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 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
National Library 191 0fCa"ada B i b J i a T nationale du Cana
The author has granteci a non- exclusive licence aiîowirig the National Library of Canada to reproduce, loan, distribute or seU copies of this thesis in microforni, paper or electronic formats.
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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
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)-
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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