Determinants of High Affinity Ligand Binding to the Group III Metabotropic Glutamate Receptors
Mark Naples
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Phannacology University of Toronto
O Copyright by Mark Naples 2001
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Determinants of High Afanity Ligand Binding to the Group III M+tabotropfe GtutamateRtccptors
Mark Naples Master of Science 2001
Graduate Department of Pharmacology University of Toronto
Abstract
Metabotropic glutamate receptors (mGluRs) comprise a family of G-protein-coupled
receptors (GPCRs) that modulate fast excitatory transmission in the marnmalian nervous
system. The initial purpose of my study was to compare the ligand-binding selectivity
profiles of the mGluR agonist [)H]L-AP~ and the novel radiolabeled antagonist ['HICPPG at
al1 eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. My
results indicate that at low nanomolar concentrations, [ 3 ~ ] ~ - ~ ~ 4 labels mGluRs 4 and 8
while ['HICPPG is selective for mGluR8. The ability of [ 3 w ~ ~ ~ ~ to label a single mGluR
subtype demonstrates that this compound and its related phenylglycine derivatives are useful
for studying ligand recognition differences arnong the highly homologous group iII receptors.
Another goal of my study was to investigate the possibility that mutation of a conserved
amino acid residue in mGluR4 (R78A) promotes closure of this receptor. Subsequent
experirnents indicated that this mutation might instead interfere with receptor glycosylation.
Acknowledgements
Firstly, 1 would like to thank my supervisor Dr. David Hampson for his guidance and
support over the course of my graduate studies. 1 have leamed a lot about research (and
everything 1 know about glutamate receptors) dunng the past two years and am grateful for
the opponunity to work in his lab. 1 would dso like to thank the members of my advisory
cornmittee, Drs. Susan George and Christine Bear, for their comments and suggestions on this
project.
1 want to thank my family and fnends for their encouragement, especially my parents and
grandparents for their generosity whenever 1 ran out of money (which seemed to be often).
Thanks also to my CO-workers and fellow students: Guangming Han, Vanya Peltekova, Nima
Soleymanlou, Gein Wong, Erica Rosemond, Dawn Kuang, and Xi-Ping Huang for making
my stay in the lab enjoyable and relatively stress-free.
My work on this project was supported by the Canadian Institutes of Health Research and
an Ontario Graduate Scholarship.
iii
Table of Contents
Abs trac t
Acknowledgements
Table of Contents
List of Abbreviations
List of Tables
List of Figures
List of Appendices
1. INTRODUCTION
1.1 Glutamate and its Target Receptors
1.2 Metabotropic Glutamate Receptors
(a) Structural Characteristics
(b) Functional Properties
1.3 Synaptic Locdization and Regional Distribution of Group III mGluRs
1.4 Regulation of Neurotransrnitter Release by Group III mGluRs
1.5 Group III Receptor Pharmacology
1.6 Therapeutic Potentid for Group III Receptor Ligands
1.7 Research Objectives and Rationale
2. MATERIALS AND METHODS
2.1 Chetnicals and Reagents
2.2 Standard Procedures in Molecular Biology
(a) Bacterial Cultures and Transformations
v
viii
X
xi
xii
(b) Preparation of Plasrnid DNA
(c) Restriction Endonuclease Digestion a d Agarose Gel Electrophoresis
(d) Gel Extraction and Ligation of DNA Fragments
2.3 Expression of Recombinant Roteins in Marnmalian Cells
(a) cDNA Expression Constructs
(b) Site-Directed Mutagenesis
(c) DNA Sequencing
(d) Culture and Transfection of Human Embryonic Kidney Cells
2.4 Membrane Preparation from Transfected Cells
2.5 Sodium Dodecylsulfate Polyacrylarnide Gel Electrophoresis and Immunoblotting
2.6 I~H]L-AP~ and [ 3 ~ ] ~ ~ ~ ~ Binding Assays and Data Analysis
2.7 Deglycosylation of mGluR4 and mGluR4 R78A
3. RESULTS
3.1 Expression of Recombinant mGluRs in HEK-293 Cells
3.2 Radioligand Binding to mGluR-Expressing HEK Ce11 Membranes
3.3 Characterization of [ 3 ~ ] ~ ~ ~ ~ as a High Affinity Robe for mGluR8
3.4 Effect of mGluR8 Point Mutations on Rotein Expression and Radioligand Binding
3.5 h u n o b l o t s of an mGluR4 Point Mutation, rnGluR4 R78A
3.5 Expression and Binding Roperties of the mGluR4 K 3 l7A and K3 17E Mutants
3.7 Deglycosylation of mGluR4 and mGluR4 R78A
4, DISCUSSION
4.1 Phamacological Profiles of the Metabotropic Glutamate Receptor Ligands [ 3 ~ ] ~ ~ ~ 4 and [ 3 ~ ~ ~ ~ ~
4.2 Amino Acids Mediating High Affinity Ligand Binding to mGluR8 78
4.3 Characterization of an Immunoreactive Band Unique to mGluR4 R78A 81
4.4 Conclusions and Future Studies 87
5. REFERENCES 90
List of Abbreviations
AMPA
BHK
Bmpx
BSA
CAMP
CaR
cDNA
CHO
CNS
CPPG
ECso
GABA
GPCR
G-protein
HEK
ICso
iGIuR
p 3
KD
kDa
KI
L-AP4
-amino-3- hydroxy-5-methy l-4-isoxazole propionate
baby hamster kidney
maximal number of binding sites
bovine semm albumin
cyclic adenosine monophosphate
ca2'sensing receptor
complementary DNA
Chinese hamster ovary
central nervous system
(R,S)-• -cyclopropyl-4-phosphonophenylglycine
half-maximal effective concentration
-aminobutyric acid
O-protein-coupled receptor
guanine-nucleotide-binding protein
hurnan embryonic kidney
ha1 f-maximal inhi bi tory concentration
ionotropic glutamate receptor
inositol- l,4,5-triphosphate
equilibrium dissociation constant
kilodal tons
equilibnum inhibition constant
L-2-amino-4-phosphonobutyrate
vii
LB
L-CCG-1
LIVBP
L-SOP
MAP4
MCPG
mGluR
MPPG
M r
mRNA
NMDA
PBP
PBS
PCR
PKA
PKC
PLC
(R,s)-PPG
SDS-PAGE
SEM
TMD
Lwia-Bertani
(2s. 1 'S.2'S)-2-(carboxycyclopropyl)gIycine
leucine, isoleucine, valine binding protein
L-senne-O-phosphate
-methyl-LAP4
(R,S)-• -methyl-4-carboxyphenylglycine
metabotropic glutamate receptor
-methyl4phosphonophenylglycine
relative rnolecular weight
messenger RNA
N-methy 1-D-aspartate
periplasmic binding protein
phosphate-buffered saline
polymerase chah reaction
protein kinase A
protein kinase C
phospholipase C
(R,S)4phos phonophenylglycine
sodium dodecylsulfate polyacrylarnide gel electmphoresis
standard emor of the rnean
transmembrane domain
viii
List of Tables
Tabie - Page
1. Comprehensive summary of radiolabeled probes available for studying mGIuRs 23
2. Cornpetition for [ 3 ~ ] ~ ~ ~ ~ binding to mGluR8 by various metabotropic ((R,S)-MCPG) and ionotropic glutamate receptor ligands (AMPA, kainic acid, NMDA, ibotenic acid), and the glutamate uptake inhibitor L-trans-PDC 52
3. Inhibition constants for agonists and antagonists cornpeting at [ 3 ~ ] ~ ~ ~ ~
binding sites on mGluR8 56
4. Affinities of [ 3 ~ ] ~ - ~ ~ 4 and [ 3 ~ ] ~ ~ ~ ~ for wild-type and mutant group III receptors 61
Lit of Figures
Figure Page
1. Phylogenetic tree displaying the sequence identity among selected mamrnalian family 3 GPCRs
2. Diagram of the proposed activation mechanism for mGluRs
3. Agonists and competitive antagonists acting at group IïI rnGluRs
4. Residues mediating glutamate binding to mGluRl and the group IU receptors mOluR4 and mGluR8
5. Irnmunoblots demonstrating expression of mGluR subtypes
6. Comparison of [ 3 ~ ] ~ - ~ ~ 4 and [ 3 ~ ] ~ ~ ~ ~ binding to mGluRs
7. Time-courses for the association and dissociation of [ 3 ~ ] ~ ~ ~ ~ to mGluR8 at 0°C
8. Cornpetition for [ 3 ~ ] ~ ~ ~ ~ binding to mGluR8 by the agonists L-SOP, L-AP4, D-AP4, L-glutamate. and (R,S)-PPG and the competitive antagonists CPPG, and MPPO
9. Comparison of [ 3 ~ ] ~ - ~ ~ 4 and L~H]CPPG binding to a series of mGluR8 mutant receptors
10. Immunoblots of the mGluR4 R78A and mGIuR8 R75A mutants
1 1. Immunoblots showing the effects of PNGase F-mediated deglycosylation of mGluR4 and mGluR4 R78A
12. Comparison of expression levels and [ 3 ~ ] ~ - ~ ~ 4 binding to the mGIuR4 K317A and K3 17E mutants
List of Appendices
Amendix
1. Primary antibodies used in this study
2. Sensitivity of [ 3 ~ ] ~ ~ ~ ~ binding to changes in pH
3. Analysis of binding results
Page
101
102
104
1. INTRODUCTION
1.1 Glutamate and i ts Target Receptors
L-Glutamate is the primary excitatory neuromuismitter in the mammalian central
nervous system (CNS). Glutamate is released fiom presynaptic nerve teminals and exerts its
eflects via two distinct classes of receptors: the ionotropic glutamate recepton (iGluRs) and
the metabotropic glutamate receptors (mGluRs). The iGluRs comprise a heterogeneous
family of glutamate-gated cation channels which have been subdivided into three broad
categones called the N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-S-rnethyl-4-
isoxazole propionate (AMPA), and kainate recepton (see Hol i rna~ and Heinemann, 1994;
Ozawa et al., 1998 for reviews). Most iGluRs are located on postsynaptic nerve teminals.
These receptors are found at the majority of synapses in the mammalian CNS and play a
fundamental role in fast excitatory transmission.
It was initially believed that the effects of glutamate were mediated solely by the activity
of iGluRs. Modulation of excitatory transmission (i.e. ce11 excitability) was thought to
involve activation of guanine-nucleotide-binding protein (G-protein) coupled receptors by
neuromodulators released fiom non-glutamatergic (extrinsic) afferents (e.g. acetylcholine,
serotonin, dopamine, and norepinephnne; Corn and Pin, 1997). The notion that control of
glutamatergic neurotransmission required extrinsic modulation was challenged in 1985, when
Sladeczek et al. observed that application of glutamate to striatal neurons stimulated the
production of inositol phosphates. Shortly thereafler, it was found that Xenopus oocytes
injected with rat cerebral messenger RNA (mRNA) exhibit an oscillatory Cl- current upon
glutamate application resulting from inositol- 1,4,5-triphosphate (IP+mediated intracellula.
ca2+ release (Sugiyama et al., 1987; Murphy and Miller, 1988). Consequently, the existence
of a novel G-protein-coupled glutamate receptor was proposed. In 1991, the first mGluR
complementary DNA (cDNA) was cloned independently by two groups (Masu et al., 1991;
Houamed et al., 1991) using the same expression cloning technique pioneered by Hollmann et
al. in 1989 to clone the first iGluR cDNA. Since that time, seven other genes and several
splice vanants encoding mGl uRs have been isolated and charac terized. These receptors,
named mGluRl through mGluR8, have been subdivided into three groups based on sequence
homology, pharmacology, and signal transduction properties (Conn and Pin, 1997). Group 1
receptors (mGluR I and mGluR5) couple to phosphatidylinositol hydrolysis, whereas group 11
(mGluR2 and mGluR3) and group 111 receptors (mGluR4, mGluR6, mGluR7, and mGluR8)
are negatively coupled to adenylyl cyclase. Amino acid sequence identity within the receptor
groups is between 60-70%, but this percentage decreases to about 45% between groups (Fig.
1). Overall, there is little sequence homology between the mGluRs and iGluRs, with the
exception of two stretches in the amino(N)-terminal region of mGluRl (amino acids 2 15-352
and 453-597) that were noted by Masu et al. (1991) to exhibit low level homology with
cloned AMPA receptor subunits. This homology may reflect conserveci sequences within the
ligand-binding domain of the two glutamate receptor classes. The structural features and
functional properties of mGluRs are reviewed in further detail below with emphasis on the
group III receptors.
The heterogeneity and ubiquitous distribution of glutamate receptors within the CNS
underlies the ability of glutamate to influence a variety of neuronal processes. In addition to
mediating fast synaptic transmission, glutamate receptor activation has been implicated in the
neuronal changes involved in memory acquisition and learning. the formation of neural
networks during development, and the pathology of some neurodegenerative disorders (see
Nakanishi, 1992; Pin and Duvoisin, 1995; Corn and Pin, 1997; Ozawa et al., 1998 for
Figure 1. Phylogenetic tree displaying the sequence identity amoag selected mammalian
family 3 GPCRs. Metabotropic glutamate receptors share sequence homology with the CaR
and GABAe receptors. The division of mGluRs into three groups and the major signal
transduction pathway associated with each of these groups is shown.
PLC = phospholipase C
AC = adenylyl cyclase
CaR
I
II
III
Transducticm pathway
+ PLC
O 20 40 60 80 100
Percent identity
reviews). The discovery of G-protein-coupled receptors (GPCRs) activated in response to
glutamate provides a mechanism whereby glutamatergic synapses cm regulate or 'fine-tune'
their activity by influencing secondary messenger production.
1.2 Metabotropic Glutamate Receptors
(a) Structural Characteristics
All mammalien mGluR cDNAs cloned to date encode very large proteins ranging
in size fiom 87 1 to 1 199 amino acids. The general smicture of mGluRs is similar to other
GPCRs in that they possess an extracellular N-terminus and an intracellular carboxyl(C)-
terminal region separated by seven putative transmembrane domains (TMDs). Despite this
structural resemblance, mGtuRs do not exhibit any sequence similarity with either the
rhodopsin-like GPCRs (family 1, e.g. adrenergic recepton) or the large-peptide receptors
(family 2, e.g. glucagon receptors). As such, mGluRs represent a distinct family of GPCRs
(family 3) that includes the metabotropic y-aminobutyric acid receptors (GABABR), the ca2+-
sensing receptor (CaR) of the parathyroid, and the putative pheromone and taste receptors
(Kaupmann et al., 1997; Brown et al., 1993; Matsunami and Buck. 1997; Hoon et al., 1999).
Among the family 3 GPCRs, the CaR exhibits the highest degree of homoiogy with the
mGluRs (30% sequence identity; Brown et al., 1993), followed by the GABAB receptors at
-20% (Kaupmann et al., 1997; refer to Fig. 1).
The most stnking feature of the family 3 GPCRs is the enormous size of their extracellular
N-terminal domains (-550 amino acids for the mGluRs) compared to other GPCRs.
Functional and phartnacological analysis of mGluR2ll (Takahashi et al., 1993), mGluR4/ 1
(Tones et al., 1995), and mGluR3/1 (Wroblewska et al., 1997) chimeras in which the N-
terminal domains were swapped indicate that this large extracellular region is involved in the
selective recognition of agonists and competitive antagonists. This is in contrast to family 1
GPCRs many of which bind ligands in a crevice formed by their transmernbrane helices. The
ensuing production of soluble proteins of mGluRI, mGluR4, and mGluR8 that encompass
most of the N-terminal region and retain binding of group selective ligands has provided
fbrther evidence that binding is mediated predominantly by this domain (Okamoto et al.,
1998; Han and Hampson, 1999; Peltekova et al., 2000). The large N-teminal regions of
GABAeRl and the CaR have likewise been shown to contain the primas, detenninants
required for ligand binding (Malitschek et al., 1999; Galvez et al., 1999; Hammerland et al.,
1 999).
In 1993, O'Hara et al. reporteci that the N-terminal domains of mGluRs share weak
sequence homology (-20%) with the extracellular domains of bacterial periplasmic binding
proteins (PBPs). PBPs mediate the high amnity transport of sugars, amino acids, and ions
into bacteriai cytoplasm. The crystal structures of many of the PBPs have been detennined.
By analogy with their three-dimensional structures, in particular the leucine, isoleucine, valine
binding protein (LIVBP) from Escherichia coli (E. coli), a homology model of the N-terminal
domain of mGluRl was constnicted (O'Hara et al., 1993). This model, coupled with results
from site-directed mutagenesis experiments, predicted that the amino acid residues important
for glutamate binding to mGluR1 are equivalent to the residues mediating amino acid binding
to LIVBP. The rccent crystallization of the N-terminal domain of mGluRl has validated the
model established by O'Hara et al. by confirming these interactions (Kunishima et al., 2000).
The conformation of the extracellular domain of mGluRs and the specific residues mediating
ligand binding to group III mGluRs will be discussed further in Section 1.7.
In addition to containing the ligand-binding region, the N-terminal domain and
extracellular loops of al1 eight mGluR subtypes also possess 19 conserved cysteine residues.
The majority of these are clustered in a short stretch of amino acids located immediately
upstream of the first TMD (referred to as the cysteine-rich region). Consenation of these
residues in al1 members of the mGluR family suggests that they sente important roles in
stabilizing the three-dimensional structure of these receptors (Ozawa et al., 1998; Pin et al.,
1999). Moreover, it has been proposeci that the cysteine-rich region plays a fundamental role
in the receptor activation process (Conn and Pin, 1997; Pin et al., 1999) given that glutamate-
stirnulated phosphatidylinositol hydrolysis is highly sensitive to reducing agents (Vignes et
al., 1993). The current mGluR-activation hypothesis is that agonist binding induces and
stabilizes a conformational change in the receptor that is transmitted through the cysteine-rich
region to the hluismembrane domains. Subsequent movement of the TMDs is thought to
expose G-protein binding sites on the intracellular portion of the receptor (Fig. 2).
Coupling of G-proteins to mGluRs likely involves al1 four intracellular segments of the
receptor. The second intracellular loop (i2) has been shown to detemine O-protein coupling
specificity while the other intracellular loops and the C-terminal tail appear to control the
efficricy of coupling (Pin et al., 1 994; Gomeza et al., 1 996a). Evidence that the C-terminal tail
affects agonist potency cornes fiom the observation that naturally occumng splice variants of
mGluR 1 possessing truncated C-termini (mGluR 1 b and mGluR 1 c) decrease agonist potency
in a heterologous expression system (Flor et al., 1996).
In addition to influencing G-protein interactions, the C-terminal region has been implicated
in protein kinase C (pK)-dependent phosphorylation of the group 1 mGluRs (Alaluf et al.,
1995; Gereau and Heinemann, 1998) and observed to control coupling of these receptors to
the N-terminal domain of various Homer proteins (Brakeman et al., 1997; Kato et al., 1998;
Tu et al., 1998). Homer proteins contain an Ena-VASP homology (EVH 1) domain that
recognizes a proline-rich consensus sequence (PPxxFR) common to the group 1 mGluRs and
Figure 2. Diagrim of the proposed activation mechanism for mGluRs. The upper panel -
is a simplified representation of mGluR structure. The large, bilobed extracellular amino-
teminal domain is analogous to those of PBPs. Based on the activation mechanism of these
bactenal proteins, it has been proposed that agonist binding within the clefi formed by the two
lobes of the receptor results in a large conformational change, trapping the ligand within this
domain. This is referred to as the Venus-flytrap' model. It is thought that the conformational
change associated with agonist binding is conveyed to the extracellular loops and TMDs via a
stretch of conserved cysteine residues. Subsequent movement of the TMDs could unmask G-
protein binding sites on the second intracellular loop of the receptor resulting in G-protein
activation (adapted fiom Pin et al., 1999).
Transmembrane region
Intracellular loops and C-terminal tail
~ p & ein
the IP3 receptors. The majority of Homer proteins are constitutively expressed in postsynaptic
densities within the CNS with highest levels found at excitatory synapses. Of the Homer
proteins identified, three splice variants of Homerl (Homerla, Ib, and lc) have been
proposed to play an important role in mGluR signaling. With the exception of Homerla,
these proteins possess a C-terminal coiled-coi1 domain that mediates the formation of Homer
dimers (homomers and heteromers). Accordingly, Homer l b and Homer 1 c promote coupling
between group 1 mGluRs and the IP3 receptor while the inability of Homerla to dimerize
prevents this interaction (Tu et al., 1998; Fagni et al., 2000). Whereas phosphorylation of the
C-terminus of group I mGluRs mediates receptor desensitization, interactions with Homer
proteins can either facilitate (e.g. Homerlb or Homerlc) or hinder (e.g. Homer 1 a) coupling of
group I receptors to their downstream effectors (Le. IP3-meàiated ca2* release; Pin et al.,
1999). The potential importance of Homer la as an inhibitor of group 1 receptor fhction is
underscored by the observation that expression of this protein is induced following synaptic
stimulation (Brakeman et al., 1997; Kato et al., 1998). Although group III mGluRs do not
couple to Homer proteins, activation of PICC has been demonstrateci to disrupt the functional
response mediated by these receptors (Macek et al., 1999). Evidence also exists that protein
kinase A (PU)-induced phosphorylation of group II1 receptors can regulate their activity (De
Blasi et al., 2001).
Other studies have proposed that the C-terminal region may also be responsible for
targeting mGluRs to different intracellular compartments. For example, it has been shown
that specific targeting of mGluR7 to the active zone of presynaptic newe terminais is
controlled by its C-terminus (Stowell and Craig, 1999). The precise role of the C-terminal
regions of mGluR4 and mGluR8 remain to be elucidated. The existence of C-terminal splice
variants for both receptors (mGluR4a, mGluR4b, mGluR8a, mGluR8b) suggests an
involvement in receptor trafficking a d o r desensitization (Thomsen et al., 1997; Corti et al.,
1998; De Blasi et al., 200 1 ).
(b) Funetional Properties
In heterologous expression systems, group 1 mGIuRs stimulate phospholipase C
(PLC) activity. The subsequent formation of IP3 prornotes ca2' release fiom intracellular
stores (Masu et al., 199 1; Abe et al., 1992; Aramon and Nakanishi, 1992). In most cases,
activation of group 1 receptors increases ce11 excitability by inhibiting K' channel activity, an
effect that may or may not be G-protein dependent. Evidence for a G-protein independent
signaling pathway mediated by mGluR 1 comes from a study demonstrating that an Src-family
tyrosine kinase is responsible for the production of excitatory postsynaptic currents at mossy
fibre synapses following mGluRl activation (Heuss et al., 1999). In ce11 lines, al1 other
mGluR subtypes (group II and group III receptors) are negatively coupled to adenylyl cyclase.
Inhibition of cyclic adenosine monophosphate (CAMP) production in response to activation of
these receptors is sensitive to pertussis toxin, suggesting that the G-proteins involved in this
signaling cascade belong to the Gi family (Conn and Pin, 1997). The ability of presynaptic
group IlA1I receptors to inhibit glutamate release is discussed in Section 1.4.
1.3 Synaptic Localization and Regional Distribution of Group III mCluRs
The ability of individual mGluR subtypes to regulate neurotransmitter release is
influenced by their synaptic localization (Ottersen and Landsend, 1997). A number of studies
have shown that group 1 mGluRs are located away fiom active zones (see Cartmell and
Schoepp, 2000 for review) and are generally restricted to postsynaptic teminals (Shigemoto
et al., 1997). The group II mGluRs exhibit both pre- and postsynaptic distributions but,
similar to the group 1 receptors, they seern to be concentrated towards the penphery of the
synapse (Petralia et al., 1996). Shigemoto et al. (1997) demonstrated that mGluR2 -
immunoreactivity was located predominantly in presynaptic terminals of the hippocampus,
while others have shown mGluR3 to be highly expressed in glial cells (Ohishi et al., 1993b;
Mineff and Valtschanoff, 1999). The involvement of glial cells in glutamate uptake and
synthesis suggests an important role for mGluR3 in mediating these physiological processes.
Group III receptors in the brain (mGluR4, mGluR7, and mGluR8) appear to be located in or
near presynaptic active zones. Hippocarnpal immunoreactivity for mGluR7 reveals an
exclusive localization of this subtype at glutamatergic nerve terminals, whereas mGluR4 has
been localized presynaptically at both glutamatergic and non-glutarnatergic synapses (Bradley
et al., 1996; Mitchell and Silver, 2000; Semyenov and Kullman, 2000).
The diflerential localization of mGluRs within neuronal compartments likely explains the
differences in glutamate potency among receptor subtypes. Receptors located fùrther away
from synaptic active zones generally respond to lower concentrations of glutamate than do
those found at these regions. For example, glutamate exhibits a much higher potency at
mGluR4 (E& -3-20 PM) than mGluR7 (ECSo >500 PM; Conn and Pin, 1997). This
observation could explain the lack of mGluR7 immunostaining at non-glutamatergic synapses
as it is unlikely that 'spillover' glutamate could reach high enough concentrations to activate
this receptor subtype. The ability of group 111 mGluRs located at non-glutarnatergic synapses
(e.g. presynaptic heteroreceptors at GABAergic synapses) to influence the release of other
neurotransmitters will be discussed in Section 1.4.
In situ hybridization studies have revealed that rnRNAs coding for group III mGluRs are
widely distributed throughout both rat and human brains (Shigemoto et al., 1992; Ohishi et
al., 1993a,b, 1995a; Fotuhi et al., 1994; Saugstad et al., 1994) with the exception of mGluR6
mRNA, which is restricted to the imer nuclear layer of the retina (Nakajima et al., 1993). As
wodd be expected, expression of mGluR6 is limited to this region (Ueda et al., 1997).
Immunocytochemical studies show mGluR4 expression to be highest in the cerebellum
(Kinoshita et al., 1996, Mateos et al., 1998). This finding is supported by evidence from an
autoradiographic study perfonned on mOluM knock-out mice using the group III selective
agonist [J~]~-2-amino-4-phosphonobutyrate (L-AP4). A significant decrease in [)H]L-AP~
binding was observed in the molecular layer of the cerebellum of mGluR4 knock-outs
compared to wild-type mice (Thomsen and Hampson, 1999). Moderate expression of
mGluR4a has also been observed in the hi ppocam pus ( predominantl y in the molecular layer
of the dentate gynis and the lateral pedorant path of the CA3 region), striatum, amygdala,
thalamus, olfactory bulb, and cortical areas (Risso-Bradley et al., 1996, 1999; Phillips et al.,
1997; Shigemoto et al. 1997). Highest levels of mGluR8 gene expression have bcen observed
in the pontine grey, olfactory bulb, and the lateral reticular nucleus of the thalamus. Overall,
the expression of mGluR8 shows considerable overlap with mGluR4, though levels of
mGluR8a mRNA appear to be lower than those of mGluR4a in the cerebral cortex,
hippocarnpus, and cerebellum (Duvoisin et al., 1995; Saugstad et al., 1997).
Studies on mGluR7 expression reveal that this is the most abundant group III receptor in
the CNS, with highest levels found in the olfactory bulb, hippocampus, and cerebral cortex.
In addition, low to moderate levels of mGluR7a immunoreactivity have been observed in the
amygdala, basal ganglia, thalamus, hypothalamus, superior colliculus, and the spinal cord
(Ohishi et al., 1995b; Phillips et al., 1998; Kosinski et al., 1999).
1.4 Regulrtion of Neurotransmitter Release by Croup III mGluRs
The presynaptic localization of group 111 mGluRs at excitatory synapses throughout
the CNS implies that these receptors play an important role in regulating neurotransmitter
release. Accordingly, inhibition of neuroûansmission at glutamatergic synapses by L-AP4 (a
group 111 selective agonist) has been well characterized in several brain regions including the
hippocampal CA I region, media1 and lateral perforant paths of the dentate gynis, striahun,
and olfactory bulb (for a complete review see Cartmell and Schoepp, 2000). Although the
mechanisms underlying this inhibitory efiect are not fblly understood, evidence exists that
suppression of presynaptic voltage-dependent ~ a " channels is responsible for the reduction in
glutamate release observed following activation of group III receptors (see Ozawa et al.,
1998). Moreover, the finding that L-AP4-mediated inhibition of ca2' currents in
cerebrocortical neurons is independent of changes in CAMP levels suggests that activated G-
protein subunits can directly inhibit cal+ channel activity (Herrero et al., 1996). In addition to
exerting effects on glutamate release, activation of presynaptic mGluRs has been shown to
reduce GABA release and suppress inhibitory neurotransmission in several brain areas (Conn
and Pin, 1997; Semyanov and Kullmann, 2000). For exarnple, Schaflhauser et al. (1998)
showed that application of L-AP4 inhibits KCI-evoked ['HIGABA release in primary cortical
cell cultures.
A recent study by Mitchell and Silver (2000) demonstrated that low concentrations of
glutamate released fiom excitatory mossy fibres within the cerebellum ('spillover' glutamate)
inhibited GABA release fiom Golgi ce11 terminais, an effect that was blocked by treatment
with group WiII selective antagonists. This study established that heterosynaptic mGluRs
play a physiologically relevant role in inhibitory neurotransmission by allowing GABAergic
neurons to respond to the activity of neighbounng excitatory synapses. As well as inhibiting
GABAergic activity, L-AP4 treatment has k e n s h o w to suppress the release of various other
neurotransmitters, including dopamine (Hu et al., 1999), acetylcholine (Caramel0 et al.,
1999), and substance P (Cuesta et al., 1999), presumably through the activity of presynaptic
mGluRs. Whether or not these functional interactions are mediated by heterosynaptic
mechanisms remains to be clari fied.
1 .S Croup III Reeeptor Pharmacology
This section will focus on the phamacology of the group III receptors with emphasis
on mGluRs 4 and 8. Attempts to characterize mGluR pharmacology have led to the synthesis
of several glutamate derivatives. As a result, the majority of ligands reported to exhibit
activity at mGluRs retain the glycine moiety and distal acidic fwictional group characteristic
of glutamate. Unfortunately, many of these compounds, though identified as being group
selective, are unable to discriminate between mGluR subtypes. Due to the widespread and
overlapping distributions of mGluRs within the CNS, the development of high affinity,
subtype selective compounds is cntical to a further understanding of the physiological roles
mediated by specific mGluR subtypes. Despite progress made in the identification of subtype
selective ligands over recent years, group III receptor phannacology remains relatively poorly
developed (for reviews see Pin and Duvoisin, 1995; Conn and Pin, 1997; Pin et al., 1999;
Schoepp et al., 1999). Phamacological profiles of mGluR subtypes have been characterized
using a variety of heterologous expression systerns including Xenopus oocytes, Chinese
hamster ovary (CHO) cells baby hamster kidney (BHK) cells and human embryonic kidney
(HEK) cells (Ozawa et al., 1998). The structures of selected agonists and competitive
antagonists acting at group 111 mGluRs and discussed below are depicted in Fig. 3.
A distinguishing characteristic of the group III receptors, with the exception of mGluR7, is
their sensitivity to the agonist L-AP4 (Conn and Pin 1997; Pin et al., 1999). Agonist
potencies at mGluR7 are vexy low, typically on the order of 100 to 1000 times lower than
potencies observed at mGluR4. For example, the agonists L-serine-O-phosphate (L-SOP;
Figure 3. Agonigts and cornpetitive antagonists acting at group III mGluRs. -2 .
Agonists
L-SOP
MPPG CPPG
ECn = 2-5 p M at mGluR4) and L-AP4 (ECSo = 0.4-1.2 pM at mGluM) are considerably less
potent at mGluR7 (ECSo > 160 pM and ECso = 160-500 PM, respectively; Conn and Pin,
1997). This may reflect the synaptic localization of mGluR7 compared to the other group III
receptors, as mentioned previously (Section 1.3). In general, the rank order of potency of
agonists acting at group III receptors is L-AP4 2 (R,S)-4-phosphonophenylglycine ((R,S)-
PPG) 2 L-SOP > (2S, 1 'S,2'S)-2-(carboxycyc1opropyl)glycine (L-CCG- 1) > L-glutamate >
1 S,3R- l -amino- l,3-cyclopentanedicarboxylate ( 1 S,3R-ACPD; Schoepp et al., 1999).
Radioligand binding studies perfonned on cloned mGluR4 and mGluR8 receptors expressed
in BHK and HEK cells have confirmed that this order of agonist potencies is reflective of the
relative ability of these compounds to displace [)H]L-AP~ binding (Eriksen and Thomsen,
1995; Hampson et al., 1999; Peltekova et al., 2000). In addition to being potent group III
agonists, LAP4 and L-SOP are among the most specific ligands for these receptors; both
compounds are inactive on other glutamate receptors at concentrations up to 1 m M (Pin et al,
1999; Schoepp et al., 1999). Interestingly, L-SOP is found at micromolar concentrations in
some brain regions (Klunk et al., 199 1 ). This observation, coupled with the selectivity of this
compound for group III receptors suggests that it may function as an endogenous
neurotransmi tter.
Substitution of the y-carboxyl group of L-glutamate with a phosphonate moiety appears to
underlie the selectivity of L-AP4 and L-SOP for the group III receptors. Consequently, other
phosphonate-containing compounds have been developed in the search for even more
selective group III agonists. These include (2)-cyclopropyl-AP4 (2-CPAP4), a
confonnationally constrained analog of L-AP4 equipotent to L-AP4 at mGluR4 (ECso = 580
nM; Kroona et al., 1991) and (R,S)-PPG, which has been shown to potently and selectively
activate human mGluR8 receptors (ECso = 0.2 PM; Gasparini et al., 1999).
Cornpetitive antagonists for the group III receptors include a-methyl-L-AP4 (MAP4), an
L-AP4 analogue, and a-methyl-4-phosphonophenylglycine (MPPG), which also exhibits
antagonist activity at mGluR2 (Sane et al., 1995; Pin et al., 1999). (R,S)-a-Cyclopropyl-4-
phosphonophenylglycine (CPPG) has likewise been reported to antagonize group III receptors
(Toms et al., 1996), and a ment study on cloned mGluR subtypes indicates that this
compound exhibits selectivity towards mGluR8 at low nanomolar concentrations (Naples and
Hampson, 2001; refer to later sections for details). MPPG and CPPG, together with the
agonist (R,S)-PPG, belong to a class of phosphonophenylglycine derivatives that appear to be
particularly potent at group III mGluRs and which may have the potential to discnminate
between mGluR4 and mGluR8 (De Colle et al.. 2000; Naples and Hampson, 2001).
Non-cornpetitive agonists for mGluRl and mGluR5, named 7-
(hydroxyirnino)cyclopopa[b]chromcn-l acarboxylate ethyl ester (CPCCOEt) and 2-methyl-6-
(phenylethyny1)pyridine (MPEP), respectively, have recently been reported. These
compounds bear no structural resemblance to glutamate and appear to inhibit the mGluR
activation process via interactions with the TMD region rather than the N-teminal domain
(Hermans et al., 1998a; Litschig et al., 1999; Brauner-Osborne et al., 1999; Pagano et al.,
2000). The existence of similar compounds acting at allosteric sites on group III receptors has
not been reported.
1.6 Therapeutic Potential for Group III Receptor Ligands
At present, no drugs acting on glutamate receptors have been approved for clinical
use. Drugs acting on WMDA receptors have been the focus of research for many years as
numerous studies have demonstrated that antagonists of these receptors are neuroprotective in
animal models of focal ischernia. Unfortunately, clinical trials perfomed on a variety of
NMDA antagonists for the treatrnent of stroke and other brain injuries have been unsuccessfÙ1
due to pronounced toxic side effects or a lack of efficacy (see Brauner-Osborne et al., 2000
for review). Although AMPA and kainate receptors have recently emerged as promising
therapeutic targets, the capability of mGluRs to 'fine-tune' neuronal activity rather than
completely shut down excitatory neurotransmission makes these recepton an attractive target
for drug design. Furthemore, the heterogeneous distribution of mGluR subtypes within the
CNS (compared to iOluRs) allows receptors mediating specific processes to be targeted by
selective agents. The ability of mGluRs to modulate excitatory neuromuismission has wide-
ranging implications for the treatment of a variety of pathological conditions including
ischemia, epilepsy, and some psychiatric and neurodegenerative disorders (COM and Pin,
1997; Brauner-Osborne et al., 2000).
It is well established that glutamate can be toxic to neurons, particularly when supplies of
oxygen and glucose are restricted as occun following epileptic seinires or strokes (Greene
and Greenamyre, 1996). Over-stimulation of the glutamatergic system is the underlying
cause of neuronal injury, and may play a role in the progression of neurodegenerative
disorders such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis,
and Parkinson's disease. Consequently, it has been proposed that activation of presynaptic
mGluRs promotes neuroprotection by inhibiting glutamate release (Maiese et al., 1995;
Buisson et al., 1996; Nicoletti et al., 1996). Conversely, treatment with agonists acting at
group I receptors (postsynaptic) has been s h o w to potentiate excitotoxic damage (Bruno et
al., 1995).
The involvement of specific group III receptors in neuroprotection remains to be clarifieci,
though studies have show that L-AP4 treatrnent can reduce ce11 death following chernically
or physically induced neuronal injuries (Faden et al., 1997; Lafon-Cazal et al., 1999).
Recently, Gasparini et al. ( 1999) demonstrated that the group III selective agonist (R,S)-PPG
protects cultured cortical neurons fiom NMDA-induced toxicity. A subsequent study on
mGluR4 knock-out mice with (R,S)-PPG revealed that selective activation of mGluR4 is
responsible for the neuroprotective effect of this compound (Bruno et al., 2000). In addition
to promoting neuroprotection, (R,S)-PPG has been shown to possess anticonvulsive properties
in vivo (Gasparini et al., 1999). This finding conesponds with previous studies demonstrating
that activation of group III mGluRs by L-AN or L-SOP can decrease the severity of epileptic
seinires (Tang et al., 1997; Abdul-Ghani et al., 1997). Collectively, these findings suggest
that group III mGluRs may constitute a target for the development of novel anticonvulsive
and/or neuroprotective agents.
1.7 Research Objectives and Rationale
Part 1 - Characterization of ['HICPPG, a novef radiolabeled antagonist
Until recently, the pharmacological characterization of mGIuRs has been hindered by
a lack of high affinity radiolabeled probes exhibiting selectivity for a single receptor subtype.
Exacerbating this problem is the fact that many of the radiolabeled and unlabeled mGluR
ligands have not been tested on a11 eight mGluR subtypes. Consequently, compounds that
have initially been reported to be subtype or group selective are often observed to exert effects
on other glutamate receptors at higher concentrations. For exarnple, the group II selective
antagonist [ ' ~ ] ~ ~ 3 4 1 4 9 5 (Johnson et al., 1999) has recently been shown to label ce11
membranes expressing recombinant group III recepton (Wright et al., 2000), albeit at 20 to
100-fold higher concentrations. Accordingly, to obtain a comprehensive pichire of receptor
selectivity it has become increasingly imperative to assess ligands across the spectrum of
mGluR subtypes.
Several radiolabeled ligands have become available for studying mGluRs in ment years
(see Pin et al., 1999; Schoepp et al., 1999 for reviews). These are describeci in detail in Table
1. As of late 1999, [ 3 ~ ] ~ - ~ ~ 4 was the only radioligand suitable for studying the
pharmacological propertin of the group III receptors. However, the recent synthesis of
['HICPPG may provide investigators with another tool to characterize group III
pharmacology as the unlabeled fom of this drug has been reported to be a cornpetitive
antagonist of group III receptors at low micromolar concentrations (Toms et al., 1996; refer to
Section 1 S). Moreover, this drug belongs to a class of phenylglycine derivatives that appear
to exhibit selectivity for mGluR8 over mGluR4 (De Colle et al., 2000).
The fint objective of my study was to investigate the selectivity of two radioligands,
['HIL-AP~ and ['HICPPG, using al1 eight cloned rat mGluRs expressed individually in HEK-
293 cells. [)H]L-AP~ has previously been shown to label mGluR4 and rnGluR8 but has not
been tested on al1 eight receptor subtypes. ['HICPPG is a novel radioligand and,
consequently, the ability of this compound to bind to mGluRs has not been reported. Based
on studies performed using unlabeled CPPG, it was expected that this compound would
selectively label the group III mGluRs with the exception of mGluR7, which is a low affinity
L-AP4 receptor.
The discovery of additional subtype selective ligands is essential for elucidating the
physiological roles mediated by specific mG1uRs. Moreover, the availability of high affinity,
selective radioligands is fundamental to the pharrnacological chamterkation of receptor
subtypes. These compounds are necessary tools for binding-pocket studies and are required
for autoradiographic studies of receptor localization. Ultimately, the results of such
experiments can assist in the design of higher affinity subtype specific ligands.
Table 1. Comprehensive summary of radiolabeled probes available for studying mGluRs.
Radioligand Reported Selectivity Amnity (K,)* References
Group II receptors
Group II receptors
Group II receptors
[3H]Quisqualk acid Group l receptors mGluRla = 27 nM mGluRSa = 81 nM
mGluR2 = 160 nM
mGluR2 = 20 nM mGluR3 = 53 nM
mGluR2 = 1.67 nM mGluR3 = 0.75 nM
Group III mcepton = 14-73 nM
[3H]L-AP4 mGluR4 and mGIuR8 mGluR4a = 44 t -504 nM mGluR8a = 866 nM
[3H]@utanute None
PH]CPPG mGIuR8 mGluRSa = 183 nM
Mutel et al., 2000; Okamoto et al., 1998
Cartmell et al., 1998; Mutel et al., 1998
Schweitzer et al., 2000; Schaffhauser et al., 1998
a Johnson et al., 1999; Wright et al., 2000
Enksen and Thomsen, 1995; Hampson et al., 1999; Peltekova et al., 2000
Naples and Hampson, 2001
* Affinities of radiolabeled compounds are from experiments performeà on cloned receptors expresseci in heterologous ce11 lines
Part II - Examination of the ligand binding pocket of mGluR8
Based on the binding pocket model established by analogy with the LIVBP fiom E. d i ,
residues important for binding of glutamate receptor ligands to mGluR 1 have been identified
(O'Hara et al., 1993; see Section 1.2(a)). Of these residues, three in particular ser16',
and in mGluR1) were predicted to be essential for glutamate binding due to their
conservation in al1 eight members of the mGluR fmily. Moreover, the equivalent residues in
mGluR4 ( ~ r ~ ~ ~ , sertS9, ~ h r ' ~ ~ ) were anticipated to interact directly with mGluR ligands by a
model in which the glutamate analog L-SOP was 'docked' into the binding pocket of mGluR4
(Hampson et al., 1999). Subsequent mutagenesis and radioligand binding studies conducted
on mGluR4 using ['HIL-AP~ proposed that hydroxyl groups on the side chains of serIs9 and
~ h r " ~ form hydrogen bonds with the a-carboxyl and a-amino groups of glutamate ligands,
respectively, whereas ~r~~~ participates in an electrostatic interaction with ligand side chains
(Hampson et al., 1999). Recent publication of a homodimeric crystal structure of the N-
terminal domain of mGIuRl in complex with glutamate supports this hypothesis as the
analogous residues were shown to participate in these interactions (Kunishima et al., 2000;
see Fig. 4A). The crystal structure of mGluRl also validates the hypothesis that the primary
deteminants of glutamate binding are located within a hinge region separating the two
extracellular lobes of the receptor.
Expenments conducted in our laboratory have focused on determining the residues that
mediate high affinity binding of L-AP4 to mGluM. The objective of this part of my project
was to extend this analysis to rnGluR8. This receptor also binds ['HIL-AP~ with high affinity
(Peltekova et al., 2000) and is highly homologous to mGluR4 within the region of the binding
poc ket proposed
perfonned on a
to interact with ligand side chains (Fig. 48). Site-directed mutagenesis was
series of residues within this region and mutant rcceptors tested for their
Figure 4. Residues mediating glutamate binding to mGluRl and the group III receptors .- -
mGluR4 and mGluR8. Panel A is a representation of the binding site of rnGluR1 in
complex with glutamate (from Kunishima et al., 2000). Colour is used to differentiate
between the two lobes of the receptor that comprise the ligand-binding site. Polar interactions
of glutamate with amino acid residues are denoted by dotted lines with distances (in A).
Circles indicate residues consewed among al1 eight mGluR subtypes and predicted to be
essential for glutamate binding. Note the proximity of ~ r ~ ' ' to ~r~~~~ in mGluR4) in
the closed form of the receptor.
Panel B shows a partial sequence alignment among mGluR1, mGluR4, and mGluR8
adapted fiom a multiple sequence alignment performed by Duvoisin et al. (1995). Amino
acid residues in rnGluRl interacting with the glutamate side chah ( ~ ~ r ' ~ and A%'') and the
analogous residues in mGluR4 and mGluR8 are shown in bold. Conserved residues among
al1 three receptors are boxed.
ability to bind either ['HJL-AP~ or ['HJCPPG, which was characterized to be a high affinity
probe selective for mGluR8 as part of this thesis work (Naples and Hampson, 2001 ; see later
sections for details). Mutagenesis experiments were pided by a homology model of the
glutamate-binding site of mGluR4 and the resul ts of mGluR4 mutagenesis experiments
(Hampson et al, 1999; D. R. Hampson and V. Peltekova, unpublished observations). Despite
the high degree of sequence homology between mGluR4 and mGluR8 (-75%; Fig. 1 .), slight
differences in phamacology exist. The ability of [)H]CPPG to selectively label mGluR8 may
provide insight into the molecular basis for these differences and allow the detenninants of
agonist and antagonist binding to be difierentiated. Ultimately, the results of site-directed
mutagenesis expenments performed on mGluR8 c m be used to test and refine the molecular
model of the ligand-binding pocket.
Part III - Identification of residues involved in maintainina the open conformation of the
' Venus-flytap'
The impetus for this part of my project comes fiom the observation that a single point
mutation in mGluR4 (R78A) not only abolishes ['HIL-AP~ binding, but shows three distinct
bands when run on an SDS-PAGE gel. These include the expected immunoreactive bands at
relative molecular weights (Mr) of 100,000 and 200,000 representing monomeric and dimeric
forms of the receptor, respectively, and an additional lower band at approximately M, =
85,000 (Hampson et al., 1999). This lower molecular weight band has not been observed in
any of the other mGluR4 point mutations made to date (Hampson et al., 1999; D. R. Hampson
and V. Peltekova, unpublished observations). Furthemore, this band can be detected by
antibodies raised against both the N- and C-terminal ends of the protein suggesting that it is
not the product of receptor proteolysis. My goal in this project was to attempt to discern the
nature of this lower molecular weight band.
The elucidation of the crystal structure for mGluR1 has lent support to the hypothesis that
mammalian mGluRs exist in two primary conformational states, the open and closed states.
This hypothesis was originally based on analogies with crystallized PBPs, but Kunishima et
al. (2000) were able to solve 'closed' crystal structures of mGluRl in both the presence and
absence of glutamate. A cornparison of the unliganded 'open' crystal structure, with the
glutamate-complexed 'closed' structure demonstrates that a significant conformational change
is associated with ligand binding. As mentioned previously, it is thought that the
conformational change induced by closure of the two lobes of the N-terminus is responsible
for transduction of the binding signal ('Venus-flytnip' model; see Fig. 2). Interestingly,
although both monomenc units of the dimeric receptor bind glutamate, only one monomenc
unit closes to trap the ligand. The significance of this is still unclear, but it is possible that the
orientation of monomeric units with respect to each other is the detemining factor for
receptor activation (De Blasi et al., 2001). Kunishima et al. (2000) concluded that mGluRs
exist in equilibnum between their open and closed foms and that glutamate binding stabilizes
the closed form of the receptor.
We proposed that the R78A point mutation in the glutamate-binding site of mGluR4
mimics agonist activation of the receptor. This, in turn, promotes closure of the 'Venus-
flytnip' causing a large perturbation in receptor structure. Experimentally, this perturbation
manifests itself as a shift to a lower molecular weight on an SDS-PAGE gel. Therefore, we
speculated that the band observed at MI = 85,000 when the mGluM R78A mutant is
subjected to SDS-PAGE represents a closed fom of mGluR4. The analogous point mutation
was made in mGluR8 to test this hypothesis.
Examination of the mGluRl crystal structure reveals that upon ligand binding and closure
of the two lobes, kg7' aligns itself across the binding pocket h m ~r~~~~ (Fig. 4A). Both of
these positively charged residues make an electrostatic interaction with the negatively charged
glutamate side chain, stabilizing the closed fotm of the receptor. We hypothesize that the
mechanism of lobe closure is similar in mGluR4 and mGluR1. Specifically, we propose that
the repulsive force between these two positive charges is responsible for stabilizing the open
conformation of the receptor and that ligand occupation of these charges allows the 'Venus-
flytrap' to close. Alignment of mGluR4 with mGluRl reveals a lysine ( L ~ S " ~ ) at this
position. In fact, a positive charge (arginine or lysine) is conserved at this position in al1
mGluRs except for the group II receptors. If this semi-conserved positively charged residue is
indeed involved in 'holding' the receptor in an open conformation, we speculate that mutating
it should shift the equilibrium towards the closed form of the receptor. Accordingly, we
hypothesize that an mGluR4 mutant lacking this positive charge will not bind [)H]L-AP~ but
will display an additional lower molecular weight band when run on an SDS-PAGE gel
(similar to the R78A mutation).
The potential identification of residues that, when mutated, promote receptor closure
analogous to agonist binding will provide insight into the heretofore unknown activation
rnechanism of mGluRs.
In summary, my work in this thesis consists of three inter-related projects:
1. The characterization of a novel mGluR radioligand, ['HICPPG
2. Examination of the potential roles of selected residues in mediating the binding of ligands
to mGluR8
3. Investigation of the amino acids responsible for maintaining the open (unliganded)
conformation of group II1 mGluRs
2. MATERIALS AND METHODS
2.1 Chemicak and Reagents
Unless specified otherwise, al1 chemicals and reagents were putchased fiom BDH Inc.
or Sigma.
2 3 Standard Procedures in Molecular Biology
(a) Bacterial Cultures and Transformations
Al1 bactenal cultures were prepared by inoculating 3 ml of Luria-Bertani (LB)
medium ((wlv): 1.0% bacto-tryptone, 0.5% yeast extract, 1.0% NaCI; pH 7.0) with single
colonies of E. coIi grown on LB-agar plates (LB medium supplemented with 1.5% bacto-
agar). Liquid cultures were incubated for 14- 16 h at 37°C with constant shaking (225 rpm) in
a New Brunswick orbital shaker. Bacteria transformed with plasmids encoding ampicillin-
resistance genes were grown in LB media supplemented with ampicillin (100 pg/ml,
Boehringer Mannheim) to sustain selective pressure during the growth penod.
To prepare competent cells for transformations, a colony of DHSa E. coli (GibcoBRL) was
grown in LB media as described above. Afler the incubation period, 250 pl of this ovemight
culture was diluted into 25 ml of fresh LB media and grown with shaking at 37°C until
reaching an ODm of 0.3-0.4 (measured using a Beckman DU530 UVNis
Specttophotometer). The bacterial cells were harvested by centrifugation ( 1380 x g, 10 min,
4°C) and made competent using the calcium chioride method described by Sarnbrook and
Russell (2001). Transformations were perfonned by adding 200 pI of competent cells to
tubes containing between 1 and 10 ng of plasmid DNA. The cells were incubated on ice for
40 min, heat-shocked at 42°C for 2 min, and then cooled on ice for an additional 2 min. One
ml of LE3 medium was added to the cells, and they were incubated with shaking at 37T for 1
h. Following this growth period, 50-300 pl of cells were spread ont0 LB-agar plates
containing 100 pg/ml ampicillin (to select for ampicillin-resistant transformants) and the
plates were incubated at 37OC for approximately 16 h.
(b) Preparation of Plasmid DNA
Ampicillin-resistant bactenal colonies were grown ovemight as described in
Section 2.2(a) and the cells were collected by centrifugation (3000 x g, 4-10 min). The
plasmid DNA was extracted fiom harvested cells using either the QIAprep Spin Miniprep Kit
(small-scale) or the QIAGEN Plasmid Maxi Kit (large-scale) according to protocols descnbed
by the manufacturer (Qiagen). Large-scale preparations of plasmid DNA were performed
whenever multiple transfections of a single plasmid were required. Purified plasmid DNA
was suspended in 10 mM TrisCl (pH 8.5) and its concentration measured by UV
spectroscopy at a wavelength of 280 nrn. The A2000nso ratio was used as an indicator of the
purity of the plasmid preparation. Only plasmid DNA with an A280RM) ratio of 21.8 was used
for transfections (see Section 2.3(d)).
(c) Restriction Endonuclease Digestion and Agarose Gel Electrophoresis
Al1 restriction enzymes were purchased From New England BioLabs or Gibco
BRL. Restriction enzyme digests were carried out on purified plasmid DNA: (i) to verify the
identity of DNAs prior to transfection; (ii) to generate DNA fragments for subcloning
purposes; and (iii) to check the orientation of ligated DNA ftagrnents following non-
directional subcloning. Restriction digests were typically performed using 5-10 units of
enzyme in reaction volumes of 10-50 pl containing 0.2-6 pg of plasmid DNA. Each reaction
was supplemented with sterile deionized water, 10x reaction buffer (supplied with each
enzyme), and 1 mg/ml bovine semm albumin (BSA; if necessary). Al1 reaction mixtures were
incubated for 2 h at temperatures required for optimal activity of the specific enzyme being
used (usually 37°C).
Following restriction endonuclease digestion, agarose gel electrophoresis was used to
separate, visually analyze, and approximate the concentration of resulting DNA fragments.
Digested samples were prepared for electrophoresis by adding 0.2 volumes of 6 x DNA-gel
loading buffer ((wlv): 0.25% bromophenol blw, 0.25% xylene cyan01 FF, 30% glycerol in
water). Prepared samples were loaded ont0 1.0% agarose gels made in 0 . 5 ~ Tris-
acetatelEDTA buffer (TAE; 20 mM Tris-acetate, 0.5 mM EDTA) supplemented with 0.5
pglrnl ethidium bromide. Electrophoresis was perfomed at 100 V using a Mupid-21 Mini-
Gel apparatus (Helixx Technologies) until sufficient separation of DNA fragments was
achieveà. The DNA fiagments were visuaiized as fluorescent bands using an ultraviolet
transilluminator (UVP Inc.) and their sizes (in bp) were estimated by comparison with a 1 kb
DNA Ladder (Gibco BRL).
(d) Gel Extraction and Ligation of DNA Fragments
Ligation reactions were perfomed to attac h DNA Fragments possessing cohesive
ends generated by restriction enzyme digestion. This was done either to constmct cassettes to
be used as templates for site-directed mutagenesis or to subclone small segments of DNA
between vectors. Following electrophoresis. DNA fragments to be ligated together were
excised fiom agarose gels with a sterile razor blade and punfied using the QIAquick Gel
Extraction Kit (Qiagen). Prior to setting up the ligation reaction, purified DNA fragments
were re-run on 1% agarose gels and their concentrations approximated by comparing the
intensity of the bands to that produced by known amounts of Hind III digested LDNA (Gibco
BRL). A typical ligation mixture contained 100-400 ng of DNA (- 1 : 1 ratio of insertvector),
1 p1 10x ligation buffer, and 1 y1 T4 DNA ligase (1 unit/pl. Roche) in a 10 pl volume. Al1
ligations were carried out ovemight at 10'C then transformeâ the following day. For non-
directional cloning of DNA fiagments, vector DNA was always dephosphorylated using calf
intestinal alkaline phosphatase (CiAP; 23 units/pl, Pharmacia) prior to electrophoresis.
Dephosphorylation of vector DNA suppresses self-ligation and, as a result, increases the
efficiency of the ligation reaction and eliminates the need to screen large numbers of
transformants. A typical dephosphorylation reaction contained 6 pg of DNA, 5 pl of IOx
CiAP buffer, and 1 unit of CiAP in a 50 pl volume. This reaction mixture was incubated at
37°C for 40 min at which time an additional unit of CiAP was added. After a final 40 min
incubation at 50°C, the reaction mixture was loaded ont0 an agarose gel and subjected to
electrophoresis.
2.3 Expression of Recombinant Proteins in Mammalian Cells
(a) cDNA Expression Constructs
cDNA coding for rat mGluR 1 a (in the pCis mammalian expression vector), as well
as cDNAs for mGluR2 and rnGluR3 were kindly provided by Drs. Y. Tanabe and S.
Nakanishi. The cDNAs for mGluR2 and mGluR3 were received in the pBluescript II KS'
vector (Stratagene) and subcloneâ into pcDNA3 (Invitrogen Inc.) at the EcoR 1 site (non-
directional). Correct orientations of these consmicts were confirmeci by restriction digestion.
Constructs containing cDNAs coding for rat mGluR4a and mGluR8a were assembled in the
pcDNA3 and pcDNA3. I (Invitrogen Inc.) expression vectors, respectively, as described
previously (Peltekova et al., 2000). Al1 other mGluR cDNAs (mGluRSa, mGluR6, and
mGluR7a) were assembled in pcDNA3. The mGluRSa in pcDNA3 construct was provided
by Dr. C. Romano; mGluR6 and mGluR7 cDNAs were provided by Dr. S. Nakanishi and
subcloned into pcDNA3 by Xi-Ping Huang and Geofiey Homby.
(b) Site-D f rected Mutagenais
Two different approaches were used to generate mGluR point-mutations. The first
method required isolation of single-stranded DNA; the second was a polymerase chain
reaction (PCR)-based approach. Al1 mGluR8 mutants (K68NK69A' K7 1 A, K7 1 Y, R75A)
were made using the Muta-Gene phagemid in vitro mutagenesis kit (BioRad). This kit is
based on a method described by Kunkel et al. (1987) in which mutations are made by
hybridizing a mutagenic primer to a single-stranded copy of the DNA.
The first step was to subclone a fiagment of mGluRSa containing the region to be mutated
into a phagemid capable of producing single-stranded DNA. To make this cassette, mOluR8a
in pcDNA 3.1 was digested with Kpn I. The resulting 1827 bp fiagment was ligated into Kpn
Icut pBluescript SC (Stratagene) and transfomed into the C5236 strain of E coli. This is a
mutant bactenal strain lacking both a functional dUTPase and uracil N-glycosylase.
Consequently, DNA synthesized within this strain contains a number of uracils substituted for
thymines. Single-stranded DNA was produced (by infection with M 1 3K07 helper phage) and
extracted according to the procedure detailed by the manufacturer of the kit. Mutagenic
oligonucleotides coding for the K68AK69A (5'-ATC CCC TTT TCC GCC GCC AGC TCC
CCA CAA-3'); K7 1 A (5'-AGT CTG TGG ATC CCC GCT TCC TTC TTC A-3 '); K7 1 Y (5'-
AGT CTG TGG ATC CCG TAT TCC TTC TTC AG-3'); and R75A (5'-ATG GCC TCA
AGT GCG TGG ATC CCC 'MT'-3') mutations were a ~ e a l e d to this single-stranded
template. Following in vitro synthesis of the complernentary strand, double-stranded DNA
was tnuisformed into DH5a E. coli. The presence of a functional uracil N-glycosylase in this
strain efficiently inactivates the non-mutated strand, leaving only the newly synthesized non-
uracil-containing strand to be replicated. The resulting transfonants were screened for the
mutations of intrrest by direct sequencing of punfied plasmid DNA (refer to Section 2.3(c)).
Mutated cassettes were excised h m pBluescript SR by digestion with Kpn I and ligated
back into the mGluR8a cDNA in pcDNA 3.1. The correct orientations of mutated inserts
within this vector were confirmed by restriction digests using either Sma 1 or BstE II.
Mutations to mGluR4a (W17A, K317E) were made using a protocol based on the
QuikChange Si te-Directed Mutagenesis method (Stratagene). This PCR-based approach
utilizes a high-tidelity Pfi DNA polymerase for DNA synthesis. The Pfi DNA polymerase
exhibits the lowest error rate of any thermostable polperase studied (Lundberg et al., 1991;
Flarnan et al., 1994; Cha et al., 1995) making it ideal for site-directed mutagenesis. To further
limit the chance of introducing an unwanted point-mutation, a segment of the mGluR4a
cDNA encompassing the region to be mutated was subcloned into pBluescript SK'. Digestion
of mGluR4a (or mGluR4 R78A) in pcDNA3 with Kpn I created a 179 1 bp fragment that was
subsequently ligated into Kpn I-digested pBluescript SC. The following mutations were
made with the primer pairs listed below using the mGluR4 cassette as a template:
K3 17A
forward primer (5'-GAT AGC TGG GGC TCC GCG AGT GCC CCT GTG CTGJ')
reverse primer (5'-CAC AGG GGC ACT CGC GGA GCC CCA GCT ATC-3')
K317E
forward primer (5'-GAT AGC TGG GGC TCC GAG AGT GCC CCT GTG CTG-3')
reverse primer (5'-CAC AGG GGC ACT CTC GGA GCC CCA GCT ATC-3')
The PCR reactions used to generate each of these mutants were identical. The total
reaction volume was 20 pl and contained the following components: 10-100 ng of double-
stranded DNA template, 125 ng of each primer, 1.6 pl of 2 m M dNTPs, stenle deionized
water. and 0.5 pl of Pfu Turbo DNA polymerase (2.5 unitslpl, Stratagene). Each reaction was
overlaid with 30 pl of minera1 oil and cycling parameters for the PCR reaction were as
follows: primers were denatured at 9S°C for 30 s, followed by 16 cycles consisting of three -
steps (i) 30 s at 95°C; (ii) 1 min at 55°C (for annealing of the primers to the template); and
(iii) 10 min extension time at 68OC. Following temperature cycling, tubes were placed on ice
for 2 min to cool the reaction below 37°C. A small aliquot (5 pl) of each PCR reaction was
subjected to agarose gel electrophoresis to check for DNA amplification. Each reaction was
then mixed with 1 pl Dpn 1 and incubated for 1 h at 37OC to digest the parental (methylated)
DNA pnor to transformation.
Afier transformation of the PCR product, colonies were screened for the mutations of
interest by direct sequencing of plasmid DNA. Due to the chance of a proofreading error by
the DNA polymerase, the entire cassette was sequenced (see below) before being ligated back
into the mGluR4a cDNA in pcDNA 3 at the Kpn I site. Correct orientations of inserts within
this vector were confirmed by restriction digestion using Xho 1.
(c) DNA Sequenclng
Ali DNA sequencing was performed by The Centre for Applied Genomics at the
Hospital for Sick Children. Purified plasmid DNA samples (2-3 pg at a minimum
concentration of 200 pg/ml) were sequenced on a LiCor LongReadIR using standard
sequencing primers.
(d) Culture and Transfection of Human Embryoaic Kidney Cells
HEK-293-tsA201 cells were used for al1 transfections. This cell line stably
expresses the SV40 promoter and T-antigen allowing for enhanced expression of transiently
transfected cDNAs (Margolskee et al., 1993). HEK cells were cultured in minimal essential
medium (GibcoBRL) supplemented with 6% fetal bovine serum and an antibiotic solution
containing a mixture of penicillin and streptomycin (GibcoBRL). Al1 cell cultures were kept
in an incubator at 37OC with COz levels maintained at 5%. Prior to transfection, the cells were
grown in 100 mm tissue culture dishes (Nunclon) to a confluency of approximately 80%.
Transient transfections were perfomed according to the calcium phosphate precipitation
method described by Gorman (1990) using 20 pg of punfieâ plasmid DNA to transfect each
100 mm plate.
Three hours prior to transfection, 10 ml of fresh minimal essential medium was added to
each plate of confluent HEK cells. The plasmid DNA was mixed with 450 pl of 0.1 x TE (1
m M TrisCl, O. 1 mM EDTA; pH 8.0) and 50 pl of 2.5 M CaC12 then added to 500 pl of 2x
HEPES-buffered saline (50 mM HEPES, 280 mM NaCl, 3 m M Na2HP04) causing
precipitation of the plasmid DNA. This solution was mixed again then added drop-wise to
each plate of HEK cells. Following a 4 h incubation, the media was aspirated fiom each dish
and cells were exposed to 6 ml of a 15% glycerol solution made in 1 x HEPES-buffered saline
(25 mM HEPES, 140 mM NaCI, 1.5 mM Na2HP04). Afier 30 s, the glycerol solution was
aspirateci and cells were washed once for 30 s with phosphate-buffered saline (PBS; 10 mM
Na2HPO~, 3 mM KH2P04, 120 mM NaCl; pH 7.2). The PBS was aspirated, 10 ml of fresh
minimal essential medium was added to each plate of transfected cells, and they were
incubated ovemight. Twenty-four hours pst-transfection. cells were subcultured into two
separate 100 mm plates using minimal essential medium and incubated at 37'C for an
additional 24 h pnor to being collected. Ce11 cultures were maintained, and al1 transfections
perfomed by Xi-Ping Huang.
2.4 Membrane Preparation from Transfected Celk
HEK-293 cells transiently transfected with cDNAs encoding wild-type or mutant rat
mGluRs were harvested 48 h pst-tnuisfection and collected by centrifugation (3840 x g, 20
min, 4°C). Membranes were prepared fiom HEK cells using the method described by Enksen
and Thomson (1995). Pelleted cells were suspended in 20 ml of ice-cold lysis buffer (30 rnM
HEPES, 5 mM MgCI2*6H2O, 1 mM EDTA, 0.1 m M PMSF; pH 7.4) and homogenized with a
Polytron prior to centrifugation (48,400 x g, 20 min, 4°C). The cell pellets were resuspended
in 1 5 ml lysis buffer supplemented with 0.08% Triton X- 100 and incubated at 37°C for 10
min. Following incubation, an additional 15 ml lysis buffer was added and the membranes
were re-centrifugecl at 48,400 x g for 15 min. Cell membranes were washed by resuspension
in 15 ml assay buffer (30 m M HEPES, 1 10 mM NaCI, 1.2 m M MgC12-6H20, 5 mM KCl, 2.5
mM CaCI2, 0.1 mM PMSF; pH 8.0), centrifuged (48,400 x g, 15 min, 4OC), and homogenized
in 1-5 ml of assay buffer. Protein concentrations were detemined as described by Bradford
(1976) using a dye reagent from BioRad and BSA as a protein standard. AI1 membranes were
diluted in assay buffer to a final concentration ranging fiom 320-625 pg/ml, divided into 2-5
ml aliquots, and stored at -70°C.
2.5 Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and
Immunoblotting
SDS-PAGE and subsequent immunoblotting were performed: (i) to confinn the
expression of mGluRs in membranes prepared fiom transfected cells pnor to perfoming
binding experiments; (ii) to compare the expression levels of mutant consûucts with their
corresponding wild-type receptors; and (iii) to assess the results of deglycosylation
experiments (see Section 2.7). Protein samples were prepared for electrophoresis by adding
25 pl of 4 x SDS sample buffer (62.5 mM TrisHCl, 2% SDS, 10% glycerol; pH 6.8) and 10 pl
of 1 M dithiothreitol (BioShop Canada Inc.) containing 1% bromophenol blue to 75 pl of
membrane protein of known concentration. Samples were partially denatured by incubation
at 37°C for 15 min before being loaded ont0 8% polyacrylarnide gels. Receptor expression
levels were compared by loading equivalent arnounts of membrane protein (5- 10 pg).
Proteins were separateci under a constant current of 25-30 mA using a Mighty Small II SE 250
vertical gel apparatus (Hoeffer Scientific Instnunents) then transferred ont0 Nitrocellulose-1
membranes (0.45 pm pore size, Gibco BRL) by transblotting (225 mA, 2 h, 4OC). Following
the transfer, nitrocellulose membranes were blocked ovemight at 4OC in washing buffer (10
mM Tris*HCI, 150 mM NaCl, 0.2% Tween 20; pH 7.6) containing 5% powdered milk.
The next day, blots were washed with washing buffer (3 x 1 O min) then incubated with the
appropriate pnmary antibody (see Appendix 1 for a complete description of ptimary
antibodies used in this study) for 2 h at room temperature with gentle rocking. Afier 2 h, the
primary antibody was removed and the blots washed again (3 x 10 min). Fnllowing the
second set of washes, blots were incubated for 2 h with either a horseradish peroxidase-
conjugated anti-mouse or anti-rabbit IgG secondary antibody (Amersham) depending on the
primary antibody used. The anti-mouse and anti-rabbit secondary antibodies were made in
wash buffer at dilutions of 1 : 10,000 and 1:5,000, respectively. When incubation with the
secondary antibody was complete, blots were washed a final time (3 x 10 min) and incubated
with a chemiluminescent substrate (Enhancecl chemiluminescent (ECL) Western blotting
detection reagents fkom Arnersham or Supersignal West Pico Cherniluminescent Substrate
from Pierce) as per the manufacturer's instructions. lmmunoreactive bands were visualized
by exposing Hypefilm ECL (Amersham) or Kodak X-OMAT Scientific Imaging film
(Kodak) to the nitrocellulose blots. Relative molecular weights (MJ of proteins were
estimated by comparing immunoreactive bands with Protein Molecular Weight Standards
(High Range, Gibco B U ) prepared according to the manufacturer's instructions.
2.6 I ~ H ~ L - A P ~ and PHICPPG Binding Assays and Data Analysis
['HJL-AP~ (specific activity = 54 Ci/mmol), ['HJCPPG (specific activity = 16
Ci/mmol) and al1 amino acids and amino acid analogues used in the radioligand binding
assays were fiom Tocris Cookson Inc. The assays for ['HJL-AP~ and ['HJCPPG binding
were carried out on membranes prepared fiorn HEK cells as described by Eriksen and
Thomsen (1995) for ['HIL-AP~ binding. For al1 binding assays, 200 pl of membranes
(concentration 320-625 pghl), 25 pl assay buffer or competing drug, and 25 pl of
radioligand were used per microcentrifuge tube for a total volume of 250 pl. All experiments
were perforrned on ice using either 30 nM ['HIL-AP~ or 20 nM ['HICPPG. Non-specific
binding of radioligands to ce11 membranes was determineci using group selective ligands at
concentrations sufficient to block al1 target receptors (2100 x Ko). Accordingly, 300 pM L-
SOP was used to define non-specific binding to membranes expressing group III mGluRs and
500 pM L-glutamate and 100 pM L-CCG- 1 were used to define non-specific binding to group
1 and group II mGluRs, respectively. Following a 30 min incubation on ice with constant
shaking at 160 rpm, bound and fiee radioligand were separated by centrifugation (1 4,000 x g,
4 min). Supematants were aspirated immediately and pelleted membranes were washed once
with cold assay buffer (without PMSF), then solubilized ovemight in 500 pl of I M NaOH.
The solubilized samples were transferred to Pony liquid scintillation vials (Packard) and 4.5
ml of scintillation fluid (Ultima Gold, Packard) was added. The vials were left to equilibrate
at room temperature for at least 4 h prior to being counted on a Tri-Carb 21OOTR liquid
scintillation counter (Canberra-Packard).
Means of triplicate or quadruplicate values were used for al1 calculations. For single point
binding assays, total specific binding was calculated as the difierence between total binding
(binding in the absence of any inhibitor) and non-specific binding. Inhibition curves were
generated by subtracting non-specific binding fiom binding observed at each concentration of
competing drug and expressing these values as a percentage of total specific binding.
Cornpetition curves were analyzed, and ICso values detennined by non-linear curve fits using
GraphPad Prism software. Binding constants (Ko, B-) describing the interaction of ['HIL-
AP4 or ['HICPPG with prepared ce11 membranes expressing wild-type or mutant receptors
were calculated from auto-competition experiments using unlabeled ligands. Inhibition
constants (Ki) for competing drugs were calculated from ICso values using the Cheng-Pnisoff
equation (Appendix 3). To compare affinities between wild-type and mutant receptors,
unpaired t-tests were perfonned using Pnsm software and a significance level of 0.05.
2.7 Deglycosylation of mCIuR4 and mGluR4 R78A
HEL293 cells transiently transfected with cDNAs encoding either mGluR4 or mGluR4
R78A were collected 48 h pst-transfection in 500 pl PBS and protein concentrations were
detennined as described previously (Section 2.4). Twenty pg of membrane protein was
denatured in 10x Denaturing Buffer (5% SDS, 1% P-mercaptoethanol, New England
BioLabs) by heating the sample for 15 min at 37'C. Following denaturation. 0.1 volumes of
1Ox G7 buffer (0.5 M Na2P04; pH 7.5) and 10% Nonidet P-40 were added to the samples
along with 4 pl of PNGase F (500 unitdpl). PNGase F catalyses the cleavage of asparagine-
linked oligosaccharides fiom proteins. The reaction mixture was incubated at 37'C for I h.
Following this incubation period, protein samples were prepared, and electrophoresis and
immunoblotting camed out as described in Section 2.5.
3. RESULTS
3.1 Expression of Recombinant mGlnRs in HEK-293 Ceiis
My initial studies entailed an examination of the selectivity profiles of the radiolabeled
agonist [ 'HIL-AP~ and the novel radiolabeleâ antagonist [)H]CPPG in HEK-293 cells
transiently transfected with cDNAs encoding each of the eight mGluR subtypes. Pnor to
performing radioligand binding experiments (Section 3.2), expression of individual mGluR
subtypes in membranes prepared fiom transfected cells were confirmai by immunoblotting
using subtype-specific or group-selective antibodies (see Appendix 1 for a description of the
antibodies used). For each receptor, the appropriate molecular weight bands were detected in
cDNA-transfected cells but not in mock-transfected cells (Fig. 5). Expression of mGluRla,
mGluRSa, mGluR6, and mGluR7a were identified using receptor-specific C-terminal
antibodies, whereas the C-terminal anti-mGluR2B antibody labeled both mGluR2 and
mGluR3. The anti-mGluR4/8 antibody was raised against a sequence in the N-terminal
domain of mGluR4 located approximately 40 amino acids downstream of the putative signal
peptide cleavage site (see Naples and Hampson, 2001 for details). This antibody recognized
both mGluR4a and mGluRSa, as expected, but also showed some cross-reactivity with
rnGluR6 and very limited recognition of mGluR7a (data not shown). The rank order of anti-
mGluR4/8 recognition corresponds to the sequence similarity of the four group II1 receptors
in this region. A pubiished sequence alignment for the group III receptors (Duvoisin et al.,
1995) indicates that this segment of polypeptide is nearly identical in mGluR4 and mGluR8
(one amino acid difference), whereas mGluR6 shows a lower level of sequence similarity; the
similarity in this region is even lower for mGluR7. The higher molecular weight bands seen
on some blots (most noticeably for the group II and group III receptors) represent mGluR-
Figure S. Immunoblots demonstrating expression of mGluR subtypes. Each lane
contains 8 pg of membrane protein from mGluR-transfected or mock-transfected HEK-293
cells. The proteins were separated on 8% SDS-PAGE gels, iransfmed to nitrocellulose, and
probed with subtype specific antibodies. The positions of molecular weight standards (in
kilodaltons) are indicated to the right.
C. Croup III
receptor dimers. Collectively, the results of the immunoblotting experiments confirm the
expression of each mGluR subtype in transfected HEK-293 cells.
3.2 Radioligand Binding to mCluR-Expressing HEK CeII Membranes
Following confirmation of receptor expression via immunoblotting, ['HIL-AP~ and
['HJCPPG binding experiments were conducted on membranes prepared fkom transfected
cells. Non-specific binding of radioligands to mûlul-expressing membranes was defined in
the presence of 500 pM glutamate, 100 pM L-CCG-1, or 300 pM L-SOP for group I,11, and
III receptors, respectively. At a concentration of 30 nM, no specific ["IL-AP~ binding was
observed in membranes prepared fiom mock-transfected cells (data not shown). Furthemore,
virtually no specific binding of ['HIL-AP~ was detected in membranes expressing mGluR1,
mGluR2, mGluR3, mGluR5, mGluR6, or mGluR7. In fact, of the eight mGluR subtypes,
[%IL-AP~ displayed a high level of specitic binding only to membranes expressing either
mGluR4 or mGluR8 (Fig. 6, top panel). Specific ['HJL-AP~ binding was 1.2 0.3 and 1 .O I
0.1 pmol boundlmg protein for mGluR4 and mGluR8, respectively (mean SEM; n = 4-6).
The amount of specific binding as a percentage of total binding was 84 & 2% for mGluR4 (n =
4), and 76 st: 4% for mGluR8 (n = 6).
The same assay used for ['HIL-AP~ binding experiments was employed to test the
mGluR-selectivity profile of [ 3 ~ ] ~ ~ ~ ~ , except that a concentration of 20 nM was used. As
with [.'H]L-AP~, no specific [-'H]CPPG binding was detected in HEK ce11 membranes
expressing mGluR1, mGluR2, mGIuR3, mGluR5, mGluR6, mGluR7, or in mock-transfected
ce11 membranes. In contrast to ['HIL-AP~, [ 3 ~ ] ~ ~ ~ ~ binding in membranes expressing
mGluR4 was barely detectable (Fig. 6, bottom panel). Only mGluR8-expressing membranes
exhibited high levels of ['HICPPG specific binding (4.0 * 0.4 pmol b o u d m g protein; n = 4).
Figure 6. Cornparison of [ 3 ~ ~ ~ - ~ ~ 4 (top panel) and ['H~CPPG (bottom panel) binding
to mCluRs. Binding assays were performed on membranes prepared fiom HEL293 cells
transiently expressing mGluRs using either 30 nM ['HIL-AP~ or 20 nM ['HICPPG. Specific
binding of tritiated ligands to ce11 membranes (pmol ligadmg protein) was nomalized to
levels observed in mGluR8-expressing membranes. In most expenments, the ['HIL-AP~ and
['HICPPG binding assays were conducted in parallel using the same batch of prepared
membranes and dilution of radioligand. Each assay was performed in quadruplicate 3-5 times
on membranes prepared fiom 2 to 4 separate transfections.
mGluR subtype
mGluR subtype
The amount of specific binding to mGluR8 was identical whether using 300 MM of the group
III agonist L-SOP or 50 )iM of the unlabeled antagonist CPPG to define non-specific binding
(data not show). In addition, the signal to noise ratio of ['HJCPPG was very high; specific
binding represented 96 k 0.4% of total binding (n = 4). The high level of ['HICPPG specific
binding observed to mGluR8 compared to ['HIL-AP~ (approximately 4-fold p a t e r ) suggests
that ['HICPPG possesses a higher affinity than ['HIL-AP~ for mGluR8. This observation is
supported by a previous study showing that the affinity of unlabeled CPPG for mGluR8 is
higher than that of L-AP4 (Peltekova et al., 2000). The absence of ['HICPPG specific
binding to any other mGluR subtype demonstrates that this novel radiolabeled antagonist
selectively labels mGluR8 at a concentration of 20 nM.
3.3 Characterization of [ 3 ~ ~ ~ ~ ~ ~ as a High Affinity Probe for mCluR8
Due to the high level of ['HICPPG binding to mGluR8, additional experiments were
conducted to characterize the interaction of ['HICPPG with this receptor. Accordingly, the
association and dissociation profiles, as well as the effects of varying the pH of the binding
assay buffer were examined for [)H]CPPG binding to mGluR8.
The association of ['HICPPG to mGluR8 at O°C was very rapid; maximal specific binding
was achieved within approximately 20 min (Fig. 7). Dissociation of bound ['HICPPG
following the addition of 100 pM unlabeled CPPG was also extremely fast. Virtually no
specific binding was observed in samples centrifùged immediately after the addition of excess
unlabeled ligand, meaning that more than 95% of bound ['HICPPG had dissociated within 4
min (the time required to separate bound fiom fiee radioligand by centrifugation).
Consequently, the rapid binding kinetics of [ 3 ~ ] ~ ~ ~ ~ precluded an accurate estimate of the
dissociation constant, kaif. The kinetics of [ 3 ~ ] ~ ~ ~ ~ binding to mGluR8a were very similar
Figure 7. Time-counes for the association (m) and dissociation (r) of [ 3 ~ ] ~ ~ ~ ~ to
mGluR8 at O°C. Association was initiated by the addition of 20 nM [)H]CPPG to ceIl
membranes expressing mGluR8. Samples were centrifiiged (14,000 x g, 4 min) following O,
2, 5, 10, 20, 45, 60, and 90 min of incubation to separate bound and fiee L~H]CPPG. Non-
specific binding was defined for each time point as binding in the presence of 300 pM L-SOP.
For dissociation expenments, membranes were incubated with 20 nM ['HJCPPG for 30 min
at 0°C in the presence of either assay buffer or L-SOP. Dissociation was initiated by the
addition of 100 PM uniabeled CPPG (vertical arrow) and specific binding was measured
following centrifugation of samples at 0.5, 1, 2, 5, 10, and 20 min. Results are presented as
the percentage of maximal specific binding of ['HJCPPG to mGluRS and represent the mean
* SEM of three separate experiments perfomed in triplicate.
Time (min)
to those observed by Eriksen and Thomsen (1995) for r 3 ~ ] ~ - A P 4 binding in membranes
prepared from mGluR4a-expressing BHK cells. They too were unable to measure the off-rate
of radioligand because of its rapid dissociation profile.
In addition to these experiments, the effkct of pH on [ 3 ~ ] ~ ~ ~ ~ binding was investigated.
Optimal specific binding of ['HJCPPG to mGluR8 was observed at pH 8.0 (see Appendix 2),
whereas non-specific binding was unaffected by changes in pH. Although the relevance of a
pH optimum for ['HICPPG binding higher than physiological pH (7.4) is unclear, this
phenomenon has been observed for other recombinant GPCRs (Jockers et al., 1994; Eriksen
and Thomsen, 1995). The results of the kinetic binding and pH experirnents were used to
refine the ['HJCPPG binding assay so that the phamacological profile of mGIuR8 could be
accurately examined using this radioligand.
To examine the ligand selectivity of mGluR8, several glutamate receptor ligands were
tested for their ability to displace [ 3 ~ ] ~ ~ ~ ~ binding. These compounds included the non-
selective mGluR antagonist (R,S)-a-methyl-4-carboxyphenylglycine ((R,S)-MCPG), the
ionotropic glutamate receptor ligands AMPA, kainic acid, NMDA, and ibotenic acid, and the
glutamate uptake inhibitor L-trans-PDC. At a concentration of 100 PM, AMPA, kainate, and
L-tmiis-PDC showed only minimal inhibition of ['HICPPG binding (<12%) while NMDA
and ibotenic acid inhibited binding by approximately 20% (Table 2; n = 3). Only the mGluR
antagonist (R,S)-MCPG caused a substantial (about 50%) reduction in specific [ 3 ~ l C ~ ~ ~
binding to mGluR8. (R,S)-MCPG was initially characterized as a cornpetitive antagonist at
group I receptors (Jane et al., 1993) but has since been show to antagonize group II and
group III agonist-induced effects at micromolar concentrations (KD = 227-479 pM; Schoepp
et al., 1999). The results of rny single-point displacement binding experiments are in
Table 2. Competition for I~H]CPPG binding to mGluR8 by various metabotropic
((R,S)-MCPG) and ionotropic glutamate receptor ligands (AMPA, k f nic acid, NMDA,
ibotenic acid), and the glutamate uptake inhibitor L-lmns-PDC.
Test eompound Percent of (3H]CPPG control binding
(R,S)-MCPG
AMPA
Kainic acid
NMDA
Ibotenic acid
L-&ans- PDC
Each drug was tested at a concentration of 100 PM. The results are the mean t SEM of three
separate experiments and are expressed as percent of control binding where 100% is the
specific binding of 20 nM I3KJCPPO to mGluR8-expressing membranes in the absence of any
inhi bi tor.
accordance with these studies and indicate that the affinity (Ki) of (R,S)-MCPG for mGluR8
is approximately 100 FM.
Homologous cornpetition experiments were conducted to calculate the equilibnum
dissociation constant (Ko) and maximum binding capacity (B-) of ['HJCPPG to mGluR8.
These values were 183 4 nM and 32 k 1 pmoVmg protein, respectively (n = 3). The high
level of mGluR8 expression reflected by this B,, value is similar to that measured previously
using the results of [.'H]L-AP~ auto-cornpetition expenments (29 * 4 pmol/mg protein;
Peltekova et al., 2000). Additional [ 3 ~ ] ~ ~ ~ ~ cornpetition expenments were performed to
fùrther characterke the pharmacological profile of mGluR8. The most potent inhibitor of
[)H]CPPG binding was unlabeled CPPG followed by the phosphonophenylglycine antagonist
MPPG (Fig. 8 and Table 3). The phosphonophenylglycine agonist (R,S)-PPG, and the
phosphonate-containing compound L-SOP possessed the highest afinities for mGluR8
among the agonists tested, whereas L-glutamate was substantially less potent at inhibiting
[ 3 ~ ] ~ ~ ~ ~ binding. L-AP4 was also a strong inhibitor of antagonist binding and the
stereospecificity of this compound was demonstrated by the observation that D-AP4 was 10-
fold less potent in its ability to displace [ 3 ~ ] ~ ~ ~ ~ binding. Overall, the rank order of
potency of agonists for competing with [ 3 ~ ] ~ ~ ~ ~ binding to mGluR8 (L-SOP = (R,S)-PPG >
L-AP4 > L-glutamate; Table 3) was different fiom the order of potency observed previously
to inhibit [ 3 ~ ] ~ - ~ ~ 4 binding to mGluR8 (L-AP4 > L-SOP > L-glutamate = (R,S)-PPG;
Peltekova et al., 2000).
3.4 Effect of mCluR8 Point Mutations on Protein Expression and Radioiigand Bhding
The ability of L~H]CPPG to label mGluR8 with high affinity provides another
experimental tool (in addition to [ 3 ~ ] ~ - ~ ~ 4 ) for the identification of amino acid residues
Figure 8. Cornpetition for ['HJCPPG binding to mGluR8 by the agonists LSOP, L- &-
AP4, D-AP4, L-glutamate, and (R,S)-PPG (top panel) and the cornpetitive antagonists
CPPC, and MPPG (bottom panel). The data are expressed as a percentage of total specific
['HICPPG binding; each point represents the mean I SEM of three expenments perfomed in
triplicate. Sigrnoidal dose-response curves were fitted using non-linear regression analysis.
Inhibition constants (Ki) calculated from these values are presented in Table 3.
log [agonist] (M)
log [antagonist] (Rd)
+ L-SOP A L-AP4 r L-glutamate A PPG Q D-AP4
+ CPPG MPPG
Table 3. Inhibition constants for agonlsts and antagonists eompeting at [3mCPPC
binding sites on mGIuR8.
Limlim L-AP4 D-AP4 L-SOP PPG L-glutamate
Antanonists CPPG MPPC MCPG
Inhibition constants (4) were cdculated from IC, values obtained from displacernent binding
experiments (Fig. 8) using the Cheng-Prusoff equation (Appendix 3). The KD and B, values
(183 + 4 nM and 32.3 I 0.9 pmollmg protein, respectively) for [ 3 w ~ P P G binding to mGluR8
were calculated from auto-competition experiments using unlabeled CPPG. Results are
expressed as the mean SEM (n = 3).
*Esiimated value based on data presented in Table 2.
mediating high af'fmity ligand binding. Of particular interest are residues that may confer
ligand selectivity and thus account for the pharmacological differences observed between
mGluR groups or receptor subtypes. The binding-pocket mode1 established by homology
with LIVBP (O'Hara et al., 1993) identified fundamental glutamate recognition residues that
are conserved arnong al1 mGluR subtypes (see Section 1.7 for details). The ment
crystallization of mGluR 1 in complex with glutamate has confimed the importance of these
conserved residues (Kunishima et al., 2000; see Fig. 4A). Because the majority of mGluR
ligands that have been characterized are glutamate derivatives, it appears that ligand
selectivity is determined primarily by residues meditating binding of ligand side chains. One
such residue is a conserved arginine ai position 78 of mGluRl that interacts with the
negatively charged side chain of glutamate (Fig. 4A). Mutation of this residue in mGluRl
and mOluR4 has been shown to abolish binding of group selective ligands to both receptors
(Hampson et al., 1999; Jensen et al., 2000). To extend this analysis to mGluR8, a senes of
positively charged residues (arginines or lysines) were targeted for mutagenesis based on their
proximity to this conserved arginine residue ( ~ r ~ ' ' in mGluR8; refer to Fig. 48) and the
ability of mutant receptors to bind either ['HIL-AP~ or ['HICPPG was examined.
Prior to perfoming radioligand binding experiments, HEK-293 cells transiently
transfected with mGluR8a or with the mGluR8 K68A/K69A, K7 1 A, K7 1 Y, or R75A mutants
were subjected to immunoblotting to ensure that none of these point mutations caused a
significant change in receptor expression levels relative to wild-type mGluR8. AI1 mGluR8
constructs were probed with an anti-c-myc antibody that recognizes a peptide inserted at the
extreme carboxyl-terminal end of the protein (preceding a 6 x His tag). Insertion of this C-
terminal sequence has previously been shown to have no effect on the affinity of ['HIL-AP~
for wild-type mGluR8 (Peltekova et al., 2000). As expected, the anti-c-myc antibody labeled
bands corresponding to the predicted molecutar weights of mGluR8 monomers and dimers
(M, = 100,000 and M, = 200,000, respectively) for mGluR8a and each of the mGluR8 mutants
(Fig. 9A). No immunoreactivity was observed in membranes prepared fiom mock-transfected
cells at these molecular weights. The intensity of the monomer band for each mutant was
roughly equivalent to that observed in membranes expressing wild-type mGluR8. This
suggests that any differences in mutant receptor binding compared to wild-type binding
cannot be attributed to differences in expression levels (see below).
Following confirmation of receptor expression by immunoblotting, binding assays were
perforrned on ceIl membranes using either 30 nM ['HIL-AP~ or 20 nM ['HICPPG. Al1
single-point assays on mutant receptors were camed out in parallel with membranes
expressing wild-type mGluR8. This allowed any specific binding observed to the mutant
recepton to be expressed as a percentage of wild-type receptor binding. Only very low levels
of ['HIL-AP~ binding were detected in membranes expressing the K71Y or R75A mutants
(both less than 5% of control; Fig 9B, n = 3). The K68AK69A double mutant also displayed
a large decrease in ['HIL-AP~ specific binding compared to the wild-type receptor (>85%
decrease; n = 3). Of the positively charged residues targeted for mutagenesis, only the K7 1 A
mutation did not cause a substantial reduction in the ability of mGluR8 to bind ['HIL-AP~. In
fact, the levels of specific binding in membranes expressing this mutant were almost twice as
high as those observed to the wild-type receptor and therefore sufficient to permit competition
c w e analysis. Auto-inhibition curves performed on mGluR8 K7 1A using [%]L-AP~
revealed that this mutation caused a small, albeit insigniticant (p = 0.08; unpaired t-test),
increase in affinity (Ko = 0.36 it 0.02 FM; Table 4, n = 3) compared to wild-type mGluR8
(Ko = 0.87 0.22 FM; fiom Peltekova et al., 2000). The increase in affinity of [)H]L-AP~
for mGluR8 K7 1A likely explains the difference in specific binding between the K7 1A
Figure 9. Cornparison of ['HJL-AP~ and ['HJCPPG bindhg to a series of mCluR8 = -
mutant receptors. Panel A is an immunoblot demonstrating the relative expression levels of
the mutant and wild-type receptors. Each lane contains 8 pg of membrane protein subjected
to SDS-PAGE, transferred to nitrocellulose, and probed with an anti-c-myc monoclonal
antibody. Positions of molecular weight standards (in kDa) are indicated to the lefi and
amws to the right point to relative positions of receptor monomen (M) and dimers (D) at M,
= 100,000 and M, = 200,000, respectively. Al1 binding assays were perfonned on membranes
prepared fiom transfected HEK-293 cells using either 30 nM ['HIL-AP~ (Panel B) or 20 nM
['HICPPG (Panel C). Specific binding was normalized to concomitant levels observed in
membranes expressing wild-type mGluR8. Results are expressed as the mean SEM (n = 3).
#,*Indicate that levels of ['HIL-AP~ and [ 3 ~ ] ~ ~ ~ ~ binding, respectively, were sufficient to
generate competition curves. Afinity constants for this construct (mGluR8 K71A) are
presented in Table 4.
Tm-
Table 4. AMnities of ['WL-AP~ and ['HjcPPG for wild-type and mutant group III
receptors.
Construct K, CPW
Dissociation constants (KD) were calculated from IC, vaiues obtained from auto-competition
experiments and represent the mean t SEM of 3-4 separate experiments perfonned in
triplicate. Dashed lines indicate that binding levels were too low for phannacological
analysis. Included for cornparison are the affinities of wild-type rnGluR4 and mGluR8. KD
values were taken frorn the rollowing references:
'Harnpson et al., 1999
b ~ e l tekovâ et al., 2000
'Naples and Hampson, 2001
*Affinity is significantly different from wild-type at p < 0.05 (unpaired t-test)
mutant and wild-type mGluR8 displayed in Fig. 9B. For cornparison, the analogous mutation
in mGluR4 (K74A) was also tested for its ability to bind [ 'HJL-AP~ (Table 4). As with
mGluR8, no significant difference was observed in the affinity of this receptor for [)H]L-AP~
(Ko = 0.47 * 0.3 1 CM; n = 4) compared to wild-type mGluR4a (Ko = 0.50 * 0.10 FM; from
Hampson et al., 1999).
Similar to the pattern observed for ['HIL-AP~, virtually no specific binding of [)H]CPPG
was detected in membranes expressing the K71Y or R75A mutants (both less than 5% of
control; Fig. 9C, n = 3). The K68AK69A double mutant also showed a considerable
decrease in [ 3 ~ ] ~ ~ ~ ~ speci fic binding compared to the wild-type receptor (285% decrease; n
= 3). These results suggest that the primary determinants of high affinity [)H]L-AP~ and
[)H]CPPG binding are related. Interestingly, the effect of the K71A mutation on ['HJL-AW
binding was different fiom its effect on [)H]CPPG binding. Whereas ['HIL-AP~ had a higher
number of specific counts and increased affinity for the K71A mutation, the afinity of
["ICPPG for the K71A mutant (Ko = 0.84 * 0.02 PM; n = 3) was approximately 4-fold
lower than for the wild-type receptor (Ko = 0.18 * 0.0; Table 4, n = 3). This difference was
statistically significant at p < 0.05 (unpaired t-test).
3.5 Immunoblots of an mGluR4 Point Mutation, mCluR4 R78A
As described previously (Section 1.7), a single point mutation in mGluR4 (R78A) that
abolishes [)H]L-AP~ binding shows three distinct bands when nin on an SDS-PAGE gel. In
addition to bands at M, = 100,000 and M, = 200,000 representing monomeric and dimeric
forms of the receptor, respectively, this constnict displays an additional lower molecular
weight band at approximately M, = 85,000. The presence of an additional band at this
position following SDS-PAGE is unique to the R78A mutation (Hampson et al., 1999; D. R.
Harnpson and V. Peltekova, unpublished observations). Molecular rnodeling of mGluR4
indicates that ~r~~~ lies within the hinge region of the binding-pocket that separates the two
lobes of the receptor (Hampson et al., 1999). Based on the position of this residue and its
fundamental role in ligand recognition among al1 mGluR subtypes, we speculated that the
lower molecular weight band observed in R78A-expressing cells represents a closed fomi of
the receptor produced by removal of this positive charge.
To confirm that the appearance of this lower molecular weight band does not result fiom
N- or C-terminal proteolysis of the receptor, immunoblotting experiments were conducted
using three antibodies directed at different regions of mGluR4 (Fig. 1 OB). The anti-mGluR4a
antibody recognizes the extreme C-teminal sequence of mGluR4a, whereas the anti-
mGIuR4/8 antibody recognizes a sequence located approximately 40 amino acids downstream
of the proposed signal peptide cleavage site. It is unlikely that proteolysis of these 40 amino
acids could account for the dramatic shift in molecular weight (-15 kDa) observed in the
mGluR4 R78A mutant. To venfy this assumption, a construct of mGluR4 R78A containing a
c-myc epitope tag inserted immediately downstream of the signal peptide (c-myc-mGluR4a)
was also expressed in HEK cells and subjected to immunoblotting using the anti-c-myc
antibody. The three antibodies described above were tested for their ability to label mGluR4
R78A; membranes expressing wild-type mGluR4 (or c-rnyc-mGluR4) were used as a control.
Both the anti-mGluR4a antibody, and the anti-mGluR4/8 antibody labeled an immunoreactive
band at approximately M, = 85,000 in membranes expressing the mGluR4 R78A mutant.
Detection of this lower molecular weight band in c-myc-mGluR4 R78A-expressing cells
provides additional evidence that the R78A mutation does not promote N-terminal proteolysis
of mGluR4 (Fig. 1 OA). The band at M, = 85,000 was absent from mGluR4-transfected, c-
myc-mGluR4-trans fected, and mock-trans fected ce11 S. Furthennore, the dimeric fom of
Figure 10. Immunoblots of the mGluR4 R78A and mGluRû R75A mutants. Panel A
shows three representative Western blots of the mGluR4 R78A mutant, one of which also
presents the analogous mGluR8 mutant (R75A). Each lane contains 6-8 pg of membrane
protein separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots
were probed with the anti-mGluR4a specific C-terminal antibody, the anti-mGluR4/8 N-
terminal antibody, or the anti-c-myc antibody (c-myc-mGluR4a expressing cells). Relative
positions of molecular weight standards (in kDa) are indicated to the left. Included for
cornparison are wild-type mGluR4a, c-myc-mGluR4a, and mGluR8a showing 'normal'
monomer and dimer bands that run at approximately M, = 100,000 and MI = 200,000,
respectively. Note the presence of an additional lower molecular weight band in the mGluR4
R78A, c-myc-mGluR4 R78A, and mGluR8 R75A mutants compareci to their respective wild-
type receptors (indicated by arrows). This band appears at approximately M, = 85,000 and is
evident for mGluR4 R78A regardless of the antibody used for detection.
Panel B is a schematic representation of mGluR4 indicating the recognition sites of the
antibodies used in these experiments (arrows). The position of the c-myc tag in c-myc-
mGluR4a is also indicated. Putative transmembrane domains are denoted by rectangles and
the proposed signal peptide (SP) is boxed.
c-myc (c-myc-mGluR4a only)
65
rnGluR4 R78A also displayed a corresponding shifi to a lower molecular weight (-30 m a )
indicating that mutation of this residue does not affect receptor dimerization. Because of the
high sequence homology between mGluR4 and mGluR8, the analogous residue (R75A) was
mutated in mGluR8. This mutation produced a similar banding pattern when probed with the
anti-mGluR4/8 antibody and, like the mGluR4 R78A mutant, did not bind ['HIL-AP~
(Section 3.4; see Fig. 9B). Moreover, the immunoreactive band at M, = 85,000 was present in
immunoblots perfonned on mGluR8 R75A using the anti-c-myc antibody (refer to Fig. 9A).
Nevertheless, it should be noted that the band at M, = 85,000 in mGluR8 R7SA was very faint
compared to the corresponding mGluR4 mutant and that a noticeable shifi in the molecular
weight of receptor dimers was absent.
The results of the immunoblotting expenments conducted on mGluR4 R78A and mGluR8
R75A suggest that the additional bands observed at M, = 85,000 are not due to receptor
proteolysis. Therefore, it is possible that the lower band observed in this mutant may
represent a confomationally constrained or differentially processed fonn of the receptor.
3.6 Deglycosylation of mGluR4 and mCluR4 R78A
Wild-type mGluR4a and mGluR4 R78A were treated with PNGase F to examine the
possibility that the presence of the lower molecular weight band in the R78A mutant results
fiom inhibition of asparagine-linked receptor glycosylation. Following SDS-PAGE and
subsequent immunoblotting, monomers of mGIuR4 can be observed to migrate as a doublet
consisting of bands at M, = 100,000 and M, = 93,000 (Fig. 1 1). This banding pattern has been
proposed to represent glycosylated and deglycosylated fonns of the receptor, respectively
(Hampson et al., 1999). However, treatment of mGluR4 and mGluR4 R78A with PNGase F
to cleave al1 asparagine-linked carbohydrates produced a much larger shifi than expected
Figure 11. Immunoblots showlng the effects of PNGase F-mediated degiycosylation of
mGluR4 and mGluR4 R78A. Relative positions of molecular weight standards (in kDa) are
indicated to the left. Control reactions were performed in parallel with deglycosylation
reactions. The reaction conditions were identical except that PNGase F was not added to
control samples (see Section 2.7 for details). Untreated samples were prepared for SDS-
PAGE as described in Section 2.5. The blot s h o w here is representative of three independent
experiments; each lane contains approximately 6 pg of protein probed with the anti-
mG1uR4/8 antibody. Arrows to the right indicate the positions of the three immunoreactive
bands that run at the approximate molecular weights:
' M, = 100,000
* MI = 93,000
MI = 80,000-85,000
(-1 5-20 ma; Fig. 1 1). The fully deglycosylated fom of both receptors appear to run at the
same molecular weight (Mr = 80-85,000) as the lower molecular weight band observed in the
untreated R78A mutant. Therefore, it is possible that the R78A mutation in mGluR4
interferes with glycosylation of this receptor. An alternative, though less likely, explanation
is that the 'closed' form of mGluR4 runs at a similar molecular weight as the deglycosylated
receptor.
3.7 Expression and Binding Properties of the mGluR4 K317A and K317E Mutants
To further examine the possibility that the band observed at Mr = 85,000 in mGluR4
R78A-expressing cells represents a closed form of the receptor, a second positively charged
residue in mGluR4 ( L ~ S " ~ ) was targeted for site-directed mutagenesis. The crystal structure
of mGluRl (Kunishima et al., 2000) shows that upon glutamate binding, ~r~~~ aligns itself
across the binding pocket fiom ( L ~ S ) ' ' in mGl uR4; refer to Fig . 4A and Section 1.7).
~r~~~~ is on the opposite lobe (lobe 2) of the receptor frmn kg7' (lobe 1) and is thought to
stabilize the negatively charged side chain of glutamate upon ligand binding. We
hypothesized that the opposing forces of these two positively charged residues might
contribute to maintaining the open conformation of the receptor in the absence of ligand.
Therefore, one might expect that mutation of L ~ s ~ ' ' in mGluR4 could produce similar effects
as the R78A mutation (loss of ['HJL-AP~ binding and appearance of an additional lower
molecular weight band following SDS-PAGE). The presence of an immunoreactive band at
M, = 85,000 following mutation of this residue would imply that the conformational change
induced by receptor closure produces a similar shifi in molecular weight as that observeà
following receptor deglycosylation.
Imrnunoblot analysis conducted on the mGluR4 K3 17A and K3 17E mutants did not reveai
the presence of an immunoreactive band at M, = 85,000 (Fig. 12A). The ability of these
mutants to bind [)H]L-AP~ was subsequently tested with al1 data normalized to levels of
specific binding observed in membranes expressing wild-type mGluR4. The amount of
specific ['HIL-AP~ binding to mGluR4 K317A was nearly identical to that observed to the
wild-type receptor (Fig. 12B). Subsequent cornpetition curve analysis conducted on this
mutant revealed that its affinity for [ 3 ~ ] ~ - ~ ~ 4 (Ko = 0.33 i 0.14 PM; n = 3) was comparable
to the wild-type receptor (Ko = 0.50 i 0. I O PM; refer back to Table 4). In contrast, mutation
of this positive residue to a negatively charged glutamate (K3 17E) resulted in a large decrease
in ['HIL-AP~ binding ( 4 0 % of control; n = 3). Although this mutant was expressed at lower
Ievels than the wild-type receptor, it is unlikely that slight diffetences in expression could
account for the drastic effect of this mutation on radioligand binding (Fig. 12A). Togethet,
these results indicate that L ~ S ~ " , while not required for high affinity ligand binding to
mGluR4, plays an important role in stabilizing the conformation of the binding pocket.
Moreover, the absence of an additional lower molecular weight band on immunoblots of the
mGluR4 K3 17A and K3 17E mutants suggests that the band observed at M, = 85,000 in the
R78A mutant represents a deglycosylated form of the receptor.
Figure 12. Comparlsoa of expression levels and ['AIL-APQ binding to the mGluR4 c - -
K3l7A and K317E mutants. Panel A is an immunoblot showing the relative expression
levels of the two mutants compared to the wild-type receptor. Each lane contains 6 pg of
membrane protein subjected to SDS-PAGE and probed with the anti-mGluR418 antibody.
Specific binding of [ 'HJL-AP~ to the K3 17A and K3 17E mutants (Panel 8) was nonnalized
to levels detected in membranes expressing wild-type mGluR4. Al1 binding experiments were
conducted in parallel on membranes prepared from two separate transfections (n = 3).
#Cornpetition cuves were generated for K 3 17A. The KD for K3 17A was 0.33 + 0.14 pM
compared to 0.50 * O. I O pM for the wild-type receptor (see Table 4). ['HIL-AP~ binding to
K3 17E was too Iow to conduct cornpetition analysis.
4. DISCUSSION
4.1 Pharmacological Profiles of the Metabotropic Glutamate Receptor Ligands ['H]L-
AP4 and ['HICPPG
The aim of the first part of my sîudy was to characterize the mGluR-selectivity
profiles of two radioligands: the agonist ['HIL-AP~, and the novel antagonist [ 3 ~ ] ~ ~ ~ ~ .
Previous studies have shown that ['HIL-AP~ binds to mGluR4a and mGluR8a with high
affinity (KD = 400-800 nM; Eriksen and Thomsen, 1995; Han and Hampson, 1999; Hampson
et al., 1999; Peltekova et al., 2000). This analysis has now been extended to other mGluR
subtypes. Because L-AP4 has been reported to exhibit selectivity towards the group III
mGluRs, we did not expect to see appreciable binding of this radioligand in membranes fiom
HEL293 cells expressing either the group 1 or group II mGluRs. Furthemore, it has been
reported that activation of mGluR7 in functional assays requires much higher agonist
concentrations than the other group III receptors (ECso for L-AP4 > 150 pM; Okamoto et al.,
1994; Wu et al., 1998). Based on these observations, we did not anticipate [ 3 ~ ] ~ - ~ ~ 4
binding in ce11 membranes expressing mGluR7 at the concentration used in out assay (30
nM). As expected, no specific ['HJL-AP~ binding was observed in cells expressing the group
1 or group II receptors, or in cells expressing mGluR7 (Fig. 6).
However, the absence of [ 3 ~ ] ~ - ~ ~ 4 binding in membranes prepared from cells expressing
mGluR6 was surprising, in view of the fact that severai studies have reported that this
receptor is activated in biochemical assays at similar agonist concentrations as are required for
activation of mGluR4 or mGluR8. mGluR6 is highly homologous to rnGluR4 (-70%
sequence identity; Nakajima et al., 1993) and consistent with this similarity, potencies of L-
SOP or L-AP4 towards inhibition of forskolin-stimulated CAMP formation are almost
identical between the two receptors (EC50 for L-SOP = 2-5 PM, ECSo for L-AP4 = 0.40.9
PM; Nakajima et al., 1993; Laurie et al., 1997; Pin and Conn, 1997). It is unlikely that the
lack of binding observed to mGluR6 was due to low expression levels because
immunoblotting results indicated that this receptor was highly expressed in transfected cells
(Fig. 5). Furthemore, receptor misfolding is an unlikely explanation for the absence of
binding to mGluR6 because responses to 100 pM L-AP4 were observed in live mGluR6-
expressing HEK-293 cells (E. Rosemond and D. R. Hampson, unpublished observations).
These responses were measured as increases in intracellular calcium release using the ca2'
indicator dye fura-2 following CO-transfection with the chimeric G-protein a-subunit Gqig.
This chimeric G-protein corresponds to the a-subunit of G, with its last 9 residues replaced by
those of Gaiz and switches the mGluR6 signal transduction pathway fiom inhibition of CAMP
production to activation of PLC and subsequent IPa-mediated ca2' release (Conklin et al.,
1993). A previous study examining mGluR2 and mGluR4 using this assay detennined that
the phannacological profiles of these receptors were identical to those reported by measuring
inhibition of forskolin-stimulated adeiiylyl cyclase activity (Gomeza et al., 1996b). My
results were similar to those observed with human mGluR6 expressed in CHO cells; Laurie et
al. (1997) were unable to detect specific binding of ['HIL-AP~ despite the ability to measure
L-APCmediated inhibition of forskolin-stimulated adenylyl cyclase. One possible
explanation for these tindings is that mGluR6 may have an intemediate affinity for agonists
that is higher than that of mGluR7 but lower than either mGluR4 or mGluR8. Accordingly,
increasing the concentration of [ 3 ~ ] ~ - ~ ~ 4 up to 300 nM remlts in small increases in the
amount of specific binding to mGluR6-expressing membranes (E. Rosemond and D. R.
Hampson, unpublished observations). Therefore it is likely that the affinity of mGluR6 for
['HIL-AP~ is too low to measure at the radioligand concentration used in our binding assay
(30 nM). If this is indeed the case, then finctional data on mGluR6 suggests that this receptor
might couple more efïiciently to G-proteins. An alternative explanation is that the optimal
requirements for ligand binding to mGluR6 (i.e. ionic composition of the assay buffer, pH,
etc.) may be different fkom those for the other group III receptors. These differences could be
related to the restricted distribution of mGluR6 within the retina and its physiological role in
mediating ON-responses of retinal bipolar cells.
The results of ['HJCPPG binding to cloned mGluRs showed that, at the concentration used
(20 nM), a high level of binding was seen only in cells expressing mGluR8 (Fig. 6). Under
the same conditions, binding to mGluR4 was barely detectable. The drarnatic differences in
['HJCPPG binding to mGluR4 and mGluR8 are not likely due to differences in receptor
expression levels because (a) immunoblots using the same antibody to detect mGluR4 and
mGluR8 showed a similar level of expression for the two receptors (Fig. 5) and, (b) the
[ 3 ~ ] ~ ~ ~ ~ and ['HJL-AP~ binding assays were perfomed in parallel using the same batches
of membranes and the amount of ['HIL-AP~ binding was roughly similar for mGluR4 and
mGluR8 (Fig. 6). We expect that at concentrations above 20 nM ['HICPPG, binding to
mGluR4 would become more prominent while at still higher concentrations, [ 3 ~ ] ~ ~ ~ ~ might
interact with mGluR6 and mGluR7. In accordance with this hypothesis, increasing the
concentration of [ 3 ~ ] ~ ~ ~ ~ to 150 nM resulted in an approximately IO-fold increase in
specific binding of this ligand to mGluR4 (data not shown). However, the arnount of specific
binding was still not sufficient to permit competition curve analysis. Functional assays
conducted on brain cells and tissues have shown that CPPG has the ability to block group II
mGluR-mediated responses, albeit at substantially higher concentrations than those used in
our binding assay (Jane et al., 1995). Although it remains to be determinecl whether or not
both the specific activity and the affinity of ['HJCPPG are high enough for use in radioligand
binding assays for other mGluRs, my results suggest that, at low concentrations, this
radioligand is best suited for studies on mGluR8.
The affinity constant (KD) calculated for ['HICPPG binding to mGluR8 was 183 nM. The
phosphono-substituted phenylglycine compounds that interact with group III mGluRs appear
to have higher aflinity for mGluR8 than the other members of this subgroup. This
observation is supportd by previous radioligand binding experiments with [)H]L-AP~ and
functional data. For example, the higher affinity of ['HICPPG for mGIuR8 compared to
mGluR4 is compatible with previous results examining the binding of ['HIL-AP~ to these
receptors; unlabeled CPPG was approximately 25 to 50-fold more potent at inhibiting [)H]L-
AP4 binding to mGluR8 than to mGluR4 (Hampson et al., 1999; Peltekova et al., 2000). In a
study measuring the abilities of dmgs to inhibit fonkolin-stimulated CAMP production in
cells expressing human mGluR4, mGluR7, or mGluR8, the phosphonophenylglycine
antagonist MPPG (a close structural analogue of CPPG; see Fig. 3) was much more potent at
mGluR8 than at mGluR4 or mGluR7 (Wu et al., 1998). Similarly, Gasparini et al. (1999)
have demonstrateâ that the phosphonophenylglycine agonist (R,S)-PPG is 20 times more
potent at mGluR8 than mGluR4. Moreover, recent characterization of the novel
phenylglycine agonist (S)-3,4-dicarboxyphenylglycine ((S)-DCPG) has show that this
compound potently activates mGluR8 (ECso = 31 nM) and is at least 1000 times more
selective for this receptor than other mGluR subtypes (Thomas et al., 200 1 ).
A tentative conclusion that can be drawn fiom these collective observations is that the
binding pocket in mGluR8 is more accommodating for the phenylglycine derivatives
compared to other members of the gmup III mGluRs. L-AP4 and the cornpetitive antagonist
CPPG likely bind within the same binding pocket of mGluRs and the molecular interactions
of the two dmgs within the pocket may overlap one another, but they are not identical. The
requirement for a phosphonate-containing moiety among group III agonists is well
established. The results from ['HJCPPG cornpetition experiments indicate that this
requirement also applies to the phenylglycine derivatives (Fig. 8; Table 3). We observed that
two phosphonate-containing phenylglycine compounds, CPPG and MPPG, were the most
potent inhibitors of ['HJCPPG binding, while MCPG, a phenylglycine compound identical to
MPPG but lacking a phosphonate group was approximately 200 and 500-fold less potent at
displacing [ 3 ~ ] ~ ~ ~ ~ binding compared to MPPG and CPPG, respectively (Table 3). The
very low potency of MCPG in antagonizing L-AP4-induced responses has previously been
demonstrated in electrophysiological experiments on rat hippocampal slices (Vignes et al.,
1995) and with mGluR8 CO-expressed in oocytes with potassium channels (IClro = 320 PM;
Saugstad et al., 1997).
My data shows that although rnGluR4 and mGluR8 have similar afinities for L-AP4 and
other non-phenylglycine agonists, mGluR8 possesses higher affinity for CPPG and MPPG
than mGluR4. These results are in accordance with a recent functional study demonstrating
that MPPG is more potent at inhibiting mGluR8 agonist-mediated responses than those of
mGluR4 (De Colle et al., 2000). One interpretation of these findings is that the ligand
recognition site of mGluR8 is more hydrophobic than that of mGluR4. The binding pocket
within mGluR8 may provide a better microenvironment with a higher degree of
hydrophobicity that is more accommodating for the hydrophobic phenyl ring present in the
phenylglycine denvatives. In support of this suggestion, preliminary results from
mutagenesis experiments in which specific residues in the ligand-binding pocket of mGluR4
have been mutated to the equivalent (and more hydrophobic) residues in mGluR8 have shown
an increase in affinity of CPPG to the mutant mGluR4 receptor (V. Pektekova, O. Hornby,
and D. R. Hampson, unpublished observations). Likewise, a cornparison between mGluR4
and mGluR8 phamacology by De Colle et al. (2000) has suggested that the binding site of
mGluR8 might be more hydrophobic or less sterically-hindered than that of mGluR4. This
hypothesis was based on their finding that compounds containing alkyl rings were often more
potent on mGluR8 than mGluR4.
Despite the similarities in phamacology among the highly homologous group III mGluRs,
my study suggests that a series of phenylglycine compounds may have the potential for
discnminating between receptor subtypes. Speci ficall y, we have demonstrated that the
phenylglycine antagonist CPPG exhibits selectivity towards mGluR8 at low nanomolar
concentrations. Furthemore, ['HICPPG is the first commercially available radioligand that is
selective for a single mGluR subtype. Collectively, these findings should encourage the
development and identification of compounds (such as (S)-DCPG; Thomas et al., 2001) that
exhibit greater affinity and selectivity towards individual mGluR subtypes.
4.2 Amino Acids Meditating High ACnnity Ligand Blnding to mGluR8
A series of point mutations were made in mGluR8a. The mutants were express4 in
HEK cells and tested for their ability to bind either [ 3 ~ ] ~ - ~ ~ 4 or [ 3 ~ ] ~ ~ ~ ~ . Both
radioligands have previously been shown to bind to mGluR8 with high afinity (Peltekova et
al., 2000; Naples and Hampson, 2001, see above). The objective of this part of my study was
to detennine the amino acid residues in mGluR8 that mediate these high affinity interactions.
kkg7' was initially targeted for mutagenesis due to the conservation of this residue arnong
al1 mGluR subtypes. It has previously been established that mutation of the analogous residue
in mGluR1 (Arg'') and mGluR4 abolishes the binding of group-selective radioligands
(Jensen et al., 2000; Hampson et al., 1999). Moreover, the crystal structure of mGluRl
demonstrates that hg7' participates in a polar interaction wi th the y -carboxyl group of
glutamate (Kunishima et al., 2000). As expected, mutation of this conserved residue to an
alanine in mGluR8 eliminated ['HIL-AP~ and ('HICPPG binding (Fig. 9). This result
provides confirmation that ~r~~~ is a fundamental ligand recognition residue among group III
receptors. By analogy with the crystal structure of mGluR1, one can infer that Arg''
participates in an electrostatic interaction with the y-phosphonate group common to both L-
AP4 and CPPG.
Due to the reported selectivity of phosphonate-containing compounds for the group III
receptors, we hypothesized that an additional positively charged residue within the binding
pocket is required for high affinity binding of these compounds. Therefore, other positively
charged residues in mGluR8 were targeted for inutagenesis based on their proximity to ~ r ~ ' ' .
Additionally, it was expected that residues mediating the binding of phosphonated compounds
would not be conserved among the group 1 or group II receptors. One residue that fit these
critena was LYS". This residue is present only in mGluR4 ( L ~ S ' ~ ) and mGluR8 and, as such,
may explain the high afinity of L-AP4 for these receptor subtypes. Mutation of L ~ S " to the
equivalent residue in mGluRl (tyrosine; refer to Fig. 48) eliminated ['HIL-AP~ and
[)H]CPPG binding (Fig. 9). We suggest that the lack of ['HJL-AP~ and ['HICPPG binding
observed to mGluR8 K7 1 Y results fiom steric hindrance. It is probable that the introduction
of a tyrosine residue at this position interferes with binding of the relatively large (compared
to glutamate) phosphonate-containing compounds. This may provide an explanation for the
inability of mGluRl to bind either L-AP4 or CPPG.
Interestingly, when LYS'' was mutated to an alanine in mGluR8, no significant effect was
observed on the affinity of ['HIL-AP~, while the affinity of ['HJCPPG for this mutant was
decreased significantly (-4-fold; Table 4). In this case, steric hindrance is not a plausible
explanation for the decrease in affinity of ['HJCPPG for mGluR8 K71A. Furthemore, this
effect is not likely attributable to a direct interaction of CPPG with L ~ S " because this residue
is conserveci in mGluR4, a low affinity receptor for CPPG. A possible explanation is that
mutation of L ~ S ~ ' affects the relative positions of nearby residues unique to mGluR8.
Consequently, polar interactions with the phenyl group of CPPG may be disrupted resulting in
l es efficient binding of this ligand. My results suggest that L ~ S " does not mediate the high
affinity binding of phosphonated compounds to mGluR8. In support of this conclusion, the
affinity of ['HIL-AP~ for the analogous mGluR4 mutant (K74A) was not significantly
different fiom the aflnity of ['HIL-AP~ for wild-type mGluR4 (Table 4; V. Peltekova and D.
R. Hampson, unpublished observations). This differs from results with mGluRl where a
tyrosine at this position appears to be required for the binding of the agonist [3~]quisqualic
acid (Kunishima et al., 2000).
In contrast to our results, previous modeling studies have predicted that L~s''' in mOluR4
is important for ligand binding. Bessis et al. (2000) constnicted a homology mode1 of the N-
terminal domain of mGluR4 bas4 on LIVBP into which the selective agonist I -
aminocyclopentane 1,3,4-tricarboxylic acid (ACPT-I) was docked. The model proposes that
the ammonium function of L ~ S " interacts with an oxygen atom on one of the acidic groups of
ACPT-I and indicates that this residue is a deteminant of subtype or group specific
phamacology. Unfortunately, mutagenesis studies were not perfonned to clarify the role of
~~s~~ in ACPT-1 binding (Bessis et al., 2000). A subsequent study involving a detailed
cornparison of mGluR4 and mGluR8 phamacology has proposed that the model established
by Bessis et al. (2000) is valid fot mGluR8 as well (De Colle et al., 2000). Regardless, my
results imply that residues involved in binding of the phosphonated compounds L-AP4 and
CPPG differ from those required for ACPT-1 binding.
Two other amino acid residues targeted for mutagenesis were ~~s~~ and With the
exception of mGluR7 (a low affinity L-AP4 receptor), ~~s~~ and Lysb9 are conserved among
the group III mGluRs. Previous experiments on mGluR4 demonstrated that mutation of either
of these lysine residues produced a modest decrease (-50%) in ['H]L-AP~ binding (V.
Peltekova and D. R. Hampson, unpublished observations). The K68AK69A double mutant
in mGluR8 produced a similar, albeit more pronounced, effect on ['HIL-AP~ and ['HJCPPG
binding (Fig. 9). It i s possible that high affinity binding of phosphonated compounds requires
one of these residues, but not both. However, it should be noted that the LIVBP-homology
models of mGluR4 do not implicate these residues in ligand binding, despite their
conservation in this receptor subtype (Hampson et al., 1999; Bessis et al., 2000). Therefore,
an alternative explanation for this result is that these residues do not participate directly in
ligand binding but contribute to the conformation of the binding pocket. Mutation of a single
lysine may affect the conformation such that the ability of the receptor to bind ligand is
compromised. As might be expected, mutation of both residues could produce effects that are
more drastic.
Together with previous modeling studies, my results suggest that the requirernents for high
afinity binding to mGluR8 are ligand specific. Unfortunately, the interpretation of site-
directed mutagenesis experiments is complicated by the possibility that point-mutations can
produce confonnational changes in the ligand binding pocket or global misfolding of the
protein. For this reason, mutations that abolish radioligand binding must be carefùlly
considered,
4.3 Cbaracterization of an Immunoreactive Band Unique to mCluR4 R78A
When subjected to SDS-PAGE, monomers and dimers of mGluR4 and mGluR8 - -
migrate at Mr = 1 00,000 and M, = 200,000 (Fig. 5). If less protein is used or expression levels
are low, the immunoreactive band at M, = 100,000 can be visualized as a doublet of bands at
M, = 100,000 and M, = 93,000 (Fig. 1 1). Mutation of a conserved arginine residue (hg7') to
an alanine in mGluR4 results in the appearance of an additional lower molecular weight band
at M, = 85,000 following SDS-PAGE. Based on the fundamental role of this positively
charged residue in ligand binding and its position within the hinge region of the binding
pocket, we speculated that the additional band observed in the R78A mutant may represent a
closed fonn of the receptor (see Section 1.7).
Evidence exists that dimerization of mGluR subtypes is mediated by a combination of
disulfide bonds and hydrophobic interactions (Romano et al., 1996; Tsuji et al., 2000).
However, my results demonstrate thst mGluR dimers (particularly of the gtoup III receptors;
Fig. 5) are still present following treatment with reducing agents and detergents. This is a
common observation among mGluRs (Hampson et al., 1999; Peltekova et al., 2000; Tsuji et
al., 2000). Therefore, it is possible that the confonnational change associated with receptor
closure could remain stable under similar denaturing conditions.
Detection of the immunoreactive band at Mr = 85,000 by N-terminal or C-terminal
antibodies to mGluR4 suggests that this band does not represent a proteolytic fiagrnent of
mGluR4 R78A (Fig. 10). Proteolysis of the 67 residues upstream of the N-terminal anti-
mGluR4/8 antibody recognition site would be expected to produce only a modest shift in
molecular weight (-7 kDa). Furthemore, the band at Mr = 85,000 was also present in a
consmict of mGluR4 R78A containing a c-myc epitope inserted downstrearn of the signal
peptide (c-myc-mGluR4a). The ability of the anti-c-myc antibody to label this lower
molecular weight band provides additional evidence against N-terminal proteolysis. With
respect to receptor proteolysis, it is a remote possibility that the R78A mutation promotes self-
splicing of mGluR4 at an interna1 site. However, it is unclear why the R78A mutation would
encourage this process while other mutations made to mGluR4 do not. Mutation of the
analogous residue in mGluR8 (kg7') produced a similar banding pattern following SDS-
PAGE (Fig. 10). However, unlike the mGluR4 mutant, the additional band at MI = 85,000
was very faint. Moreover, this band was occasionally observed in the wild-type receptor,
albeit at a lesser intensity. The faint appearance of this lower molecular weight band in
immunoblots of wild-type mGluR4 and mGluR8 might be explained by the observation that
mGluRs exist in dynamic equilibriurn between two primary conformational States, the 'open'
and 'closed' States (Kunishima et al., 2000). As a result, it is expected that a small proportion
of wild-type receptors will exist in the closed conformation. This population of receptors may
or may not be noticeable on immunoblots depending on receptor expression levels and/or
developing conditions (i.e. exposure times).
Nonetheless, one would expect the lower band for mGluR8 R75A to be more pronounced
if it indeed represents a closed f o m of mGluR8 due to the high degree of sequence homology
between mGluR4 and mGluR8 in this region. It should also be noted that mutation of ~r~~~
in a soluble construct of mGluR4 failed to duplicate the banding pattern observed with the
full-length receptor (data not shown). The soluble construct of mGluR4 is truncated 39 amino
acids upstream of the first putative transmembrane domain and displays a similar
phamacological profile as the full-length receptor (Han and Hampson, 1999). It is possible
that the lower molecular weight band is absent in this construct because missing downstream
portions of the receptor are required to stabilize receptor closure.
Examination of the mGluR1 crystal structure reveals that upon ligand binding, ~r~~~ aligns
itself across the binding pocket fiom ~ r ~ j * (present on the opposite lobe; Kunishima et al.,
2000). Like ~rg'*, this residue participates in an electrostatic interaction with the negatively
charged side chah of glutamate and serves to stabilize the closed form of the receptor.
Furthemore, this arginine is the only positively charged residue on the second lobe of the
receptor that interacts with glutamate. Since a positive charge (lysine) is conserved at this
position in the group III mGluRs, ( L ~ S ) ' ~ in mGluR4; Duvoisin et al., 1995) we hypothesized
that the repulsive force between these positive charges on opposite lobes is responsible for
holding the receptor in its 'open' conformation in the absence of ligand. Therefore, it was
expected that if the mGluR4 R78A mutant promotes a shift in equilibrium towards the closed
form of the receptor, then mutation of L ~ s ' ' ~ might pmduce a similar result.
Interestingly, the mGluR4 K3 17A mutant possessed a similar affinity for ['HIL-AP~ as
wild-type mGluR4 (Table 4) suggesting that this residue is not important for ligand binding.
Furthemore, the extra band at M, = 85,000 was not observed in this mutant following SDS-
PAGE (Fig. 12A). The binding results imply that removal of this positive charge on the
second lobe of the receptor does not promote receptor closure. This finding is in agreement
with the crystal structure of mGluRI, which indicates that residues on the second lobe of the
receptor, though they may interact with glutamate, may not be required for high affinity
ligand binding (Kunishima et al., 2000). The possibility that receptor closure could be
induced by mutating L~S"' to a negatively charged glutamic acid was investigated. In
contrast to the mGluR4 K317A mutant, the ability of the mGluR4 K317E mutant to bind
['HIL-AP~ was severely impaired (Fig. 128). It is likely that introduction of this negative
charge inhibited ['HIL-AN binding by foming an ion pair with a positively charged residue
on the other lobe of the receptor. Occupation of this positive charge therefore promotes
receptor closure, analogous to ligand binding. However, the additional lower molecular
weight band was also absent in irnmunoblots performed on this mutant (Fig. 12A).
Collectively, these results suggest that the band at M, = 85,000 in immunoblots of mGluR4
R78A-expressing cells does not represent a closed fom of mGluR4.
Other experiments perfonned on mGluR4 and rnGluR4 R78A have indicated that this
additional lower molecular weight band may correspond to a deglycosylated fom of the
receptor (Fig. 1 1). Excluding the putative signal peptide, the predicted molecular weight of
unglycosylated mGluR4a is approximately 98 kDa. Moreover, it has been demonstrated that
PNGase F-mediated deglycosylation of a soluble mGluR4 consmict following expression of
this receptor in HEK-293 cells produces a shift in molecular weight of 8 kDa (Han and
Hampson, 1 999). Based on these observations, i t had been suggested that mGluR4 monomer
bands at M, = 100,000 and M, = 93,000 (Fig. 1 1) represent glycosylated and unglycosylated
foms of the receptor, respectively (Hampson et al., 1999). Studies on mGluRl and the
human ca2?sensing receptor have s h o w that monomers of both receptors migrate as
doublets during SDS-PAGE (Hermans et al., 1998b; Ray et al., 1998). Additionally,
inhibition of asparagine-linked glycosylation of mGluR1 has demonstrated that the Iower
molecular weight band of this doublet represents a partially deglycosylated fonn of the
receptor (Mody et al., 1999). Similar results have been observed for the ca2+-sensing receptor
(Ray et al., 1998).
My results demonstrate that full-length mGluR4 is more heavily glycosylated than
previously expected (-15-20 D a ; Fig. II). The most likely explanation for the disparity
between glycosylation of mGluR4 and its truncated soluble form (Han and Hampson, 1999) is
the presence of an additional consensus sequence for asparagine-linked glycosylation in the
full-length receptor. Therefore, one could hypothesize that the bands at M, = 100,000 and M,
= 93,000 represent different populations of recepton. The approxirnate molecular weight of
the proposeci signal peptide (-4 D a ) might account for this difference. An alternative
explanation is that the doublet represents receptor species at different stages of post-
translational modification (Le. partially processed and mature glycoproteins). The results of a
study examining the sensitivity of the ca2'-sensing receptor to the deglycosylating enzymes
asparagine-glycosidase F and endoglycosidase H has provided evidence in support of this ides
(Ray et al., 1998). Ray et al. (1998) observed that the human ca2'-sensing receptor runs as a
doublet of bands at (-130 and 150 kDa) when expressed in HEK-293 cells. Following
treatment with the deglycosylating enzymes mentioned above, the ca2'-sensing receptor
migrated as a single band ai 1 15 kDa. Previous studies have indicated that the M, of the fully
deglycosylated mGluRl and ca2'-sensing receptors approximate the molecular weight
predicted fiom their respective primary amino acid sequences (Mody et al. 1999; Ray et al.,
1998). However, my results demonstrate that the Mr of fully deglycosylated mGluR4 is 80-
85,000 (Fig. 1 1); this is much less than its predicted molecular weight (98 kDa). The reason
for this discrepancy is unclear.
The question that remains to be answered is: if the mGluR4 R78A mutation indeed impairs
glycosylation of this receptor, then why do the partially processed and fully mature receptors
still appear? A possible explanation is that this mutation impairs proper folding of the
receptor such that a large propoition of receptors are incorrectly glycosylated. ~ r ~ ' ' is
located 20 amino acids upstream of an asparagine-linked glycosylation consensus sequence
and therefore may have the ability to influence glycosylation at this site. Interestingly,
irnmunocytochemistry experiments have demonstrated that mGluR4 R78A is targeted to the
ce11 surface in HEK cells (Hampson et al., 1999). One would expect that misfolding prior to
glycosylation or due to incomplete glycosylation would affect proper trafficking of this
receptor. Accordingly, mutation of critical asparagine residues in the human ca2+-sensing
receptor decreased its cell-surface expression (Ray et al., 1998). In contrast, prevention of
asparagine-linked glycosylation of mGluRl by tunicamycin did not affect ce11 surface
expression of this receptor in HEK cells (Mody et al., 1999). Whether or not unglycosylated
receptors would be correctly targeted to prespaptic teminals in mGluR4-expressing neurons
is unclear. Regardless, these studies suggest that the importance of receptor glycosylation on
proper trafficking can vaiy, even among closely related GPCRs.
4.4 Conclusions and Future Studies
In the first part of my study, al1 eight rat mGluR subtypes were expressed in HEL293
cells and tested for their ability to bind either ['HIL-AP~ or the novel radiolabeled compound
['HJCPPG. My results confinn the reporteci selectivity of [ 3 ~ ] ~ - ~ ~ 4 and reveal that
[ 3 ~ ] ~ ~ ~ ~ selectively labels mGluR8 at a concentration of 20 nM (Naples and Harnpson,
2001). The ability of other phenylglycine compounds (e.g. MPPG, (R,S)-PPG) to potently
compete for ['HICPPG binding to mGIuR8 compareci with their ability to inhibit ['HIL-AW
binding to mGluR4 (Hampson et al., 1999) suggests that the presence of a phenyl ring confers
selectivity for mGluR8 over mGluR4. We conclude that the binding pocket of mGluR8 is
more hydrophobic than that of mGluR4 and therefore better able to accommodate the
phenylglycine compounds. These findings should lead to the development and identification
of compounds possessing even greater affinity and selectivity for mGluR8.
[ 3 ~ ] ~ ~ ~ ~ is the first commercially available radioligand identified to be selective for a
single mGluR subtype and, as such, represents a useful tool for studying mGluR8. Presently,
little is known about the specific physiological processes mediated by this receptor. In the
marnmalian CNS expression of mGluR8 shows considerable overlap with that of mGluR7,
indicating that these receptors serve coniplementary functions (i.e. inhibition of
neurotransmitter release; Saugstad et al., 1997). Based on the relatively low potency of
glutamate for mGluR7 it has been suggested that this receptor mediates homosynaptic - -
modulation of glutamatergic neurotransmission, whereas mGluR8 might influence glutamate
release through heterosynaptic mechanisms. Future studies could exploit the selectivity of
CPPG for mGluR8 to examine the precise role of this receptor in glutarnatergic transmission.
A second conclusion of this study is that the amino acids in mGluR8 that mediate binding
of ['HIL-AP~ and ['HICPPG are similar. The only difference observed between the two
ligands was that mutation of L ~ S ~ ' to an alanine in mGluR8 did not affect the afinity of this
receptor for L-AP4, but resulted in an approximately 4-fold decrease in afinity for the
phenylglycine antagonist CPPG. At present, no molecular models have been developed
speci ficall y for mGluR8. However, based on their phamacological similarities, it has been
suggested that models of the mGluR4 binding site are valid for mGluR8 as well (De Colle et
al., 2000). Previous models of the mGluR4 ligand-binding pocket (Hampson et al., 1999;
Bessis et al., 2000) have adapted this receptor to the structural mode1 of the mGluRl binding
site (O'Hara et al., 1 993). Although these models have proven useful for identifying the key
residues involved in glutamate binding, CO-ordinates for the crystal structure of mGluR1 have
recently become available (Kunishima et al., 2000). Therefore, it is anticipated that this will
guide the production of new, more accurate models of mGluRs. Future studies utilizing these
models will shed light on the binding determinants that confer subtype-selective
phamacology. Moreover, it is likely that X-ray structures of other mGluR subtypes will be
solved in the funire. The resultant detemination of amino acids involved in ligand binding to
different mGluR subtypes should lead to rapid advances in the design of subtype specific
ligands.
In the final part of this study, we demonstrated that the R78A mutation in mGluR4 impairs
proper glycosylation of this receptor. Despite this observation, îrafficking of mGluR4 R78A
to the ce11 surface in HEL293 cells appears to be unaffected (Hampson et al., 1999)
indicating that folding and processing of this receptor is othenvise not severely compromised.
Interestingly, the M, of mGluR4 following deglycosylation with PNGase F was
approximately 85,000. This is substantially different from the predicted molecular weight of
unglycosylated mGluR4a (-98 kDa). The reason for this discrepancy is unclear but is
unlikely to be due to receptor misfolding since deglycosylation of soluble rnGluR4a has
previously been shown to have no effect on the affinity of this receptor for ['HIL-AP~ (Han
and Hampson, 1999). Future studies are needed to examine the role of asparagine-linked
oligosaccharides in mGluR trafficking and/or folding. It is probable that the requirements for
receptor trafficking in mGluR-expressing neurons are more stringent compared to those of
HEK cells.
In summary, my work in this thesis has (1 ) demonstrated that the novel radiolabeled
antagonist ['HJCPPG is selective for mGluR8 at a concentration of 20 nM, (2) characterized
residues within the binding pocket of mGluR8 that confer selectivity for ['HIL-AP~ and
[.'H]CPPG, and (3) revealed that mutation of a conserved arginine residue in mGluR4 impairs
proper glycosylation of this receptor.
5. REFERENCES
Abdul-Ghani, A-S., Attwell, P.J.E., Singh Kent, N., Bradford, H.F., Croucher, M.J., Jane, D.E. (1 997). Anti-epileptogenic and anticonvulsant activity of L-2-arnino-4- phosphonobutyrate, a presynaptic glutamate receptor agonist. Brain Res. 755,202-2 12.
Abe, T., Sugihara, H., Nawa, H., Shigemoto, R., Mizuno, N., Nakanishi, S. (1992). Molecular chanicterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/~a2+ signal transduction. J. Biol. Chem. 267, 1 336 1 - 13368.
Alaluf, S., Mulvihill, E.R., Mcllhinney, R.A. (1995). Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor la by protein kinase C in permanently transfected BHK cells. FEBS Lett. 367, 30 1-305.
Ararnori, I., Nakanishi, S. ( 1992). Signal transduction and phannacological characteristics of a metabotropic glutamate receptor, mGluRI, in transfected CHO cells. Neuron 8, 757-765.
Bessis, A-S., Bertrand, H-O., Galvez, T., De Colle, C., Pin, J-P., Acher, F. (2000). Three- dimensional mode1 of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process. Protein Science 9,220-2209.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana!. Biochem. 72, 248- 253.
Bradley, S.R., Levey, A.I., Hersch, S.M., COM, P.J. (1996). Immunocytochemical localization of group III metabotropic glutamate receptors in the hippocampus with subtype- specific antibodies. J. Netirosci. 16,2044-2056.
Brakeman, P.R., Lanahan, A.A., O'Brien, R., Roche, K., Bames, C.A., Huganir, R.L., Worley, P.F. (1 997). Homer: a protein that selectively binds metabotropic glutamate receptors. Nature 386,284-288.
Brauner-Osborne, H ., Jensen, A. A., Krogsgaard-Larsen, P. ( 1 999). Interaction of CPCCOEt, with a chimeric mGluR l b and calcium sensing receptor. Neuroreport 10,392303925.
Brauner-Osborne, H., Egebjerg, J., Nielsen, E. 0., Madsen, U., Krogsgaard-Larsen, P. (2000). Ligands for glutamate receptors: design and therapeutic prospects. J. Med. Chem. 43, 2609- 2645.
Brown, E.M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger. M.A., Lytton, J., Hebert, S.C. (1993). Cloning and characterization of an extracellular ca2'-sensing receptor from bovine parathyroid. Nature 366,575-580.
Bruno, V., Copani, A., Knopfel, T., Kuhn, R., Casabona, G. (1995). Activation of meîabotropic glutamate receptors coupled to inositol phospholipid hydrolysis amplifies NMDA-induced neuronal degeneration in cultured cortical cells. Neuropharrnacology 34, 1089- 1098.
Bruno, V., Battaglia, G., Ksiazek, I., van der Putten, H., Catania, M.V., Giufida, R., Lukic, S., Leonhart, T., Inderbitnn, W., Gasparini, F., Kuhn, R., Hampson, D.R., Nicoletti, F., Flor, P.J. (2000). Selective activation of mGluR4 metabotropic glutamate receptors is protective against excitotoxic neuronal death. J. Neiirosci. 20,64 13-6420.
Buisson, A., Yu, S.P., Choi, D.W. (1996). DCG-IV selectively attenuates rapidly triggered NMDA-induced neurotoxicity in cortical neurons. Eur. J. Neurosci. 8, 138- 143.
Caramelo, O.L., Santos, P.F.. Carvalho, A.P., Duarte, C.B. (1999). Metabotropic glutamate receptors modulate [3~]acetylcholioe release fiom cultured amacrine-like neurons. J. Neurosci. Res. 58,505-5 14.
Cartmell, S., Adam, G., Chaboz, S., Hemingsen, R., Kemp, J.A., Klingelschmidt, A., Metzler, V., Monsma, F., Schaffhauser, H., Wichrnann, J., Mutel, V. (1998). Characterization of ['Y]- (2S,2~R,3~R)-2-(2~,3~-dicarboxycyclopropyl)glycine (['HIDCO IV) binding to metabotropic mGluz receptor-transfected ce11 membranes. Br. J. Phannacol. 123,497-504.
Cartmell, J., Schoepp, D. D. (2000). Regulation of neurotransmitter release by metabotropic glutamate receptors. J. Neurochem. 75,889-907.
Cha, R.S., Tilly, W.G. (1995) PCR Primer, Cold Spnng Harbor Laboratory Press.
Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D., Boume, H.R. (1 993). Substitution of three arnino acids switches receptor specificity of Gqa to that of Gia. Nature 363,274-276.
Conn, P.J., Pin, J-P. (1997). Phannacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. To-ricol. 37,205237.
Corti, C., Restituito, S., Rimland, LM., Brabet, I., Corsi, M., Pin, J-P., Ferraguti, F. (1998). Cloning and characterization of alternative mRNA foms for the rat metabotropic glutamate receptors mGluR7 and mGluR8. Eur. J. Neurosci. 10,3629-3641.
Cuesta, M.C., Arcaya, J.L., Cano, G., Sanchez, L., Maixner, W., Suarez-Roca, H. (1 999). Opposite modulation of capsaicin-evoked substance P release by glutamate receptors. Neurochem. Int. 35,47 1-478.
De Blasi, A., Corn, P.J., Pin, J-P., Nicoletti, F. (2001). Molecular determinants of metabotropic glutamate receptor signaling. Trends Phannacoi. Sci. 22, 1 14- 1 20.
De Colle, C., Bessis, A-S., Bockaert, J., Acher, F., Pin, J-P. (2000). Phannacological chmcterization of the rat metabotropic glutamate receptor type 8a revealed strong similarities and slight differences with the type 4a receptor. Eur. J. Phamacoi. 394, 17-26.
Duvoisin, R.M., Zhang, C., Rarnonell, K. (1995). A novel metabotropic glutamate receptor expressed in the retina and olfactory bulL J. Neumsck t5,3035-3083.
Enksen, L., Thomsen, C. ( 1995). ['~]-~-2-amino-4-~hos~honobutyrate labels a metabotropic glutamate receptor, mGluR4a. Br. J. Pharmacol. 116,3279-3287.
Faden, A.I., Ivanova, S.A., Yakovlev, A.G., Mukhin, A.G. (1 997). Neuroprotective effects of group III mGluR in traumatic neuronal injury. J. Neurohauma 14,885-895.
Fagni, L., Chavis, P., Ango, F., Bockaert, J. (2000). Complex interactions between mGluRs, intracellular ca2+ stores and ion channels in neurons. Trends Neurosci. 23, 80-88.
Flaman, J.M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C., Ishioka, C., Friend, S.H., Iggo, R. (1 994). A rapid PCR fidelity assay. Nucleic Acids Res. 22,3259-3260.
Flor, P.J., Gomeza, J., Tones, M.A., Kuhn, R., Pin, &P., Knopfel, T. (1996). The C-terminal domain of the mGluRl metabotropic glutamate receptor affects sensitivity to agonists. J. Neurochern. 67,58-63.
Fotuhi, M., Standaert, D.G., Testa, C.M., Penney, J.B., Young, A.B. (1994). Differential expression of metabotropic glutamate receptors in the hippocampus and entorhinal cortex of the rat. Moi. Brain Res. 21,283-292.
Galvez, T., Parmentier, ML., Joly, C., Malitschek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., Pin, J-P. (1999). Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding. J. Biol. Chem. 274, 13362- 13369.
Gasparini, F., Bruno, V., Battaglia, G., Lukic, S., Leonhardt, T., Inderbitzin, W., Laurie, D., Sommer, B., Vamey, M.A., Hess, S.D., Johnson, E.C., Kuhn, R., Urwyler, S., Sauer, D., Portet, C., Schmutz, M., Nicoleîti, F., Flor, P.J. (1 999). (R,S)-4-Phosphonophenylglycine, a potent and selective group II1 metabotropic glutamate receptor agonist, is anticonvulsive and neuroprotective in vivo. J. Pharmacol. Exp. Ther. 290, 1 678- 1 687.
Gereau, R.W., Heinemann, S.F. (1998). Role of protein kinase C phosphorylation in rapid desensitization of metabotropic glutamate receptor 5. Natron 20, 143- 15 1.
Gomeza, J., Joly, C., Kuhn, R., Knopfel, T., Bockaert, J., Pin, J-P. (1996a). The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins. J. Biol. Chem. 271.2 199-2205.
Gomeza, J., Mary, S., Brobet, I., Parmentier, M-L., Restituito, S., Bockaert, J., Pin, J-P. ( 1998). Coupling of mGluR2 and mGluR4 to Ga1 5, Ga 16 and chimeric Gaq/i proteins: characterization of new antagonists. Mol. Pharmacol. 50, 923-930.
Goman, C.M. (1 990). Mammalian ce11 expression. Curr. Opin. Biotechnoi. 1,36047.
Greene, J.O., Greenamyre, J.T. (1996). Bioenergetics and glutamate excitotoxicity. Prog. Nesrrobioi. 48,6 1 3434.
Hammerland, L.G., Krapcho, K.J., Garrett, J.E., Alasti, N., Hung, B.C., Simin, R.T., Levinthal, C., Nemeth, E.F., Fuller, F.H. (1999). Domains detennining ligand specificity for ca2+ recepton. Mol. Phannacol. 55,642-648.
Hampson, D.R., Huang, X-P., Pekhletski, R., Peltekova, V., Homby, G., Thomsen, C., ïbgersen, H. (1999). Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor. J. Biol. Chem. 274,33488-33495.
Han, G., Hampson, D.R. (1999). Ligand binding to the arnino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor. J. Biol. Chem. 274, 1 0008- 100 13.
Hetmans, E., Nahorski, S.R., Challiss, R.A. (1998a). Reversible and non-competitive antagonist profile of CPCOOEt at the human type la metabotropic glutamate receptor. Neuropharmacology 37, 1 645- 1647.
Hemans, E., Young, K.M., Challiss, R.A.J., Nahorski, S.R. ( l998b). Effects of human type la metabotropic glutamate receptor expression level on phosphoinositide and ca2' signalling in an inducible expression system. J. Neurochem. 70, 1772- 1775.
Heuss, C., Scanziani, M., Gahwiler, B.H., Gerber, U. ( 1999). G-protein independent signaling mediated by metabotropic glutamate receptors. Nat. Neurosci. 2, 1070-1 077.
Hollmann, M., O'Shea-Greenfield, A., Rogers, S.W., Heinemann, S. (1989). Cloning by functional expression of a member of the glutamate receptor family. Nature 342,6430648.
Hollmann, M., Heinemann, S. ( 1994). Cloned glutamate recepton. Annu. Rev. Neurosci. 17, 3 1- 108.
Hoon, M.A., Adler, E., Lindemeier, J., Battey, J.F., Ryba, N.J., Zuker, CS. ( 1999). Putative rnammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell96,54 1 -55 1 .
Houamed, K.M., Kuijper, J.L., Gilbert, T.L., Haldeman, B.A., O'Hara, P.J. (1991). Cloning, expression, and gene structure of a G protein-coupled glutamate receptor fiom rat brain. Science 252, 13 18- 132 1.
Hu, G., Du@, P., Swanson, C., Ghasernzadeh, M.B., Kalivas, P.W. (1999). The regulation of dopamine transmission by metabotropic glutamate receptors. J. Pharmacol. &p. Theer. 289, 412-416.
Jane, D.E., Jones, P.L., Pook, P.C., Salt, T.E., Sunter, D.C., Watkins, J.C. (1993). Stereospecific antagonism by (+)-a-methyl-4-carboxyphenylglycine (MCPG) of (1 S,3R)- ACPD-induced effects in neonatal rat motoneurones and rat thalamic neurones. Neuropharmacologv 32,725-727.
Jane, D.E., Pittaway, K., Sunter, D.C., Thomas, N.K., Watkins, J.C. (1995). New phenylglycine derivatives with potent and selective antagonist activity at pesynaptie glutamate receptors in neonatal rat spinal cord. Neuropharmacology 34,85 1-856.
Jensen, A.A., Sheppard, P.O., O'Hara, P.J., Krogsgaard-Larsen, P., Bfiuner-Osborne, H. (2000). The role of ~ r ~ ' * in the metabotropic glutamate receptor mGlui for agonist binding and selectivity. Eur. J. Phurmacoi. 397,247-253.
Jockers, R., Linder, M.E., Hohenegger, M., Nanoff, CC., Bertin, B., Strosberg, AD., Marullo, S., Freissmuth, M. ( 1 994). Species di fferences in the G protein selectivity of the human and bovine Ai Dadenosine receptor. J. Biol. Chem. 269,32077-32084.
Johnson, B.G., Wright, R.A., Arnold, M.B., Wheeler, W.J.. Omstein, P.L., Schoepp, D.D. (1999). [ ' ~ ] ~ ~ 3 4 1 4 9 5 as a novel rapid filtration antagonist for group II metabotropic glutamate receptors: characterization of binding to membranes of mGluR receptor subtype expressing cells. Neurophaimacoiogy 34.85 1-860.
Kato, A., Fumiko, O., Saitoh, Y., Fukazawa, Y., Sugiyama, H., Inokuchi, K. (1998). Novel members of the VeslMomer family of PDZ proteins that bind metabotropic glutamate receptors. J. Biol. Chem. 273,23969023975.
Kaupmann, K., Huggel, K., Held, J., Flor, P.J., Biscoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W., Bettler, B. (1997). Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239-246.
Kinoshita, A., Ohishi, H., Nomura, S., Shigemoto, R., Nakanishi, S., Minino, N. (1996). Presynaptic localization of a metabotropic glutamate receptor, mGluR4a, in the cerebellar cortex: a light and electron microscope study in the rat. Neurosci. Lett. 207, 199-202.
Kosinski, C. M., Risso-Bradley, S., Conn, P.J., Levey, A.I., Landwehrrneyer, G.B., Pemey, J.B. Jr., Young, A.B., Standaert, D.G. (1999). Localization of metabotropic glutamate receptor 7 mRNA and mGluR7a protein in rat basal ganglia. J. Comp. N w o l . 415,266-284.
Klunk, W.E., McClure, R.J., Pettegrew, J.W. (1991). L-phosphosenne, a metabolite elevated in Alzheimer's disease, interacts with specific L-glutamate receptor subtypes. J. Narochem. 56, 1 997-2003.
Kroona, H.B., Peterson, N. L., Koemer, J.F., Johnson, R.L. ( 199 1). Synthesis of the 2-amino- 4-phosphonobutanoic acid analogues (E)- and (2)-2-amino-2,3-methano-4- phosphonobutanoic acid and their evaluation as inhibitors of hippocarnpal excitatory neurotransmission. J. Med. Chem. 41, 164 1 - 1650.
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 407,97 1-977.
Kunkel, T.A.. Roberts, J.D., Zakour, R.A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymof. 154,367-382.
Lafon-C~el, M., Fegni, L., Guiraud, M.J., May, S., temer-Natoli, M., Pin, W., Shigcrnoto, R., Bockaert, J. (1999). mGluR7-like metabotropic glutamate receptors inhibit NMDA- mediated excitotoxicity in cultured mouse cerebellar granule neurons. Eur. J. Neicrosci. 11, 663-672.
Laurie, D.J., Schoeflter, P., Wiederhold, KA., Sommer, B. (1997). Cloning, distribution and functional expression of the human mGiu6 metabotropic glutamate receptor. Netrrophannacology 38, 15 19- 1529.
Litschig, S., Gasparini, F., Rueegg, D., Stoehr, N., Flor, P.J., Vranesic, I., Prezeau, L., Pin, J- P., Thomsen, C., Kuhn, R. ( 1999). CPCOOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. Mol. Pharmacd 55,453-46 1.
Lundberg, K.S., Shoemaker, D.D., Adams, M.W.W., Short, J.M., Sorge, J.A., Mathur, E.J. ( 199 1 ). High-fidelity amplification using a thermostable DNA polymerase isolated fiom Pyrococcus funosus. Gene 108, 1-6.
Macek, T.A., Schaffhauser, H., Conn, P.J. (1999). Activation of PKC disrupts presynaptic inhibition by group II and group 111 metabotropic glutamate receptors and uncouples the receptor fiom GTP-binding proteins. Ann. N. Y. Acad. Sci. 868,554-557.
Maiese, K., Greenberg, R., Boccone, L., Swiriduk, M. (1995). Activation of the metabotropic glutamate receptor is neuroprotective during nitnc oxide toxicity in primary hippocampal neurons of rats. Netrrosci. Lett. 21, 173- 176.
Malitschek, B., Schweizer, C., Keir, M., Heid, J., Froestl, W., Mosbacher, J., Kuhn, R., Henley, J., Joly, C., Pin, J-P., Kaupmann, K., Bettler, B. (1999). The N-terminal domain of gamma-arninobutyric acid (B) receptors is sufticient to speciQ agonist and antagonist binding. Mol. Pharmacol. 56,448-454.
Margolskee, R.F., McHendry-Rinde, B., Hom, R. (1993). Panning transfected cells for electrophysiological studies. Biotechniques 15,906-9 1 1.
Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., Nakanishi, S. (1991). Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765.
Mateos, J.M., Azkue, J., Sama, R., Kuhn, R., Grandes, P., Knopfel, T. (1998). Localization of the mGluR4a metabotropic glutamate receptor in rat cerebellar cortex. Histochem. Cell Biol. 109, 135- 139.
Matsunami, H., Buck, L.B. (1997). A multigene family encoding a diverse array of putative pheremone receptors in mammals. Ce11 90, 775-784.
Mineff, E., Valtschanoff, J. (1 999). Metabotropic glutamate recepton 2 and 3 are expressed by astmcytes in rat ventmbasal thalamus. Neurosci. Leti. 270,95998.
Mitchell, S.J., Silver, R.A. (2000). Glutamate spillover suppresses inhibition by activating presyneptic mGluRs. Nahrre 101,498-501.
Mody, N., Hemans, E., Nahorski, S.R., Challiss, R.A.J. (1999). Inhibition of Nlinked glycosylation of the human type la metabotropic glutamate receptor by tunicamycin: effects on cell-surface receptor expression and fùnction. Nwopharmacologv 38, 1485- 1492.
Murphy, S.N., Miller, R.J. (1988). A glutamate receptor regulates ca2+ mobilization in hippocarnpal neurons. Proc. Nd. Acad. Sci. U.S.A. 85, 8737-874 1.
Mutel, V., Adam, G., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Wichma~, J., Woltenng, T., Richards, J.G. ( 1998). Characterization of ( 2 ~ , 2 ' ~ , 3 ' ~ ) - 2 - ( 2 ' , 3 ~ - [ ~ ~ J- dicarboxycyclopropy1)glycine binding in rat brain. J. Neurochem. 71, 2558-2564.
Mutel, V., Ellis, G.J., Adam, G., Chaboz, S., Nilly, A., Messer, J., Bleuel, Z., Metzler, V., Malherbe, P., Schlaeger, E.J., Roughley, B.S., Faull, R.L., Richards, J.G. (2000). Characterization of [ ' ~ ~ ~ u i s ~ u a l a t e binding to recombinant rat metabobopic glutamate la and Sa receptors and to rat and human brain sections. J. Neurochem. 7S,2 590-260 1.
Nakajima, Y., Iwakabe, H., Akazawa, C., Nawa, H., Shigemoto, R., Mizuno, N., Nakanishi, S. (1993). Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-arnino-4-phosphonobutyrate. J. Biol. Chem. 268, 1 1868- 1 1873.
Nakanishi, S. (1992). Molecular diversity of glutamate receptors and implications for brain fùnction. Science 258,597-603.
Naples, M.A., Hampson, D.R. (2001). Phamacological profiles of the metabotropic glutamate receptor ligands ['HI L - A P ~ and ['HICPPG. Neziropharmacologv 40. 1 70- 1 77.
Nicoletti, F., Bruno, V., Copani, A., Casabona, G., Knopfel, T. (1996). Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders? Trent& Nadrosci. 19,267-27 1.
O'Hara, P.J., Sheppard, P.O., Thegerson, H., Venezia, D., Haldeman, B.A., McGrane, V., Houamed, KM., Thomsen, C., Gilbert, T.L., Mulvihill, E.R. (1993). The ligand-binding domain of metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neirron 11,4 1 -52.
Ohishi, H., Shigemoto, R., Nakanishi, S., Minino, N. (1993a). Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Nairoscie~ice 53, 1 009- 10 1 8.
Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N. (1993b). Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study. J. Cornp. Neurol. 335,252-266.
Ohishi, H. Akazawa, C., Shigemoto, R., Nakanishi, S., Mizuno, N. (1995a). Distributions of the mRNAo for L-2-ami~phophoi~)butyratese~itive metahopic glutamate receptors, mGluR4 and mGluR7, in the rat brain. J. Cornp. Neuroi. 360,555-570.
Ohishi, H., Nomura, S., Ding, Y-Q., Shigemoto, R., Wada, E., Kinoshita, A., Li, J-L., Neki, A., Nakanishi, S., Minino, N. (1 995b). Presynaptic localization of a metabotropic glutamate receptor, mGluR7, in the primary afferent neurons: an immunohistochemical study in the rat. Neurosci. Lett. 202, 85-88.
Okamoto, N., Hori, S., Akazawa, C., Hayashi, Y., Shigemoto, R., Mizuno, N., Nakanishi, S. (1994). Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J. Biol. Chem. 269, 1 23 1 - 1236.
Okamoto, T., Sekiyama, N., Otsu, M., Shimada, Y., Sato, A., Nakanishi, S., Jingami, H. (1 998). Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1. J. Bioi. Chem. 273, 13089- 13096.
Ottersen, O.P., Landsend, A.S. (1997). Organization of glutamate receptors at the synapse. E w . J. Nerrrosci. 9, 22 19-2224.
Ozawa, S., Kamiya, H., Tsuzuki, K. (1998). Glutamate receptors in the mammalian central nervous system. Prog. Neurobiol. 5458 1 -6 1 8.
Pagano, A., Ruegg, D., Litschig, S., Stoehr, N., Stierlin, C., H e i ~ c h , M., Floersheim, P., Prézeau, L., Carroll, F., Pin, J-P., Cambria, A., Vranesic, I., Flor, P.J., Gasparini, F., Kuhn, R. (2000). The non-competitive antagonists 2-methyl-6-(pheylethyny1)pyridine and 7- hydroxyiminocyclopropan[b]chromen- l a-carboxylic acid et hyl ester interact wi th overlapping binding pockets in the transmembrane region of group 1 metabotropic glutamate receptors. J. Biol. Chem. 27, 33750-33758.
Peltekova, V., Han, G., Soleymanlou, N., Hampson, D.R. (2000). Constraints on proper folding of the arnino tenninal domains of group iii metabotropic glutamate receptors. Mol. Brain Res. 76, 1 80- 1 90.
Petralia, R.S., Wang, Y-X., Niedzielski, A.S., Wenthold, R.J. (1996). The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Nerrroscience 71, 949-976.
Phillips, T., Makoff, A., Brown, S., Rees, S., Emson, P. (1997). Localization of mGluR4 protein in the rat cerebral cortex and hippocampus. NeuroReport 8,3349-3354.
Phillips, T., Makoff, A., Mumson, E., Mimrnack, M., Waldvogel, H., Faull, R., Rees, S., Emson, P. (1998). Immunohistochemical localization of mGluR7 protein in the rodent and human cerebellar cortex using subtype speci fic antibodies. Mo I. Bruin Res. 57, 1 32- 1 4 1.
Pin, J-P., Joly, C., Heinemann, S.F., Bockaert, J. (1994). Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. EMBO J. 13,342-348.
Pia, J-P., Duvoisin, R (1995). The metabotropic gluiamaîe recepbrs: structure a d funçtions. Neuropharmacology 34, 1 -26.
Pin, J-P., De Colle, C., Bessis, A-S., Acher, F. (1 999). New perspectives for the development of selective metabotropic glutamate receptor ligands. Eur. J. Pharmacol. 375,277-294.
Ray, K., Clapp, P., Goldsmith, P.K., Speigel, A.M. (1998). Identification of the sites of N- linked glycosylation on the human calcium receptor and assessment of their role in ce11 surface expression and signal transduction. J. Biol. Chem. 273,34558-34567.
Risso-Bradley, S., Levey, A.I., Hasch, S. M., Conn, P.J. ( 1 996). Immunocytochemical localization of group III metabotropic glutamate receptors in the hippocampus with subtype- specific antibodies. J. Neurosci. 16.20442056.
Risso-Bradley, S., Standaert, D.G., Rhodes, K.J., Rees, H.D., Testa, C.M., Levey, A.I., Conn, P.J. (1999). lmmunohistochemical localization of subtype 4a metabotropic glutamate recepton in the rat and mouse basal ganglia. J. Comp. Neurol. 407,33-46.
Romano, C., Yang, W-L., O'Malley, K.L. (1996). Metabotropic glutamate receptor 5 is a disulfide-linked dimer. J. Biol. Chem. 271,286 12-286 16.
Sambrook, J., Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual Third Edition, Cold Spnng Harbor Laboratory Press.
Saugstad, S.A., Kinzie, J.M., Mulvihill, E.R., Segerson, T.P., Westbrook, G.L. (1994). Cloning and expression of a new member of the L-2-amino-4-phosphonobutyric acid- sensitive class of metabotropic glutamate receptors. Mol. Pharmacol. 45,367-372.
Saugstad, S.A., Kinzie, J.M., Shinohara, M.M., Segerson, T.P., Westbrook, G.L. (1997). Cloning and expression of rat metabotropic glutamate receptor 8 reveals a distinct pharmacological profile. Mol. Pharmacol. 51, 1 19- 125.
Schaflhauser, H., Richards, J.G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Stadler, H., Woltering, T., Mutel, V. (1998). In vitro bindin characteristics of a F new selective group 11 metabotropic glutamate receptor radioligand, [ HlLY354740, in rat brain. Mol. Pharmacol. 53,228-233.
Schweitzer, C., Kratzeisen, C., Adam, G., Lundstrom, K., Malherbe, P., Ohresser, S., Stedler, H., Wichrnann, S., Woltering, T., Mutel, V. (2000). Characterization of [ 3 ~ ] - ~ ~ 3 5 4 7 4 0 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors. Neuropharmacologv 39. 1 700- 1 706.
Schoepp, D.D.. Jane, D.E., Monn, J.A. (1999). Phannacological agents acting at subtypes of metabotropic glutamate receptors. Neurophamacologv 38, 143 1 - 1476.
Semyanov, A., Kullmann, D.M. (2000). Modulation of GABAergic signaling among intemeurons by metabotropic glutamate receptors. Neuron 25,663-672.
Shigemoto, R., Nakanishi, S., Miaino, N. (1992). Distribution of the mRNA f~ a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. Neurosci. Lett. 163,53957.
Shigemoto, R., Kinoshita, A., Wade, E., Nimura, S., Ohishi, H., Takada, M., Flor, P.J., Neki, A., Abe, T., Nakanishi, S., Mizuno, N. (1997). Differential presynaptic localization of metabotropic glutamate receptor subtypes in rat hippocampus. J. Neurosci. 17,7503-7522.
Sladeczek, F., Pin, J-P.,Recasens, M., Bockaert, J., Weiss, S. (1985). Glutamate stimulates inositol phosphate formation in striatal neurons. Nature 31 7,7 17-7 1 8.
Stowell, J.N., Craig, A.M. ( 1999). Axoddendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains. Neuron 22,525-536.
Sugiyama, H., Ito, I., Hirono, C. (1987). A new type of glutamate receptot linked to inositol phospholipid metabolism. Nahtre 325,53 1-533.
Takahashi, K., Tsuchida, K., Tanabe, Y., Masu, M., Nakanishi, S. (1993). Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination. J , Biol. Chem. 268, 1934 1 - 19345.
Tang, E., Yip, P.K., Chapman, A.G., Jane, D.E., Meldrum, B.S. (1997). Prolonged anticonwlsant action of glutamate metabotropic receptor agonists in inferior colliculus of genetically epilepsy-prone rats. Eur. J . Pharmacol. 327, 10% 1 1 5.
Thomas, N.K., Wright, R.A., Howson, P.A., Kingston, A.E., Schoepp, D.D., Sane, D.E. (2001). (S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary affetent tenninals in the neonatal rat spinal cord. Neuropharmacology 40.3 1 1-3 1 8.
Thomsen, C., Pekhletski, R., Haldeman, B., Gilbert, T.A., O'Hara, P., Harnpson. D.R. (1997). Cloning and characterization of a metabotropic glutamate receptor, mGluR4b. Neuropharmacology 36,2 1-30.
Thomsen, C., Hampson, D.R. ( 1999). Contribution of metabotropic glutamate receptor mGluR4 to L-2-[3 mi no-4-phosphonobutyrate binding in mouse brain. J. Neurochem. 72, 835-840.
Toms, N.J., Jane, D.E., Kemp, M.C., Bedingtield, J.S., Roberts, P.J. (1 996). The effects of (RS)-a-cyclopropyl-4-phosphonopheny1glycine ((RS)-CPPG), a potent and selective metabotropic glutamate receptor antagonist. Br. J. PItarniacol. l W , M 1-854.
Tones, M.A., Bendali, N., Flor, P.J., Knopfel, T., Kuhn, R. (1995). The agonist selectivity of a class III metabotropic glutamate receptor, human mGluR4a, is detennined by the N-terminal extracellular domain. Neuroreport 7, 1 1 7- 120.
Tsuji, Y., Shimada, Y., Takeshita, T., Kajimura, N., Nomura, S., Sekiyama, N., Otomo, J., Usukwa, J., Nakanishi, S., Jingami, H. (2000). CIyptio dimer interface end doniain organization of the extracellular region of metabotropic glutamate receptor subtype 1. J. Biol. Chem. 275,28 144-28 15 1.
Tu, J.C., Xiao, B., Yuan, J.P., Lanahan, A.A., Leoffert, K., Li, M., Linden, D.J., Worley, P.F. (1998). Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Narron 21,717-726.
Ueda, Y., Iwakabe, H., Masu, M., Suniki, M., Nakanishi, S. (1997). The mGluR6 S'upstream transgene sequence directs a cell-specific and developmentally regulated expression in retinal rod and ON-type cone bipolar cells. J. Necrrosci. I7,30 14-3023.
Vignes, M., Guiramand, J., Sassetti, I., Recasens, M. (1993). Effect of thiol reagents on phosphoinositide hydrolysis in rat brain synaptoneurosomes. Eur. J. Neurosci. 5, 327-334.
Vignes, M., Clarke, R.J., Davies, C.H., Chambers, A., Jane, D.E., Watkins, J.C., Collingridge, G.L. (1995). Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocmpal slices. Neuropharmacology 34,973-982.
Wroblewska, B., Wroblewski, J.T., Pshenichkin, S., Surin, A., Sullivan, S.E., Neale, J.H. (1997). N-Acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J. Neurorochem. 69, 1 74- 18 1.
Wright, R.A., Arnold, M.B., Wheeler, W.J., Ornstein, P.L., Schoepp, D.D. (2000). Binding of ['H](~s, 1 'S,2 ~S)-2-(9-xanthylmethyl)-2-(2~-c~boxycyclopropyl)g1ycine (["ILY 34 1495) to ceIl membranes expressing recombinant human group II1 metabotropic glutamate receptor subtypes. Narcnyn Schmiedebergs Arch. Pharmacol. 362,546-554.
Wu, S., Wright, R.A., Rockey, P.K., Burgett, S.G., Arnold, J.S., Rosteck, P.R. Jr., Johnson, B.G., Schoepp, D.D., Belagaje, R.M. (1998). Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells. Mol. Brain Res. 53.88-97.
Appendix 2. Sensitivity of ['HJCPPG binding to ehangcs in pH. Ce11 membranes
expressing mGluR8 were prepared simultaneously under difierent pH conditions. Each data
point represen ts the mean * SEM of three independent experiments perfomed in tri plicate.
Maximal binding of [)H]CPPG was observed at pH 8.0. Accordingly, all membrane
preparations and subsequent binding experiments were perfomed at pH 8.0.
Appeaàù 3 Anaiysb of bidiag nruh
Specific radioactivitv calculation
Specific activity (SA) of radioligand (Cilmmol) was converted to cpm/pmol as follows:
X (cpmlpmol) = SA (Cilmmol) x 2.22~10'~ dpm/Ci x 1û9 mmollpmol x E (cpmldpm)
where E = efficiency of counter
Cheng-Rusoff eauation (conversion of IC, to Ki)
L I
where K, = affinity of radioligand for the receptor
