A subthreshold persistent sodium current mediates bursting in rat subfornical organ neurones. Journal of Physiology-London 529(2):359-371.
Wess J. 1997. G-protein-coupled receptors: Molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB Journal 11(5):346-354.
Wojcikiewicz RJH. 2004. Regulated ubiquitination of proteins in GPCR-initiated signaling pathways. Trends in Pharmacological Sciences 25(1):35-41.
169
Wu ZZ, Tandon R, Ziembicki J, Nagano J, Hujer KM, Miller RT, Huang CF. 2005. Role of ceramide in Ca2+-sensing receptor-induced apoptosis. Journal of Lipid Research 46(7):1396-1404.
Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gamblin SJ, Smerdon SJ, Cantley LC. 1997. The structural basis for 14-3-3 : phosphopeptide binding specificity. Cell 91(7):961-971.
Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR, Sugimoto T, Brown EM. 1998. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca-0(2+))-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells. Journal of Bone and Mineral Research 13(10):1530-1538.
Yamaguchi T, Chattopadhyay N, Kifor O, Sanders JL, Brown EM. 2000. Activation of p42/44 and p38 mitogen-activated protein kinases by extracellular calcium-sensing receptor agonists induces mitogenic responses in the mouse osteoblastic MC3T3-E1 cell line. Biochem Biophys Res Commun 279(2):363-8.
Yang SF, Freer S, Benson AA. 1967. Transphosphatidylation by Phospholipase D. Journal of Biological Chemistry 242(3):477-&.
Yang XW, Lee WH, Sobott F, Papagrigoriou E, Robinson CV, Grossmann JG, Sundstrom M, Doyle DA, Elkins JM. 2006. Structural basis for protein-protein interactions in the 14-3-3 protein family. Proceedings of the National Academy of Sciences of the United States of America 103(46):17237-17242.
Yano S, Brown E, Chattopadhyay N. 2004a. Calcium-sensing receptor in the brain. Cell Calcium 35(3):257-264.
Yano S, Macleod RJ, Chattopadhyaya N, Tfelt-Hansen J, Kifor O, Butters RR, Brown EM. 2004b. Calcium-sensing receptor activation stimulates parathyroid hormone-related protein secretion in prostate cancer cells: role of epidermal growth factor receptor transactivation. Bone 35(3):664-672.
Ye CP, HoPao CL, Kanazirska M, Quinn S, Rogers K, Seidman CE, Seidman JG, Brown EM, Vassilev PM. 1997a. Amyloid-beta proteins activate Ca2+-permeable channels through calcium-sensing receptors. Journal of Neuroscience Research 47(5):547-554.
Ye CP, HoPao CL, Kanazirska M, Quinn S, Seidman CE, Seidman G, Brown EM, Vassilev PM. 1997b. Deficient cation channel regulation in neurons from mice with targeted disruption of the extracellular Ca2+-sensing receptor gene. Brain Research Bulletin 44(1):75-84.
Yu L, Liang S, Liu X, He Q, Studholme DJ, Wu Q. 2004. NCD3G: a novel nine-cysteine domain in family 3 GPCRs. Trends Biochem Sci 29(9):458-61.
Zhang M, Breitwieser G. 2005. High affinity interaction with filamin a protects against calcium-sensing receptor degradation. Journal of Biological Chemistry 280(12):11140-11146.
Zhang W, Fu S, Lu F, Wu B, Gong D, Pan Z, Lv Y, Zhao Y, Li Q, Wang R and others. 2006. Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes. Biochemical and Biophysical Research Communications 347(4):872-881.
Zhang Z, Qiu W, Quinn S, Conigrave A, Brown E, Bai M. 2002. Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function. Journal of Biological Chemistry 277(37):33727-33735.
Zhang Z, Sun S, Quinn S, Brown E, Bai M. 2001. The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions. Journal of Biological Chemistry 276(7):5316-5322.
Zhao J. 2007. Sumoylation regulates diverse biological processes. Cellular and Molecular Life Sciences 64(23):3017-3033.
170
Zhao X, Hauache O, Goldsmith P, Collins R, Spiegel A. 1999. A missense mutation in the seventh transmembrane domain constitutively activates the human Ca2+ receptor. FEBS Letters 448(1):180-184.
Zheng Q, Zhao Y. 2007. The diverse bilofunctions of LIM domain proteins: determined by subcellular localization and protein-protein interaction. Biology of the Cell 99(9):489-502.
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Appendix 1: Oligonucleotides
Primer Sequence
CaRCTF 5' GCC ATA TGT CCC GCA ACA CC 3'
CaRCTR 5' CGT CGA CTT ATG AAT TCA CTA CG 3'
CaR923F 5' GCC ATA TGC CAT TCC CAC AGC C 3'
CaR965F 5' GCC ATA TGA AGG TCA TCT TTG G 3'
CaR987F 5' GCC ATA TGA TGG CCC ACG GGA ATT C 3'
CaR997R 5' CGT CGA CTT AGG AGT TCT GGT GCG 3'
TesF 5' GGA TCC ATG GAC CTG GAA AAC AAA GTG 3'
TesR 5' GAG CTC CTA AGA CAT CCT CTT CTT ACA TTC 3'
Tes476F 5' GTG GGC TCC TCC TGT CCA G 3'
M13F 5' GTT GTA AAA CGA CGG CCA GT 3'
923SalIF 5' CGT CGA CCT CCA TTC CCA CAG CCC 3'
965SalIF 5' CGT CGA CCT AAG GTC ATC TTT GGC 3'
987SalIF 5' CGT CGA CCT ATG GCC CAC GGG AAT TC 3'
CaRT898R 5' CGT CGA CCT ACC GCT TGC GGG AGA CGT TGC 3'
CaRT899F 5' CGT CGA CCT TCC AGC AGC CTT GGA GGC 3'
Fil36F 5' CGA ATT CCT GAC CAT TGA GAT CTG CTC GG 3'
Fil36R 5' GCT CGA GCT AAC GGT CCT GAA CGT AGG TCT CCG 3'
FilBF 5' CGG ATC CAG CCC ATC GGG CAA GAC CCA TG 3'
FilBR 5' GCT CGA GCT AGA AGG GGC TGT CGG GAA TGT G 3'
Tes(91-109)F 5' GGA TTC GAA CTG CAC TTC T 3'
Tes(91-109)R 5' AGA AGT GCA GTT CGA ATC C 3'
Tes(C271A)F 5' CTG CAG CAC CGC TGG TGA ACT CTG GTC GAC 3'
Tes(C271A)R 5' GTC GAC CAG GAG TTC ACC AGC GGT GCT GCA G 3'
Tes(H292A)F 5' GAA GCT GTA CTG TGG CAG AGC TTA CTG TGA CAG TGA
G 3'
Tes(H292A)R 5' CTC ACT GTC ACA GTA AGC TCT GCC ACA GTA CAG CTT
C 3'
Tes(ELL)F 5' GTT TTA TCT GCA GCA CCT GTG GTG CAG CCG CGG TCG
AC 3'
Tes(ELL)R 5' GTC GAC CGC GGC TGC ACC ACA GGT GCT GCA GAT AAA
AC 3'
172
Tes(VD)F 5' GCA CCT GTG GTG AAC TCC TGG CCG CCA TGA TTT ACT
TC 3'
Tes(VD)R 5' GAA GTA AAT CAT GGC GGC CAG GAG TTC ACC ACA GGT
GC 3'
Tes(M)F 5' GAA CTC CTG GTC GAC GCG ATT TAC TTC TGG AAG AAT
G 3'
Tes(M)R 5' CAT TCT TCC AGA AGT AAA TCG CGT CGA CCA GGA GTT
C 3'
Tes(IYF)F 5' GGT CGA CAT GGC TGC CGC CTG GAA GAA TGG GAA GCT
G 3'
Tes(IYF)R 5' CAG CTT CCC ATT CTT CCA GGC GGC AGC CAT GTC GAC
C 3'
Tes(WKN)F 5' CAT GAT TTA CTT CGC GGC GGC TGG GAA GCT GTA CTG 3'
Tes(WKN)R 5' CAG TAC AGC TTC CCA GCC GCC GCG AAG TAA ATC ATG 3'
Tes(GK)F 5' CTT CTG GAA GAA TGC GGC GCT GTA CTG TGG CAG 3'
Tes(GK)R 5' CTG CCA CAG TAC AGC GCC GCA TTC TTC CAG AAG 3'
Tes(LY)F 5' GAT TTA CTT CTG GAA GAA TGG GAA GGC GGC CTG TGG
CAG 3'
Tes(LY)R 5' CTG CCA CAG GCC GCC TTC CCA TTC TTC CAG AAG TAA
ATC 3'
VP16-2 5' GAG TTT GAG CAG ATG TTT ACC G 3'
173
Appendix 2: Anitbodies and Western Blotting Conditions
Antibody Blocking
Conditions (Buffer/Incubation)
Primary Antibody
Conditions (Dilution -
Buffer - Incubation)
First Washing
Conditions
Secondary Antibody
Conditions (Dilution -
Buffer - Incubation)
Final Washing
Conditions
FLAG Antibody
3% skim milk powder in TBS
30 minutes at room temperature
1 in 10,000 FLAG
Antibody 3% Skim
Milk Powder in TBS
30 minutes at room
temperature
3 times in TBST for 5 minutes
1 in 10,000 goat anti-
mouse 3% Skim
Milk Powder in TBS
30 minutes at room
temperature
8 times in TBST for 2 minutes
ERK Antibody
5% skim milk powder in PBST 1 hour at room
temperature
1 in 10,000 ERK
antibody 5% Skim
Milk Powder in PBST 1 hour at
room temperature
3 times in PBST for 5 minutes
1 in 10,000 Goat Anti-
Rabbit 5% Skim
Milk Powder in PBST 1 hour at
Room Temperature
3 times in PBST for 5 minutes
P-ERK Antibody
5% skim milk powder in PBST 1 hour at room
temperature
1 in 2,000 P-ERK
antibody 5% Skim
Milk Powder in PBST 1 hour at
room temperature
3 times in PBST for 5 minutes
1 in 10,000 Goat Anti-
Rabbit 5% Skim
Milk Powder in PBST 1 hour at
Room Temperature
3 times in PBST for 5 minutes
GFP Antibody
5% skim milk powder in TBS 1
hour at room temperature
1 in 1,000 EGFP
Antibody 3% Skim
Milk Powder in TBS
30 minutes at room
temperature
3 times in TBST for 5 minutes
1 in 10,000 Goat Anti-
Rabbit 5% Skim
Milk Powder in TBST 1 hour at
Room Temperature
3 times in TBST for 5 minutes
Testin Antibody
5% skim milk powder in TBS 1
hour at room temperature
1 in 5,000 Tesin
antibody 5% Skim
Milk Powder in TBS
1 hour at room
temperature
3 times in TBST for 5 minutes
1 in 10,000 Goat Anti-
Rabbit 5% Skim
Milk Powder in TBS
1 hour at Room
Temperature
3 times in TBST for 5 minutes
His Antibody
5% skim milk powder in PBS 1
hour at room temperature
1 in 5,000 His antibody
5% Skim Milk Powder
in PBS 1 hour at
room temperature
3 times in PBST for 5 minutes
1 in 10,000 Goat Anti-
Mouse 5% Skim
Milk Powder in PBS
1 hour at Room
Temperature
3 times in PBST for 5 minutes
174
Antibody Blocking
Conditions (Buffer/Incubation)
Primary Antibody
Conditions (Dilution -
Buffer - Incubation)
First Washing
Conditions
Secondary Antibody
Conditions (Dilution -
Buffer - Incubation)
Final Washing
Conditions
α-Tubulin
5% skim milk powder in TBS 1
hour at room temperature
1 in 5,000 α-Tubulin
Antibody 3% Skim
Milk Powder in TBS
1 hour at room
temperature
3 times in TBST for 5 minutes
1 in 10,000 goat anti-
mouse 3% Skim
Milk Powder in TBS
1 hour at room
temperature
3 times in TBST for
10 minutes
175