Protein-Protein Interactions Provide a Platform for G-Protein-Coupled Receptors to Exert Physiological and
Pathological Functions in Central Nervous System: Therapeutic Potential?
By
Kai Ying Lai
A thesis submitted in conformity with the requirements for the degree of Doctoral of Philosophy
Department of Physiology University of Toronto
© Copyright by Kai Ying Lai 2019
ii
Protein-Protein Interactions Provide a Platform for G-Protein-
Coupled Receptors to Exert Physiological and Pathological
Functions in Central Nervous System: Therapeutic Potential?
Kai Ying Lai
Doctoral of Philosophy
Department of Physiology
University of Toronto
2019
Abstract
In the central nervous system membrane receptors play an important role in neuronal
communication. Since neurons mainly communicate through the release of neurotransmitters,
membrane receptors are responsible for binding neurotransmitters and transducing their message
into an intracellular response. In general, there are two types of membrane receptors: ionotropic
and metabotropic. Ionotropic receptors are ligand-gated ion channels, which are responsible for
mediating fast synaptic responses. On the other hand, metabotropic receptors act through their
downstream signaling cascades to trigger intracellular responses.
iii
A prominent example of metabotropic receptors is the G-protein-coupled receptor
(GPCR) superfamily. In the human body this receptor superfamily consists of approximately 799
receptors. Conventionally, these GPCRs are seven-transmembrane-domain receptors which act
through their coupled G-proteins to elicit their physiological actions. There is also a growing
body of evidence that these GPCRs can physically interact with each other as well as with other
types of membrane receptors.
This present study focuses on examining whether protein-protein interactions, other than
coupled G-protein signaling cascades, are another platform for GPCRs to exert their effects or to
regulate the functions of other proteins. We also investigate whether these protein-protein
interactions involving GPCRs present any physiological or pathological implications.
iv
Acknowledgments
I would like to take this opportunity to thank the people who have helped me throughout
my Ph.D. studies. Foremost, I would like to thank my supervisor, Dr. Fang Liu, for her support
and guidance because without her, none of this work would be possible.
I would also like to thank my advisory committee: Dr. Paul Fletcher, Dr. Sheena Josselyn
and Dr. Albert Wong, who have consistently provided guidance and insightful inputs to me
experiments and data analyses.
I also want to take this opportunity to thank the people who I had the great honor to work
closely with during my Ph.D. research:
Dr. Ping Su --- for teaching me all the valuable biochemical techniques and giving me the
previous opportunities to collaborate on several projects.
Dr. Dongxu Zhai --- for not only helping me purchase the items my research required, but also
performing the animal surgeries to make my research on mGluR1-NMDAR protein complex
possible.
Dr. Andrew Abela (from Dr. Paul Fletcher’s lab) --- for training me to use the touchscreen boxes
and working with me to interpret the massive amount of data.
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Special thanks to the past and present lab members and members from other research labs:
Dr. Shupeng Li, Dr. Hailong Zhang, Dr. Haiyin Li, Mr. Anlong Jiang, Mr. Jay Boychuk, Dr.
Laura Feldcamp, Mr. Roger Raymond, and many more for making our research facility a fun
studying and working place.
Dr. Frankie Lee and Mr. Charlie Campbell for being the best people to share office with.
Ms Lori Dixon, Ms Katrina Deverell and all the staff of the animal facility: for their support and
assistance with my animal studies.
Last but not least: I sincerely thank my parents, my sister and other family members for their
unconditional love and support throughout my graduate studies.
vi
Table of Contents
Acknowledgments.......................................................................................................................... iv
Table of Contents ........................................................................................................................... vi
List of Tables ................................................................................................................................ xii
List of Figures .............................................................................................................................. xiii
List of Abbreviations .....................................................................................................................xv
Chapter 1 Introduction .....................................................................................................................1
Introduction .................................................................................................................................1
1.1 Neuronal Communication within the Central Nervous System ...........................................1
1.2 Types of Postsynaptic Membrane Receptors --- ionotropic vs metabotropic receptors ......2
1.3 G-Protein-Coupled Receptors ..............................................................................................4
1.4 Diversity of GPCR ...............................................................................................................5
1.4.1 The Rhodopsin receptor family/Class A GPCR ......................................................6
1.4.2 The Secretin receptor family/Class B GPCR ...........................................................7
1.4.3 The Adhesion receptor family/Class B GPCR.........................................................8
1.4.4 The Glutamate Receptor Family/ Class C GPCR ....................................................8
1.4.5 The Frizzled Receptor Family/ Class F GPCR ........................................................9
1.5 GPCR Dimerization .............................................................................................................9
1.6 Protein-Protein Interaction on signaling cascades .............................................................10
1.6.1 Interaction Between Members of the Same Receptor Subfamily ..........................10
1.6.2 Interaction Between Members of Different Receptor Subfamilies ........................12
1.6.3 Interaction between a GPCR and a non-GPCR .....................................................13
1.7 Rationale and Hypothesis ..................................................................................................15
Chapter 2 ........................................................................................................................................18
vii
Metabotropic Glutamate Receptor 1 Modulates the Excitotoxic Functions of N-Methyl-D-
Aspartate Receptor (NMDAR) through its Interaction with the GluN2A subunit of
NMDAR ....................................................................................................................................18
Metabotropic Glutamate Receptor 1 Modulates the Excitotoxic Functions of N-Methyl-D-Aspartate Receptor (NMDAR) through its Interaction with the GluN2A subunit of
NMDAR ....................................................................................................................................19
2.1 Introduction ........................................................................................................................19
2.1.1 Glutamate Neurotransmission ................................................................................19
2.1.2 The Implication of Glutamate in Ischemic Stroke .................................................20
2.1.3 N-methyl-D-aspartate receptor (NMDAR) ............................................................21
2.1.4 Metabotropic Glutamate Receptor 1 (mGluR1) ....................................................21
2.1.5 Rationale and Hypothesis: Functional/Physical Cross-Talk between mGluR1 and NMDAR and Their Implications in Ischemic Stroke .....................................22
2.2 Materials and Methods .......................................................................................................23
2.2.1 Co-immunoprecipitation ........................................................................................23
2.2.2 GST Protein Affinity Purification ..........................................................................24
2.2.3 Western Blotting ....................................................................................................24
2.2.4 GST Fusion Protein Constructs .............................................................................24
2.2.5 GST Fusion Protein Expression and Purification ..................................................25
2.2.6 Primary Mouse Hippocampal Culture ...................................................................26
2.2.7 NMDA-Excitotoxicity in Primary Mouse Hippocampal Culture and Confocal Imaging ..................................................................................................................27
2.2.8 Animals ..................................................................................................................28
2.2.9 Transient Middle Cerebral Artery Occlusion (tMCAO) ........................................28
2.2.10 Intracerebroventricular (ICV) Peptide Delivery ....................................................28
2.2.11 Neurological Assessment .......................................................................................29
2.2.12 Tetrazolium Chloride (TTC) Staining and Brain Infarction Analysis ...................29
2.2.13 Acute Brain Slice Treatment ..................................................................................29
2.2.14 NMDA-Stimulated ERK1/2 Phosphorylation .......................................................30
viii
2.2.15 Membrane Expression of NMDA Receptors .........................................................30
2.2.16 Statistical Analysis .................................................................................................31
2.3 Results ................................................................................................................................31
2.3.1 GluN2A subunit of NMDAR facilitates mGluR1-NMDAR complex formation ..31
2.3.2 Identification of the amino acid sequence that enables the mGluR1 to form a complex with NMDAR ..........................................................................................31
2.3.3 The development of interfering peptides to disrupt the mGluR1-NMDAR interaction ..............................................................................................................33
2.3.4 The disruption of mGluR1-NMDAR interaction attenuated NMDA-mediated excitotoxicity..........................................................................................................37
2.3.5 The neuroprotective effects of mGluR1-NMDAR disruption in animal model
of ischemic stroke ..................................................................................................40
2.3.6 The disruption of mGluR1-NMDAR complex abolished NMDA-mediated ERK1/2 Phosphorylation .......................................................................................47
2.3.7 The mGluR1-NMDAR interaction does not regulate NMDAR membrane
expression ..............................................................................................................49
2.4 Discussion ..........................................................................................................................51
Chapter 3 ........................................................................................................................................58
Prenatal Disruption of D1R-SynGAP Complex Impairs GABAergic Interneuron Migration &
Causes Behavioural Deficits in Adulthood ...............................................................................58
Prenatal Disruption of D1R-SynGAP Complex Impairs GABAergic Interneuron Migration & Causes Behavioural Deficits in Adulthood ...........................................................................59
3.1 Introduction ........................................................................................................................59
3.1.1 γ-aminobutyric acid (GABA) ................................................................................59
3.1.2 GABAergic Interneurons .......................................................................................59
3.1.3 Morphology and Synaptic Connectivity of GABAergic Interneurons ..................60
3.1.4 Biochemical Markers and Electrophysiological Properties of GABAergic Interneurons ...........................................................................................................62
3.1.5 Pathological Significance of GABAergic Interneuron Dysfunction .....................64
3.1.6 Dopaminergic System on GABAergic Interneurons .............................................65
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3.1.7 Rationale and Hypothesis ......................................................................................66
3.2 Materials and Methods .......................................................................................................67
3.2.1 Drugs and Peptides ................................................................................................67
3.2.2 Co-immunoprecipitation and GST affinity pull-down ..........................................67
3.2.3 GST fusion protein constructs ...............................................................................68
3.2.4 HEK293T Cell Culture and DNA Transfection.....................................................69
3.2.5 cAMP accumulation assay .....................................................................................69
3.2.6 Surface biotinylation assay ....................................................................................70
3.2.7 Acute mouse brain slices .......................................................................................70
3.2.8 Primary culture of MGE-derived neurons .............................................................71
3.2.9 MGE Matrigel Explant ..........................................................................................71
3.2.10 Immunohistochemistry and immunocytochemistry ...............................................71
3.2.11 Analysis of immunohistochemistry .......................................................................73
3.2.12 Animals ..................................................................................................................73
3.2.13 Open-Field Locomotor Activity ............................................................................73
3.2.14 Pre-pulse Inhibition ................................................................................................74
3.2.15 Sociability Test ......................................................................................................74
3.2.16 Touchscreen Discrimination Task .........................................................................75
3.2.17 Responding on different schedules of reinforcement ............................................78
3.2.18 Statistical analyses .................................................................................................79
3.3 Results ................................................................................................................................79
3.3.1 Activation of the D1R facilitates the D1R-SynGAP complex formation ...............79
3.3.2 Identification of the amino acid sequence that enables the D1R to form a
complex with SynGAP ..........................................................................................79
3.3.3 SynGAP upregulates D1R-mediated signaling through the D1R-SynGAP
interaction. .............................................................................................................83
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3.3.4 SynGAP enhances D1R cell surface localization through the D1R-SynGAP interaction. .............................................................................................................86
3.3.5 Disruption of the D1R-SynGAP interaction inhibits GABAergic interneuron migration. ...............................................................................................................89
3.3.6 Disruption of the D1R-SynGAP complex changes microtubule dynamics ...........95
3.3.7 Prenatal disruption of the D1R-SynGAP complex leads to more parvalbumin- and calbindin-positive GABAergic interneurons located in the lateral
neocortex of adult offspring. ................................................................................100
3.3.8 Disruption of D1R-SynGAP on animal behaviours .............................................103
3.4 Discussion ........................................................................................................................113
Chapter 4 ......................................................................................................................................120
Presynaptic Dopamine D2 Receptor Upregulates Dopamine Reuptake Actions of Dopamine
Transporter Through Their Physical Interaction .....................................................................120
Presynaptic Dopamine D2 Receptor Upregulates Dopamine Reuptake Actions of
Dopamine Transporter Through Their Physical Interaction ...................................................121
4.1 Introduction ......................................................................................................................121
4.1.1 The Dopaminergic System ...................................................................................121
4.1.2 The Four Major Pathways of the Dopaminergic System .....................................122
4.1.3 Dopamine Receptors ............................................................................................123
4.1.4 Dopamine Transporter .........................................................................................125
4.1.5 Rationale and Hypothesis ....................................................................................127
4.2 Materials and Methods .....................................................................................................131
4.2.1 Experimental Animals .........................................................................................131
4.2.2 Commercially-Synthesized Peptides ...................................................................132
4.2.3 Animal Surgery ....................................................................................................132
4.2.4 In vivo microdialysis............................................................................................132
4.2.5 High Performance Liquid Chromatography (HPLC) ..........................................133
4.2.6 Peptide-Induced Locomotor Activity in WKY and SHR Rats ............................133
4.2.7 Y-Maze Test.........................................................................................................134
xi
4.2.8 Co-immunoprecipitation and Western Blot .........................................................134
4.2.9 Data Analysis .......................................................................................................135
4.3 Results ..............................................................................................................................136
4.3.1 The Effects of D2R-DAT Disruption on Extracellular Dopamine .......................136
4.3.2 The Effects of TAT-DATNT on Hyperactivity in SHR rats .................................139
4.3.3 The Effects of TAT-DATNT on Spontaneous Alternation Behaviour (SAB) in
SHR Rats ..............................................................................................................143
4.4 Discussion ........................................................................................................................145
Chapter 5 Conclusion and Future Directions ...............................................................................152
Conclusion and Future Directions ...........................................................................................152
5.1 Conclusion .......................................................................................................................152
5.2 Future Directions .............................................................................................................156
5.2.1 mGluR1-NMDAR Interaction .............................................................................156
5.2.2 D1R-SynGAP Interaction .....................................................................................159
5.2.3 D2R-DAT Interaction ...........................................................................................161
5.3 Final Thoughts .................................................................................................................162
References ....................................................................................................................................163
Copyright Acknowledgements.....................................................................................................194
xii
List of Tables
Table 3. 1 - Habituation and Pre-training phases prior to the visual touchscreen discrimination
training. ....................................................................................................................................... 106
xiii
List of Figures
Figure 1.1 – Schematic Illustration of Synaptic Neurotransmission between Neurons ................. 2
Figure 1.2 – Structural Representation of a G-Protein-Coupled Receptor (GPCR) ....................... 4
Figure 2.1 – The mGluR1-NMDAR protein complex is mediated through two sites within the C-
terminus of mGluR1. .................................................................................................................... 34
Figure 2.2 – The pretreatment of TAT-mGluR1C1+C4
protected hippocampal neurons against
NMDA-excitotoxicity. .................................................................................................................. 39
Figure 2.3 – The pre-treatment of TAT-mGluR1C1+C4
reduced brain infarction and improved
motor recovery in animals subjected to transient middle cerebral occlusion. .............................. 43
Figure 2.4 – TAT-mGluR1C1+C4
showed beneficial effects even when administered one hour
after the onset of ischemia. ........................................................................................................... 46
Figure 2.5 - The disruption of mGluR1-GluN2A protein complex hindered the NMDAR-
mediated activation of ERK signaling. ......................................................................................... 48
Figure 2.6 - The disruption of mGluR1-NMDAR protein complex did not alter the membrane
expression of NMDAR. ................................................................................................................ 50
Figure 3.1 - Regulation of D1R-SynGAP Complex Formation .................................................... 82
Figure 3.2 - SynGAP modulates D1R-mediated signaling............................................................ 85
Figure 3.3 - SynGAP enhances cell surface membrane localization of D1R ................................ 88
Figure 3.4 - TAT-fusion peptides can cross both the placenta and blood-brain barrier, and TAT-
D1Rpep is able to disrupt the D1R-SynGAP interaction. ................................................................ 91
Figure 3.5 - Disrupted GABAergic interneuron tangential migration in TAT-D1Rpep-injected
embryonic mice ............................................................................................................................. 94
xiv
Figure 3.6 - TAT-D1Rpep changes neuronal cytoskeleton and decreases MAP2 phosphorylation.
....................................................................................................................................................... 99
Figure 3.7 - Abnormal distribution of both PV- and CB-interneurons in adult offspring from
TAT-D1Rpep-injected pregnant mice. .......................................................................................... 102
Figure 3.8 - The disruption of D1R-SynGAP during neurodevelopment causes behavioural
abnormalities. .............................................................................................................................. 105
Figure 3.9 - The disruption of D1R-SynGAP during neurodevelopment impairs associative
learning. ...................................................................................................................................... 110
Figure 3.10 – The Disruption of D1R-SynGAP does not affect motivational behaviours .......... 112
Figure 4.1 – The Disruption of D2R-DAT Protein Complex Stimulates Voluntary Movement in
Sprague-Dawley Rats.................................................................................................................. 128
Figure 4.2 – The Disruption of D2R-DAT by TAT-DATNT Rescues the Locomotor Impairment
Imposed by AMPT-Mediated Dopamine Depletion. .................................................................. 131
Figure 4.3 – The Stimulant Effect of D2R-DAT Disruption is due to the Rise of Extracellular
Dopamine Level in SD Rats. ...................................................................................................... 138
Figure 4.4 – TAT-DATNT Alleviates the Hyperactivity of Spontaneously Hypertensive Rats. . 142
Figure 4.5 –TAT-DATNT at Low Dose Improved Spontaneous Alternation Behaviour in SHR
Rats. ............................................................................................................................................ 144
xv
List of Abbreviations
3,5-DHPG 3,5-dihydroxyphenylglycine
5-HT1AR serotonin 5-HT1A receptor
5-HT2AR serotonin 5-HT2A receptor
5-HT3AR serotonin 5-HT3A receptor
aCSF artificial cerebrospinal fluid
ADHD attention-deficit hyperactivity disorder
AMPT α-methyl-p-tyrosine
ASD autism spectrum disorder
Ara-C cytarabine
ATP adenosine triphosphate
β1-AR β1-adrenoceptor
BRET bioluminescence resonance energy transfer
BSA bovine serum albumin
CB calbindin
CFP cyan fluorescent protein
CNS central nervous system
coIP coimmunoprecipitation
CR calretinin
xvi
CT carboxyl tail
D1R dopamine D1 receptor
D2R dopamine D2 receptor
D3R dopamine D3 receptor
D5R dopamine D5 receptor
DAG diacylglyercol
DAT dopamine transporter
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
EL extracellular loop
ERK1/2 extracellular signal–regulated kinases1/2
FR fixed ratio
FRET fluorescence resonance energy transfer
GABA γ-aminobutyric acid
GABAAR GABAA receptor
GAD glutamic acid decarboxylase
GalR1 galanin receptor-1
GDP guanosine diphosphate
GPCR G-protein-coupled receptor
GST glutathione-S-transferase
xvii
GTP guanosine triphosphate
HPLC high-performance liquid chromatography
ICA internal carotid artery
ICV intracerebroventricular delivery
IL intracellular loop
IP intraperitoneal
IP3 inositol-1,4,5-triphosphate
IPTG isopropyl β-D-1-thiogalactopyranoside
LB lysogeny broth
MAPK mitogen-activated protein kinase
MGE medial ganglion eminence
mGluR1 metabotropic glutamate receptor 1
NAc nucleus accumbens core
NET norepinephrine transporter
NMDAR N-methyl-D-aspartate receptors
P2RY purinergic receptor
PAR protease-activated receptor
PBS phosphate-buffered saline
PFC prefrontal cortex
PKA protein kinase A
xviii
PPI prepulse inhibition
PSD-95 postsynaptic density 95
PR progressive ratio
PV parvalbumin
SAB spontaneous alternation behaviour
SEM standard error of mean
SERT serotonin transporter
SHR Spontaneously Hypertensive rat
SST somatostatin
SynGAP synaptic Ras GTPase-activating protein
TfR transferrin receptor
TM transmembrane domain
tMCAO transient middle cerebral artery occlusion
TTC tetrazolium chloride
VIP vasoactive intestinal peptide
VTA ventral tegmental area
WKY Wistar Kyoto rat
YFP yellow fluorescent protein
1
Chapter 1 Introduction
Introduction
1.1 Neuronal Communication within the Central Nervous System
Neurons, cells within in the central nervous system (CNS), are different from other cells
in in the human body because they possess specialized thin branches knowns as dendrites and
axons. Dendrites allow neurons to receive signals from other neurons, whereas neurons send
information to neighboring neurons through their axons. This exact process of neurons
communicating with each other is known as synaptic neurotransmission. Because the cell
membrane acts as an insulator against direct electrical signaling, neuronal communication must
be made through chemical signals like neurotransmitters. This exact process of neurons
communicating with each other is known as synaptic neurotransmission.
This chemical communication occurs when the dendrite of one neuron and the axon of
another neuron are brought within 20 to 40 nm of each other, forming the synaptic cleft1. To
initiate synaptic neurotransmission a neuron must have an action potential traveling down its
axon terminal, creating an increase in the concentration of intracellular calcium ions. The
increasing calcium ions cause vesicles filled with neurotransmitters to fuse with the cell
membrane, triggering the release of neurotransmitters into the synaptic cleft. The
neurotransmitters then travel through the synaptic cleft and bind to neurotransmitter receptors on
the surface of adjacent neurons. The neuron releasing the chemical signal is called the
presynaptic neuron, whereas the neuron that receives and reacts to the released neurotransmitter
is called the postsynaptic neuron (Figure 1.1).
2
Figure 1.1 – Schematic Illustration of Synaptic Neurotransmission between Neurons
The picture depicts the connection between both presynaptic and postsynaptic neurons. The
presynaptic neuron communicates with the postsynaptic neuron by releasing neurotransmitters
into the synaptic cleft. The neurotransmitters travel through the cleft and bind to membrane
receptors on the surface of the postsynaptic neuron to trigger a response.
1.2 Types of Postsynaptic Membrane Receptors --- ionotropic vs metabotropic receptors
There are two major classes of neurotransmitter receptors sitting on the dendritic surface
of postsynaptic neurons: ligand-gated ion channel receptors and metabotropic receptors.
Ligand-gated ion channel receptors are also known as ionotropic receptors. These
receptors are integral membrane proteins that contain a pore, which provides a pathway for
specific ions to flow across the cell membrane. Upon the binding of neurotransmitters to their
3
orthosteric sites, ligand-gated ion channel receptors undergo conformational changes to open
their pores for charged ions to pass through and enter the cell1, 2. These receptors are typically
responsible for mediating fast synaptic neurotransmission on a millisecond time scale.
Ligand-gated ion channel receptors are generally classified as either excitatory or
inhibitory, depending on their effects on the postsynaptic neuron3. They are considered
excitatory when their pores allow positively charged ions (i.e. cations, usually sodium or calcium
ions). The influx of these positive ions causes the postsynaptic neuron to depolarize, making it
more likely to fire an action potential. Inhibitory ligand-gated ion channel receptors are only
permeable to negatively-charged ions (i.e. anions, usually chloride ions). Upon the binding of
neurotransmitters, these channel receptors will also open their pores, but the influx of chloride
ions causes postsynaptic neuron hyperpolarization, preventing the subsequent firing of an action
potential.
Metabotropic receptors elicit their effects on the postsynaptic neuron through their
downstream signaling cascades1. Upon the binding of neurotransmitter, metabotropic receptors
activate proteins that in turn activate “effector” proteins/enzymes. The activated effector
enzymes subsequently generate second-messenger molecules that can diffuse within a cell to
elicit effects on a variety of target proteins, greatly changing their activities. Since metabotropic
receptors undergo several molecular steps to exert their actions rather than by directly opening an
ion channel pore upon activation, the effects they produce are slower in onset and longer lasting
than those produced by ionotropic receptors, and range from hundreds of milliseconds to
minutes3. A classical example of metabotropic receptors is the G-protein-coupled receptor
superfamily.
4
1.3 G-Protein-Coupled Receptors
The G-protein-coupled receptors (GPCRs) are a receptor superfamily which all the
members share several structural similarities. Every GPCR possesses seven transmembrane
domains (TM1-7) composed of hydrophobic amino acids which are embedded in the membrane
in a way that allows the receptor to undergo conformational changes, in addition to an
extracellular amino terminus and an intracellular carboxyl tail (Figure 1.2)4.
Figure 1.2 – Structural Representation of a G-Protein-Coupled Receptor (GPCR)
The diagram illustrates the basic structural similarities shared by members of the GPCR
superfamily. Along with an extracellular amino terminus and an intracellular carboxyl tail, each
GPCR possesses seven membrane-spanning domains (TM1-7), which are connected by three
intracellular loops (IL-3) and three extracellular loops (EL1-3).
5
G-protein-coupled receptors are coupled to a heterotrimeric guanine nucleotide-binding
protein, also known as a G-protein. Each G-protein consists of three subunits: Gα, Gβ and Gγ.
Based on sequence homology, four major G-protein families (Gs/olf, Gi/o, Gq/11 and G12/13) have
been identified within the human genome5, 6.
Upon the binding of a neurotransmitter, a GPCR undergoes conformational changes to
expose its G-protein, promoting the exchange of the bound guanosine diphosphate (GDP) for
guanosine triphosphate (GTP)7-9. When bound to GDP, the G-protein is in its inactive form as its
three subunits (i.e. αβγ) remain associated. A neurotransmitter-induced conformational change of
the GPCR accelerates the dissociation of GDP from its G-protein, which is the rate-limiting step
in G-protein activation10. Due to the high concentration of GTP inside a cell, the nucleotide-free
G-protein will quickly bind to any available GTP to facilitate its own activation. Subsequently,
the displacement of GPD by GTP causes the G-protein to dissociate from the receptor and to
separate into two parts (α- and βγ- subunits). Both the dissociated α- and βγ- subunits are then
free to alter the activities of different downstream effector proteins inside the cell, leading to
changes in cellular biochemistry, physiology and even gene expression. For instance, Gα subunits
can modulate effectors such as adenylyl cyclase, cGMP phosphodiesterase, phospholipase C,
etc.11, 12. Whereas, Gβγ subunits recruit G-protein-coupled receptor kinases to the cell membrane
and regulate a variety of ion channels13, 14. These cellular responses are terminated when the Gα
subunit reunites with Gβγ upon its complete hydrolysis of GTP.
1.4 Diversity of GPCR
More than three decades ago, the rhodopsin receptor was the first G-protein-coupled
receptor (GPCR) to be structurally identified15, 16. The studies showed that the rhodopsin receptor
transduces light energy into its transcellular signaling cascades. Since the structural revelation of
6
the rhodopsin receptor, significant technical advances have been made to unveil more members
from the GPCR superfamily17. With the growing body of studies on the GPCR superfamily, it
has become evident that this receptor superfamily can react to a broad range of ligands including
small organic compounds18, 19, eicosandoids20, peptides21 and proteins22, in addition to light.
By 2007, the human GPCR superfamily has been found to consist of at least 799 unique
full-length receptors23. Fredriksson and colleagues further classified these 799 receptors into five
main families based on phylogenetic criteria: Rhodopsin, Secretin, Adhesion, Glutamate and
Frizzled24. To avoid possible confusion with specific GPCR receptors (e.g. rhodopsin receptor vs
Rhodopsin receptor family), these five GPCR family names will be written in italics with an
initial capital letter in this thesis.
1.4.1 The Rhodopsin receptor family/Class A GPCR
Within the GPCR superfamily, the Rhodopsin receptor family has the most members,
containing approximately 670 full-length receptors23. Structurally, members of the Rhodospin
receptor family possess short N-termini but diverse transmembrane domains. Given their short
N-termini, the binding of ligands is primarily mediated by the transmembrane regions and
extracellular loops 25.
The Rhodopsin receptor family can respond to a diversity of ligands including peptides,
amines and purines. Its diverse ligands involve this receptor family in a wide range of
physiological functions including the senses of vision and olfaction, neuronal signaling, and
immunological and cardiovascular functions, etc26. Consequently, the Rhodopsin receptor family
can be further divided into four sub-groups: α, β, γ, and δ24, 27.
7
The α-group contains at least 18 physiologically important receptors, including the
histamine receptors, the dopamine receptors, the serotonin receptors, the adrenoceptors, the
muscarinic receptors, the prostanoid receptors, and the cannabinoid receptors28. These receptors
are also widely studied for the development of antihistamines, antacid drugs, cardiovascular
drugs and antipsychotics. In general, the ligand-binding pocket for members from the α-group is
considered to be embedded in the transmembrane cavity, involving different transmembrane
domains 29.
The β-group mainly consists of peptide-binding receptors, notably the neuropeptide Y
receptor. It is hypothesized that the binding pocket of this receptor group lies within the
transmembrane cavity with the involvement of the extracellular loops and the N-terminus.
The γ-group includes receptors for both peptides and lipid-like ligands24. The members of
this group include opioid receptors, somatostatin receptor and angiotensin receptors. The δ-group
mostly contains the purinergic receptors (P2RYs), the glycol-protein-binding receptors, the
protease-activated receptors (PARs) and the olfactory receptors24.
1.4.2 The Secretin receptor family/Class B GPCR
Unlike the Rhodopsin family, the Secretin receptor family is a relatively small family,
consisting of only 15 members. Secretin receptors share between 21% and 67% sequence
homology, while most of the variation exists in the N-terminus region. The members of this
family are the calcitonin and calcitonin-like receptors, the corticotropin-releasing hormone
receptors, the glucagon receptor, the gastric inhibitory polypeptide receptor, the glucagon-like
peptide receptors, the growth-hormone-releasing hormone receptors, the adenylate cyclase
activating polypeptide receptor, parathyroid hormone receptors, the secretin receptor, and the
vasoactive intestinal peptide receptors30. Given the identities of its members, the Secretin
8
receptor family is mostly involved in the endocrine and metabolic system of the mammalian
body.
1.4.3 The Adhesion receptor family/Class B GPCR
The Adhesion family is the second largest receptor family within the GPCR superfamily,
having 33 members24. Despite sharing some sequence similarities with the Secretin receptor
family, there are striking differences within the N-terminal domain architecture between both
families30. The Adhesion family possesses the GPCR proteolytic (GPS) domain in its
extracellular regions, whereas the Secretin family does not. Adhesion receptors bind extracellular
matrix molecules, while Secretin receptors bind peptide hormones. It is believed that this family
likely plays a role in immunological functions and developmental biology of the central nervous
system26.
1.4.4 The Glutamate Receptor Family/ Class C GPCR
The Glutamate receptor family has 22 members: eight metabotropic glutamate receptors,
two GABAB receptors, the calcium-sensing receptor, the sweet and umami taste receptors,
GPRC6A and seven orphan receptors31. Based on its members, it is evident that this family is
important for neurobiological and gustatory functions26.
In general, members from the Glutamate family interact with their respective endogenous
ligands within their N-termini. Specifically, the crystallization of rat metabotropic glutamate
receptor 1 (mGluR1) shed light into the mechanism by which the N-terminus interacts with a
ligand. The crystallization study illustrated that the N-terminus of mGluR1 is folded into two
lobes, fixed by intraprotomeric disulphide bridges32. Its ligand-binding mechanism has been
compared to a Venus flytrap: the two lobes of its N-terminus form a cavity for the binding of
9
glutamate. Aside from their ligand-binding N-termini, metabotropic glutamate receptors can also
interact with their allosteric ligands through their TM3, TM5, TM6 and TM733-35.
1.4.5 The Frizzled Receptor Family/ Class F GPCR
The Frizzled receptor family consists of 11 members: ten frizzled receptors and one
smoothen receptor24. The frizzled receptors bind to Wnt glycoproteins36, whereas the smoothen
receptor functions in a ligand-independent manner serving as the signaling unit in the sonic-
hedgehog-and-smoothened-receptor complex37. The receptors from this family are implicated in
developmental biology, cancer, etc.26.
1.5 GPCR Dimerization
Although GPCRs can function as monomers38, a growing body of research has suggested
the possibility that GPCRs may also function as dimers (i.e. two GPCR come together to form a
protein complex). Coimmunoprecipitation of differentially epitope-tagged receptors has been one
of the commonly-used techniques to study GPCR dimerization. This technique was first used to
study the dimerization of β2 adrenergic receptors (β2-AR). Hebert et al. co-expressed HA- and
MYC-tagged β2-AR in cell lines, and detected HA immunoreactivity in samples
immunoprecipitated with anti-MYC antibodies, demonstrating the existence β2-AR dimers39. To
ensure the selectivity of the receptor-receptor interaction, the investigators repeated the study
using MYC-tagged M2 muscarinic receptor, but did not coimmunoprecipitate it along with the
HA-tagged β2-AR. Following this study, many research groups have used similar approaches and
reported the homodimerization of different GPCRs including the dopamine receptor40, the
metabotropic glutamate receptor 541, the δ-opioid receptor 42, the M3-muscarinic receptor 43, etc.
Aside from coimmunoprecipitation, dimerization can be detected using resonance energy
transfer approaches. This technique can detect dimerization in living cells as it bases on the non-
10
radiative transfer of energy between an energy donor and an energy acceptor44. With
bioluminescence resonance energy transfer (BRET) technique, upon the dimerization of two
receptors, the catalytic degradation of a luciferin molecule by a luciferase enzyme generates
resonance energy that transfers to a green fluorescent protein and enables the fluorescent protein
to emit at its characteristic wavelength45. By genetically linking GFP and Renilla luciferase to
the carboxyl tail of β2-AR, a BRET study confirmed that β2-AR dimers do exist in living cells46.
Furthermore, BRET was used to show dimerization in δ-opioid receptors47 and thyrotropin-
releasing hormone receptors48.
Another technique to detect receptor-receptor interactions in living cells is fluorescence
resonance energy transfer (FRET). In this technique, a pair of fluorescent proteins such as cyan
fluorescent protein (CFP) and yellow fluorescent protein (YFP) are used due to their unique
emission-absorption relationship49. Upon the excitation of CFP, it generates resonance energy
that is transferred to excite YFP if the two receptors are in close proximity. Stanasila et al. have
used FRET to report the homo-dimerization of α1a-adrenoceptors and α1b-adrenoceptors,
respectively50. In addition to this, the formation of heterodimer between α1a-adrenoceptor and
α1b-adrenoceptor has also been documented using FRET50.
1.6 Protein-Protein Interaction on signaling cascades
1.6.1 Interaction Between Members of the Same Receptor Subfamily
There is an increasing body of evidence demonstrating the existence of receptor-receptor
interactions between closely related receptors such as GABAB receptor 1 and GABAB receptor 2
51-53, the δ-opioid and µ-opioid receptors54, 55, and the SST3 and SST2a somatostatin receptors56.
11
GPCRs are known to exert their physiological functions through their interactions with
G-proteins. Therefore, receptor-receptor interactions may present a new means for GPCRs to
elicit their physiological actions. In the Rhodopsin receptor family, the physical interaction
between dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R) was first reported in rat
striatal tissue using coimmunoprecipitation57. The D1R-D2R interaction was later confirmed in
both primary rat striatal neuronal culture and in situ brain sections using confocal FRET
studies58.
To unveil the structures mediating the D1R-D2R interaction, Pei et al. generated GST-
fusion proteins that encoded different fragments of each dopamine receptor and performed a
series of affinity binding assays59. The experimental data concluded that the carboxyl tail of D1R
interacts with the third intracellular loop of D2R to facilitate their receptor-receptor interaction59.
A different research group further expanded that the D1R-D2R interaction was specifically
mediated through discrete amino acids within those regions: two adjacent glutamic acid residues
in the carboxyl tail of D1R and two adjacent arginine residues in the third intracellular loop of
D2R60.
In the central nervous system, five distinct dopamine receptors (i.e. D1 to D5 receptors)
have been identified, and all of them belong to the GPCR superfamily. In general, D1 receptors
activate adenylyl cyclase to increase cyclic AMP production through Gs/olf protein signaling,
whereas D2 receptors exert the opposite effects by inhibiting adenylyl cyclase through their Gi/o
protein coupling61, 62. Rather than Gs/olf or Gi/o G-proteins, the activated D1R-D2R heterodimer
couple with a completely different G-protein signaling cascade (i.e. Gq/11), which enables the
protein complex to activate phospholipase C and trigger different downstream pathways to
release calcium from endoplasmic reticulum57, 58, 63.
12
The D1R-D2R protein complex is not the only example in which a physical interaction
between two receptors can alter their signaling transduction. The physical interaction between
dopamine D2 receptor (D2R) and D5 receptor (D5R) is another example, where the carboxyl tail
of D5R interacts with the third intracellular of D2R 64, 65. The activation of D5R induces a robust
increase in intracellular calcium level, and this ability is attenuated by its interaction with D2R64.
Another example within the dopaminergic receptor family is the interaction between D2R and
dopamine D3 receptor (D3R)66. Novi et al. co-transfected COS-7 cells with both D2R and D3R to
study whether D3 receptor can alter D2 functions through their physical interaction, and revealed
that in the presence of excess D3R, partial D2R agonists such as aripiprazole and S33592 lost
their partial agonistic properties but rather produce antagonistic actions at D2R67.
1.6.2 Interaction Between Members of Different Receptor Subfamilies
Receptor-receptor interaction between members of different GPCR receptor subfamilies
can also impact their respective signaling cascades. The interaction between dopamine D2
receptor (D2R) and serotonin 5-HT2A receptor (5-HT2AR) is one example. This receptor-receptor
interaction was detected in cells using FRET, BRET, and coimmunoprecipitation techniques68-70.
The interaction is mediated by the carboxyl tail of 5-HT2AR and the third intracellular loop of the
D2R68. Using FRET, Lukasiewicz et al. determined that ligand-binding can directly influence the
formation of this heterodimer in cells: the presence of either 5-HT2AR agonists or D2R agonists
decrease the level of interaction between the two receptors, whereas their respective antagonists
increase it68. Regarding their G-protein signaling cascades, the two receptors are associated with
different G-proteins and therefore distinct downstream signaling cascades: the D2R is linked to
Gi/o protein, whereas 5-HT2AR is linked to Gq/11. Through their interaction, however, Borroto-
Escuela et al. reported that activated D2R enhances serotonin 5-HT2AR-mediated Gq/11 signaling
cascade (e.g. phospholipase C activation and intracellular Ca2+ mobilization)69. On the other
13
hand, the activation of 5-HT2AR attenuates the ability of D2R to inhibit adenylyl cyclase69. These
findings together suggested an intriguing functional cross-talk between D2R and 5-HT2AR
through their physical interaction.
Another physical interaction leading to functional cross-talk occurs between two
members from the Rhodopsin receptor family: the galanin receptor-1 (GalR1) and serotonin 5-
HT1A receptor (5-HT1AR)71. GalR1, a receptor for the neuropeptide galanin, is a member of the γ-
group in the Rhodopsin receptors family, whereas 5-HT1AR belongs to the α-group24. Both
receptors transduce their signals through the activation of Gi/o proteins, inhibiting the activity of
adenylyl cyclase72, 73. The receptor-receptor interaction between GalR1 and 5-HT1AR was first
reported in HEK293 cells using FRET, whereas the co-activation of GalR1 and 5-HT1AR did not
yield additive inhibition of adenylyl cyclase, despite both receptors being coupled to Gi/o
proteins71. Furthermore, the presence of GalR1 reduces ligand-binding affinity for 5-HT1AR
receptor, suggesting an antagonistic allosteric relationship between the receptors74.
1.6.3 Interaction between a GPCR and a non-GPCR
Aside from interacting with members from their own GPCR superfamily, GPCRs can
also interact with membrane receptors outside the GPCR superfamily, with which they share no
structural or functional similarities. One of the most intriguing examples of such GPCR/non-
GPCR interactions is the interaction between dopamine D5 receptor (D5R) and the ligand-gated
ion channel GABAA receptor (GABAAR)75. This interaction is mediated through the carboxyl
tail of D5R and the second intracellular loop of the GABAAR γ2-subunit. It was revealed in
HEK293 cells that the formation of the D5R-GABAAR protein complex requires the co-
activation of both receptors. Functionally, the presence of GABA reduces the ability of D5R to
stimulate adenylyl cyclase, but does not alter the affinity of dopamine for its receptor. The
14
activation of D5R also dampens GABAAR-mediated current in HEK293 cells and modifies the
amplitude of GABAAR-medated miniature inhibiotry postsynaptic currents in cultured
hippocampal neurons. Such antagonistic regulation is abolished when a peptide encoding the
carboxyl tail of D5R is present or when the carboxyl-tail of D5R receptor is replaced with the
carboxyl tail of dopamine D1 receptor. These data together emphasize the importance of the
binding between the carboxyl tail of D5R and the γ2 subunit of GABAAR for the expression of
functional cross-talk.
Functional cross-talk through physical interaction is not exclusively limited to receptor-
receptor interaction. Hu et al. showed that the β1-adrenoceptor (β1-AR), a GPCR, can also
interact with cytoplasmic proteins such as the postsynaptic density scaffolding protein PSD-9576.
The interaction is mediated by the carboxyl tail of β1-AR and the third PDZ domain of the PSD-
95. Particularly, the last few amino acids of the β1-AR carboxyl tail are crucial for the formation
of this interaction as mutations of these few amino acid residues abolish the binding and disrupt
the co-localization of β1-AR and PSD-95 in HEK293 cells. Although this interaction does not
alter the ability of β1-AR to induce the production of cylic AMP, the presence of PSD-95
markedly attenuates the internalization of β1-AR in HEK293 cells.
15
1.7 Rationale and Hypothesis
For many years, it is widely accepted that G-protein coupled receptors (GPCRs) function
through their G-protein signaling cascades as their name suggests. This conventional mechanism
of action involves the binding of an agonist, where GPCR activates its G-protein and triggers its
downstream signaling cascades to exert its physiological effects.
Beside G-protein signaling cascades described in Section 1.5 and 1.6, there are studies
suggesting that members from the GPCR superfamily can physically interact with one another,
and that it is perhaps their structural similarities that allow for their receptor-receptor
interactions. Many researchers have suggested that these physical interactions can have impacts
on the functionalities of the receptors involved. In other words, a GPCR can regulate/modulate
the functions of another GPCR through their physical interaction.
As protein-protein interactions become better understood within the GPCR superfamily,
several aspects of protein-protein interactions involving GPCRs remain to be explored. For
instance, it remains to be confirmed whether protein-protein interactions provide an additional
platform for GPCRs to elicit their physiological functions in the central nervous system. Another
question is whether GPCRs exclusively form protein complexes with membrane receptors within
the GPCR superfamily. Although many studies discussed in Section 1.6 reported protein-protein
interactions solely involving receptors from the GPCR superfamily, there are also reports of
protein-protein interactions between GPCR and receptors outside the GPCR superfamily. In this
thesis, we investigate whether GPCR protein-protein interactions exclusively exist at the post-
synaptic membrane. Many reports discussed in this chapter focus on GPCR protein-protein
interactions at the post-synaptic membrane, but the possibility of GPCR protein-protein
16
interactions occurring at the pre-synaptic membrane remains to be determined. To answer some
of these questions about protein-protein interactions involving GPCRs, we have investigated
three different protein-protein interactions in this dissertation:
i) metabotropic glutamate receptor 1 and N-methyl-D-aspartate receptor (Chapter 2)
ii) dopamine D1 receptor and synaptic Ras GTPase-activating protein (Chapter 3)
iii) dopamine D2 receptor and dopamine transporter (Chapter 4)
Regarding the potential protein-protein interaction between metabotropic glutamate
receptor 1 and N-methyl-D-aspartate receptor, several studies have reported a functional
connection between these two receptors, whereas metabotropic glutamate receptor 1 has been
shown to modulate the effects of N-methyl-D-aspartate receptor on excitotoxicity. Structurally,
both receptors have been reported to bind to inter-related scaffolding proteins at the postsynaptic
membrane, prompting the possibility of a physical interaction between the receptors. In Chapter
2, our hypothesis is that these two receptors physically do form a protein complex, and such
protein complex is implicated in the excitotoxic actions of N-methyl-D-aspartate receptor.
To date, many studies have reported that GPCRs can form protein complexes with
membrane receptors, but few have focused on the possibility of GPCRs interacting with
intracellular proteins. One objective explored in this thesis is to determine whether GPCRs can
physically interact with intracellular proteins. To do so we report on the investigation of the
potential interaction between dopamine D1 receptor and synaptic Ras GTPase-activating protein
in Chapter 3. Although a growing body of research has suggested the important roles of both
dopamine D1 receptor and synaptic Ras GTPase-activating protein in the development of
GABAergic interneurons within the central nervous system, the exact molecular mechanism
17
remains elusive. Therefore, we hypothesize that dopamine D1 receptor interacts with synaptic
Ras GTPase-activating protein, and more importantly, this interaction is crucially involved in the
tangential migration of GABAergic interneurons in the developing mammalian brain.
Unlike the two protein-protein interactions mentioned above, the interaction between
dopamine D2 receptor and dopamine transporter is known to occur at the pre-synaptic membrane.
This interaction is mediated through the N-terminus of dopamine transporter and the third
intracellular loop of dopamine D2 receptor. Although the previous studies have revealed that this
interaction is involved in the regulation of dopamine re-uptake in vitro, its physiological
functions remain to be confirmed in vivo. Based on the previous in vitro data, we here
hypothesize that the interaction between dopamine D2 receptor and dopamine transporter
promotes the re-uptake of dopamine in vivo, and therefore the disruption of this interaction leads
to an increase in extracellular dopamine and causes behavioural changes.
By studying these three potential protein complexes, we have examined the prospects of
GPCRs physically interacting with a membrane receptor (N-methyl-D-aspartate receptor) and an
intracellular protein (synaptic Ras GTPase-activating protein) at the post-synaptic membrane as
well as a neurotransmitter transporter (dopamine transporter) at the pre-synaptic membrane. By
disrupting these protein complexes in vitro and in vivo, we strive to investigate whether protein-
protein interactions can be another viable platform for GPCRs to fulfill their physiological roles.
This investigation aims to provide direct evidence to answer the above questions regarding
GPCR protein-protein interactions.
18
Chapter 2
Metabotropic Glutamate Receptor 1 Modulates the Excitotoxic Functions of N-Methyl-D-Aspartate Receptor (NMDAR) through
its Interaction with the GluN2A subunit of NMDAR
Author contributions for this chapter:
I performed all experiments described in this chapter with the following exceptions:
• Dr. Dongxu Zhai performed the transient middle cerebral artery occlusion surgery • Dr. Ping Su conducted the co-immunoprecipitation experiment.
• Mr. Jay Boychuk performed western blotting to examine the phosphorylation level of ERK1/2.
19
Metabotropic Glutamate Receptor 1 Modulates the Excitotoxic Functions of N-Methyl-D-Aspartate Receptor (NMDAR) through its Interaction with the GluN2A subunit of NMDAR
2.1 Introduction
2.1.1 Glutamate Neurotransmission
Glutamate is the carboxylate anion form of glutamic acid, but more importantly, it is the
major excitatory neurotransmitter in the mammalian central nervous system (CNS)77-79. In the
CNS, glutamate is about 1,000 times higher in concentration than other important
neurotransmitters such as dopamine or serotonin79.
The biosynthesis of glutamate often takes place in neuronal terminals where it is largely
synthesized from glutamine. Glial cells release glutamine into extracellular spaces, where
neurons take up and transform it into glutamate via phosphate-activated glutaminase80. After
being released into the synaptic cleft, the actions of glutamate can be terminated by reuptake into
either neurons or glial cells through specific glutamate transporters. Once in glial cells, glutamate
will be transformed back into glutamine by specific glutamine synthetase81.
As a principal neurotransmitter, glutamate mediates synaptic transmission and plays a
crucial role in synaptic plasticity, learning and memory, and cell death82-85. To exert its
physiological functions, glutamate must bind to its receptors, which are divided into two main
families ----- ionotropic and metabotropic receptors. Ionotropic glutamate receptors are ligand-
gated ion channels, which include N-methyl-D-aspartate receptors (NMDAR), α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid receptor and kainate receptors. These receptors
mediate the fast-excitatory actions of glutamate by allowing the influx of cations, mostly
Na+/Ca2+ or Na+/K+ ions, into cells. Metabotropic glutamate receptors mediate slower
20
neurotransmission through their downstream signaling cascades, and generally rather serve a
modulatory role in neurotransmission.
2.1.2 The Implication of Glutamate in Ischemic Stroke
Although glutamate is essential for the normal functions of the CNS, it is also implicated
in the pathophysiology of ischemic stroke. Stoke is one of the leading causes of mortality and
long-term disability worldwide as it affects nearly 15 million people every year86, 87. In the
United States alone, a stroke will take place on average every 40 seconds, and kills one person
every four minutes88, 89. Most stroke cases (approximately 80%) are ischemic stroke, which
arises when the systematic blood flow to the brain is severely impaired due to a thrombosis, an
embolism or systemic hypo-perfusion86, 90. During an ischemic stroke, the brain is subsequently
deprived of oxygen due to the lack of steady blood flow. As a result, neurons are unable to
generate sufficient adenosine triphosphates (ATPs) through oxidative phosphorylation. In the
lack of ATPs, Na+/K+ ATPases fail to maintain and restore the ionic gradient potential across the
plasma membrane. Consequently, neurons will continue to depolarize and lead to a massive
release of neurotransmitters, particularly glutamate91, 92. Since normal membrane potential is also
required for the proper functioning of glutamate transporters, these transporters are disabled to
overcome the massive release of glutamate, further contributing to the pathological
progression93. The excessive glutamate release will then lead to over-stimulation of ionotropic
glutamate receptors, which results in a constant influx of Ca2+ ions. The abnormal massive influx
of Ca2+ ions triggers a variety of calcium-regulated processes such as the activations of
proteases, endonucleases, nitric oxide synthase, the production of free radicals, and the
disruption of mitochondrial membrane94. Within the ionotropic glutamate receptor family,
NMDAR is the most potent in mediating Ca2+ ion influx and its antagonists have been widely
considered potential neuroprotective agents95-97. Unfortunately, many NMDAR antagonists have
21
failed in clinical trials as they had unacceptable adverse effects in patients, likely due to the
physiological significance of NMDAR95, 98, suggesting the need for an alternative approach.
2.1.3 N-methyl-D-aspartate receptor (NMDAR)
Like other ionotropic glutamate receptors, NMDAR possesses a tetrameric structure. As
of now, seven NMDAR subunits have been identified: GluN1, GluN2A-GluN2D, and GluN3A-
GluN3B99. Generally, a functional NMDAR contains two obligatory GluN1 subunits and two
GluN2/GluN3 subunits. Although a thorough structural investigation has not been conducted on
NMDAR, the X-ray crystallography of a closely-related GluA2 provides some valuable insights
to the structural properties of NMDAR100, 101. It is widely accepted that all seven NMDAR
subunits share several structural similarities: an extracellular amino terminus, a ligand-binding
domain, four transmembrane domains (note: with the second “transmembrane” domain as an
membrane re-entrant loop102), and an intracellular carboxyl tail103-107. Functionally, the GluN1
subunits provide the binding site for glycine, a co-agonist, while the GluN2 subunits construct
the binding site for glutamate/NMDA. The binding of glutamate and glycine in the ligand-
binding domains causes the receptor to undergo conformational changes, opening the receptor
pore to allow the influx of cations. Within the tetrameric NMDAR, the composition of the GluN2
subunits plays an important role in determining the receptors properties such as activation and
deactivation kinetics, ion conductance, and affinity for agonist108.
2.1.4 Metabotropic Glutamate Receptor 1 (mGluR1)
To date, there are eight metabotropic glutamate receptors (mGluR1-8) identified in the
human genome109-116. These eight metabotropic glutamate receptor subtypes are divided into
three groups based on their sequence homology, downstream signaling cascades, pharmacology,
and phylogenetics117. Group I consists of mGluR1 and mGluR5, which are generally coupled
22
with Gq/11 proteins and are activated by 3,5-dihydroxyphenylglycine (3,5-DHPG). Group II has
GluR2 and mGluR3, which are coupled with Gi/o proteins and are activated by 2R,4R-amin-
opiperidindicarboxylic acid. Group III includes mGluR4, mGluR6, mGluR7 and mGluR8, which
are also reported to couple to Gi/o in recombinant systems but are activated by 2-amino-4-
phosphonobutyrate.
As a member of the GPCR superfamily, mGluR1 shares the common seven-
transmembrane-domain structure (refer to Figure 1.2) and relies on G-proteins to trigger its
downstream signaling cascades. The second intracellular loop is the longest intracellular loop
and has been implicated in G-protein coupling and selectivity118. Studies involving chimeric
receptors have confirmed that the second intracellular loop is the essential component to mediate
the coupling between mGluR1 and G-protein, whereas the first intracellular loop, the third
intracellular loop and the carboxyl tail strengthen the coupling118, 119. Upon activation, mGluR1
activates phospholipase C through its coupling with Gq/11. Phospholipase C will in turn cleave
phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-triphosphate (IP3) and diacylglyercol
(DAG). While DAG stays at the membrane to activate protein kinase C, the soluble IP3 can
diffuse into the cell and bind to its receptors on the surface of the endoplasmic reticulum to
release Ca2+ from the intracellular calcium store.
2.1.5 Rationale and Hypothesis: Functional/Physical Cross-Talk between mGluR1 and NMDAR and Their Implications in Ischemic Stroke
Located on the post-synaptic membrane, mGluR1 may influence the actions of NMDAR,
and previous studies have suggested that mGluR1 contributes to NMDAR-mediated
excitotoxicity. For instance, pharmacological activation of mGluR1 amplifies the neurotoxic
effects of NMDAR stimulation in vitro120-122. Reports have also shown that mGluR1 antagonists
prevent neuronal damage in animal models of stroke123, 124. Since both NMDAR and mGluR1 are
23
localized on the post-synaptic membrane, there is a possibility that they may form a physical
interaction. The GluN2A subunit of NMDAR can bind scaffolding proteins such as PSD-95125,
126, whereas mGluR1 through its carboxyl tail can form protein complexes with Homer1, another
scaffolding protein127. Interestingly, the coupling to Homer1 enables mGluR1 to form protein
complexes with intracellular proteins such as PSD-95 through Shank protein128. Therefore, we
hypothesize that: 1) mGluR1 and NMDAR form a receptor-receptor interaction, and that 2) this
receptor-receptor interaction influences the excitotoxic actions of NMDAR.
2.2 Materials and Methods
2.2.1 Co-immunoprecipitation
Rat hippocampal brain tissue was lysed with 1 × RIPA lysis buffer (50 mM Tris (Bioshop
Canada Inc.; Catalogue #: TRS001), 150 mM NaCl, 1% NP-40 (Bioshop Canada Inc.; Catalogue
# NON505), 0.5% deoxycholate (Sigma-Aldrich Canada Co.; Catalogue # D6750), 2 mM
EDTA, 1% Triton X-100, 0.1% SDS, 10% glycerol and 1:100 protease inhibitor cocktail (Sigma-
Aldrich, catalogue# P8340)), and homogenized by a physical homogenizer. For co-
immunoprecipitation, 500 – 1000 μg solubilized protein extracted from rat hippocampal brain
tissue was incubated in the presence of primary antibodies or IgG (negative control) (1 – 2 μg)
together with protein A/G plus agarose (Santa Cruz Biotechnology, Dallas, TX, USA; catalogue#
sc-2003) at 4°C for 12 h. Pellets were washed with 1 × TBST (20 mM Tris, 150 mM NaCl, and
0.1% Triton X-100 at pH 7.4), boiled for 5 min in 2x Laemmli sample buffer (Bio-Rad
Laboratories, Mississauga, ON, Canada; catalogue# 161-0737) and subjected to SDS-PAGE. 50
– 100 μg of protein extracted from rat hippocampal brain tissue was also boiled in 2x Laemmli
sample buffer and used as a control in each experiment.
24
2.2.2 GST Protein Affinity Purification
For GST protein affinity purification experiments, 500 – 1000 μg of protein was
incubated with glutathione-sepharose beads (GE Healthcare Bio-Sciences, Mississauga, ON,
Canada; catalogue# 17075601) bound to the indicated GST-fusion proteins (50 – 100 μg) at 4°C
overnight. Beads were washed with PBS buffer (80mM Na2HPO4, 1.5M NaCl, 20mM
KH2PO4, 30mM KCl, and 0.5% Triton X-100 at pH 7.4), boiled for 5 min in SDS sample buffer
and subjected to SDS-PAGE.
2.2.3 Western Blotting
The SDS-PAGE was conducted in Tris-Glycine-SDS buffer (BioBasic Canada Inc.;
catalogue# A0030) and was run at 90 – 120 V for approximately two hours. Subsequently,
proteins were transferred onto nitrocellulose membrane at 400 mV for 2 hours before western
blotting with primary antibodies. The primary antibodies used in this study include anti-mGluR1
(Sigma Millipore, Etobicoke, ON, Canada; rabbit, catalogue# 07-617), ant-GluN2A (Santa Cruz
Biotechnology, Dallas, TX, USA; goat, catalogue# sc1468), anti-GluN2A (Sigma Millipore,
Etobicoke, ON, Canada; rabbit, catalogue# 07-632), anti-GluN1 (Sigma Millipore, Etobicoke,
ON, Canada; mouse, catalogue# 05-432), anti-TfR (Novus Biologicals, Centennial, CO, USA;
rabbit, catalogue# NB100-92243), anti-ERK1/2 (Cell Signaling Technology, Boston, MA, USA;
rabbit, catalogue# 9102S), and anti-pERK1/2 (Cell Signaling Technology, Boston, MA, USA;
rabbit, catalogue #9101S). The intensity of protein expression level was quantified by
densitometry (software: Image Lab, Bio-Rad Laboratories, Mississauga, ON, Canada).
2.2.4 GST Fusion Protein Constructs
GST-fusion proteins encoding truncated mGluR1 fragments were amplified by PCR from
full-length rat cDNA clones. GST-fusion proteins were prepared from bacterial lysates with
25
Glutathione Sepharose 4B beads as per the manufacturer (GE Healthcare Bio-Sciences,
Mississauga, ON, Canada; catalogue# 17075601) as previously described. To construct GST-
fusion proteins encoding truncated mGluR1, cDNA fragments were amplified by PCR with
specific primers (Life Technologies, Waltham, MA, USA). All 5´ and 3´ oligonucleotides
incorporated BamHI sites (GGATCC) and XhoI sites (CTCGAG), respectively, so that digestive
enzymes BamHI and XhoI (New England Biolabs, Whitby, ON, Canada) were used to facilitate
sub-cloning into the pGEX-4T3 vector. All constructs were sequenced to confirm appropriate
splice fusion and the absence of spurious PCR generated nucleotide errors.
2.2.5 GST Fusion Protein Expression and Purification
Upon the confirmation of DNA sequences, the purified plasmids were transformed into
BL21 competent cells (approximately 3 µL plasmids and 33 µL BL21 competent cells) and
incubated on wet ice for 30 minutes. Subsequently, samples were heat-shocked at 42°C for 90
seconds, followed by a two-minute incubation on wet ice. The samples were then cultured on
ampicillin-containing lysogeny broth (LB) agar plates overnight at 37°C.
Following the overnight incubation, bacterial colonies were collected from the
ampicillin-containing LB agar plates. Picked colonies were incubated (at 250 rpm and 37°C) in
25 mL ampicillin-containing LB medium overnight, and then the solution was transferred into a
225 mL of fresh ampicillin-containing LB medium for further incubation (at 250 rpm and 37°C)
until the culture solution reached an optical density value of 0.6 at 600 nm. To induce the
production of GST-fusion proteins, isopropyl β-D-1-thiogalactopyranoside (IPTG) at 0.5 mM
was added to the culture solution, and the incubation continued for three hours in an orbital
shaker at 250 rpm and 37°C. The solution was then centrifuged at 3000 rpm for 20 minutes, and
the resulting pellets were collected for protein extraction.
26
The pellets were re-suspended and lysed in 5 mL PBS containing 1% Triton and 1:100
protease inhibitors. To break the cells, the samples were sonicated (Vibra Cell, Sonics &
Materials Inc. Danbury, Connecticut, USA) on ice for 40 seconds, and was repeated for twice
more for each sample. Following sonication, samples were incubated on a shaker at 4°C for one
hour to allow further lysis. Subsequently, samples were centrifuged at 12, 000 rpm for 10 – 15
minutes and the resulting supernatant was collected from each sample. Supernatants were mixed
with glutathione agarose beads to purify the GST-fusion proteins and incubated on a shaker at
4°C for two hours. Following the two-hour incubation, samples were centrifuged at 3000 rpm at
4°C for one minute to remove the supernatant from each sample. Remaining beads were washed
with 1× PBS. The GST-fusion proteins were eluted from the beads by the addition of 10 mM
glutathione in 50 mM Tris-HCl, and samples were centrifuged at 14,000 rpm for 10 minutes to
collect the supernatants containing purified GST-fusion proteins.
2.2.6 Primary Mouse Hippocampal Culture
Pregnant CD-1 mice were sacrificed, and embryos were collected on E16-18. The
embryos were dissected to collect the hippocampal region. The hippocampal tissues were
subsequently subjected to 0.25% Trypsin lysis for 15 minutes at 37°C. After, trypsin was
inhibited by Dulbecco's Modified Eagle Medium containing10% fetal bovine serum. Samples
were centrifuged at 100 × g for five minutes to remove supernatant. Fresh neurobasal medium
containing 1:50 B27, 1:100 L-Glutamine and 1:100 Strepto-Penicllin (catalogue# 15140122,
Gibco by Life Technologies) were added and the tissue pellet was then mechanically dissociated
by gentle trituration through a fire-polished Pasteur pipette. The cultured neurons were then
plated into poly-D-lysine (catalogue# P1024, Sigma-Aldrich, Oakville, ON, Canada) coated 24-
well plate at a density of 1.0 × 105 cells per well. Cultures were incubated at 37°C in a 5% CO2
incubator in Neurobasal (catalogue# 12349015, Gibco by Life Technologies) + B27 (catalogue#
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17504044, Gibco by Life Technologies) + Glutamax (catalogue# 35050061, Gibco by Life
Technologies) medium. Half of the medium were replaced by fresh neurobasal medium every
three to four days until the end of experiments. Cytarabine (Ara-C) (Sigma-Aldrich, Oakville,
ON, Canada) was added to the medium from four days in vitro (DIV 4) onwards to prevent glial
cell differentiation.
2.2.7 NMDA-Excitotoxicity in Primary Mouse Hippocampal Culture and Confocal Imaging
On DIV 13-14, half the medium was collected from wells and replaced by fresh
neurobasal medium. Neurons were treated with vehicle, treatment peptides (10 μM; TAT-
mGluR1C1 YGRKKRRQRRRTFLNIFRRKKPGAGN and TAT-mGluR1C4 YGRKKRRQRRR-
PGTPGNSLRSLYPPPPPPQHLQML) or TAT control peptide (10 μM; YGRKKRRQRRR) for
one hour. Subsequently, neurons were exposed to 100 μM NMDA in the presence of 10 μM
glycine for one hour. Following NMDA exposure, medium was removed and replaced with the
original medium collected at the beginning of the experiment. 24 hours later, the neurons were
double-labelled with 50 μg/mL propidium iodide (Sigma-Aldrich, catalogue# P4170) and 20
μg/mL Hoechst 33342 (Life Technologies, Waltham, MA, USA; catalogue# H3570). Following
the histological staining, the cells were washed with 1x PBS, and then fixed with 4%
paraformaldehyde for 15 minutes. After fixation, cells were washed three times with 1x PBS,
five minutes each. Subsequently, cells were imaged with confocal laser scanning microscope
(Olympus FluoView FV1200, Center Valley, PA, USA) at 10× objective lens magnification for
primary cultured neurons. The number of propidium iodide and Hoechst 33342-stained cells was
quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA) to assess the level
of cell death.
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2.2.8 Animals
Male Sprague-Dawley rats weighing 200 – 225g were purchased from Charles River
Laboratories (Montreal, Quebec, Canada), and were acclimatized to the animal facility for a
week before being subject to any surgical and experimental procedures. All animal procedures
were approved by the Animal Care Committee at the Center for Addiction and Mental Health
and followed the relevant guidelines and regulations of the Canadian Council on Animal Care
2.2.9 Transient Middle Cerebral Artery Occlusion (tMCAO)
The intraluminal suture method was used to induce transient focal cerebral ischemia
(tMCAO) in rats as described previously129, 130. Male Sprague Dawley rats, weighing 300 – 350
grams, were anesthetized using 5% isoflurane and maintained at 2.5%, supplemented with
compressed air. tMCAO was achieved by introducing a 3–0 monofilament suture into the middle
cerebral artery via the internal carotid artery. Ischemia was maintained for 90 minutes. During
the surgical procedure, body temperature was maintained at 36.5-37.5°C with a rectal feedback
controlled homoeothermic blanket system and a heating lamp. Rats were deeply anesthetized
with isoflurane and sacrificed 24 hours after the induction of tMCAO.
2.2.10 Intracerebroventricular (ICV) Peptide Delivery
Animals were mounted onto a stereotaxic frame under anesthesia. The scalp was shaved
and swabbed with betadine, then a midline frontal incision was made in the scalp and the skin
was retracted bilaterally. A burr hole (2 mm) was drilled into the skull using a hand-drill and an
injector cannula was gently inserted into the following coordinate: 0.8 mm posterior to the
bregma, 1.5 mm lateral to the midline, and 4.5 mm ventral to the surface of the skull. After the
cannula reached its final dorsal/ventral coordinate, 3 µl of peptide solution (5 mM; 15 nmol per
animal) was slowly administered. Following the injection, the injector cannula remained in
29
position for 1 minute before being slowly removed. The opening on the skull was then closed
with bone wax, and the scalp incision was sutured with 3-0 silk suture. Animals were then
removed from the stereotaxic frame and placed underneath a heat lamp for recovery.
2.2.11 Neurological Assessment
Neurological examinations were performed at two hours and at 24 hours after the onset of
the occlusion. Neurological functions were scored on a five-point scale 131, 132: a score of 0
indicated no neurological deficits, a score of 1 (failure to extend the left forepaw fully), a mild
focal neurological deficit, a score of 2 (inconstant circling to the left) a moderate focal
neurological deficit, a score of 3 (falling to the left) a severe focal deficit, and rats with a score of
4 did not walk spontaneously and had a depressed level of consciousness.
2.2.12 Tetrazolium Chloride (TTC) Staining and Brain Infarction Analysis
TTC staining was performed as previously described with some modifications130.
Following the 24-hr assessment, animals were fully anesthetized with 5% isoflurane. Upon
achieving complete anesthesia, animals were perfused with PBS; brains were immediately
collected, dissected and cut into 1-mm-thick coronal sections. The slices were incubated in
0.25% 2,3,5-triphenyltetrazolium chloride (in PBS) at 37°C for 30 minutes, and subsequently
fixed with 4% paraformaldehyde (in PBS) overnight at 4°C. The slices were then scanned and
analyzed for infarction using ImageJ (National Institutes of Health, Bethesda, MD, USA).
2.2.13 Acute Brain Slice Treatment
Male Sprague Dawley rats, weighing 225 – 250 g, were purchased from Charles River
Laboratories (Quebec, Canada). One week after arrival, animals were completely anesthetized
with 5% isoflurane through inhalation; animals were then sacrificed, and the brains were
collected. The brains were immediately submerged in ice-cold artificial cerebrospinal fluid
30
(aCSF: 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM
NaHCO3 and 25 mM glucose at pH 7.4) with constant supply of 95% O2 and 5% CO2 for five
minutes. After the five-minute incubation in aCSF, the hippocampus region was isolated and cut
into 350-µm slices using a McIlwain Tissue Chopper. The slices were then incubated in 35°C
aCSF with constant supply of 95% O2 and 5% CO2 for 60 minutes to allow tissue recovery
2.2.14 NMDA-Stimulated ERK1/2 Phosphorylation
Following the recovery and peptide treatment procedures described above, brain slices
were subjected to 50 µM NMDA for 30 minutes, except for the control group. After NMDA
exposure, slices were lysed with 1x RIPA. One PhosSTOP pellet (Roche Diagnostics,
Indianapolis, IN, USA; catalogue# 4906845001) was dissolved in 10 mL 1x RIPA to inhibit
phosphatases and preserve phosphorylation of extracellular signal–regulated kinases1/2
(ERK1/2). The level of ERK1/2 phosphorylation was then determined through immunoblotting.
2.2.15 Membrane Expression of NMDA Receptors
Following the recovery procedures described above, the slices were divided into five
treatment groups: i) control, ii) TAT peptide (20 µM TAT peptide), iii) TAT-mGluR1C1 (10 µM
TAT and 10 µM TAT-mGluR1C1), iv) TAT-mGluR1C4 (10 µM TAT and 10 µM TAT-
mGluR1C4), and v) TAT-mGluR1C1+C4 (10 µM TAT-mGluR1C1 and 10 µM TAT-mGluR1C4).
Except for the control group, the slices were treated with their corresponding peptide treatments
for 60 minutes. Following the peptide incubation, brain slices were lysed, and membrane
proteins were extracted using a commercially available extraction kit (Product # BSP002,
BioBasic Inc.). The solubilized proteins were subsequently subjected to western blotting to
determine the membrane expression of GluN1 and GluN2A subunits of NMDAR.
31
2.2.16 Statistical Analysis
All data were expressed as mean ± standard error of mean (SEM). The significance levels
of *p < 0.05, **p < 0.01, or ***p < 0.001 were used for all an
