Pharmacological profiles of the metabotropic glutamate receptor ligands [3H]L-AP4 and [3H]CPPG

doi:10.1016/S0028-3908(00)00128-3

Neuropharmacology

Volume 40, Issue 2, 2001, Pages 170-177

https://doi.org/10.1016/S0028-3908(00)00128-3 Get rights and content

Abstract

Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors that are expressed in the central and peripheral nervous systems. The purpose of this study was to compare the ligand binding selectivity profiles of the mGluR agonist [³H]L-AP4 and the novel radiolabeled phenylglycine antagonist [³H]CPPG at all eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. At a concentration of 30 nM [³H]L-AP4, no specific binding was detected in membranes expressing the group I receptors mGluR1a or mGluR5a, or in membranes expressing the group II mGluRs, mGluR2 and mGluR3. Among the group III mGluRs, specific [³H]L-AP4 binding was detected in cells expressing mGluR4a and mGluR8a but not in cells expressing mGluR6 or mGluR7a. The binding of [³H]CPPG showed an exceptional pattern of selectivity amongst the mGluR subtypes; at a concentration of 20 nM [³H]CPPG, a high level of specific binding was seen in membranes containing mGluR8a but not in any of the other mGluR subtypes. The affinity constant (K_D) calculated for [³H]CPPG binding to mGluR8a was 183 nM. In competition experiments, the phosphono-substituted phenylglycine congeners including MPPG, (RS)-PPG, and unlabeled CPPG were the most potent inhibitors of [³H]CPPG binding while non-phosphonated compounds such as l-glutamate and MCPG were substantially less potent. These results demonstrate that [³H]L-AP4 and [³H]CPPG can be used as probes to selectively label group III mGluRs and that CPPG and related phenylglycine derivatives are useful for studying differences in the ligand recognition sites of highly homologous mGluRs.

Introduction

Most classes of neurotransmitter receptors expressed in the nervous system are composed of multiple homologous receptor subtypes. In many cases receptor subtypes possess subtle but potentially important pharmacological differences that can be assessed using suitable radiolabeled probes with sufficiently high affinity and subtype selectivity. Until recently, the pharmacological characterization of G-protein coupled metabotropic glutamate receptors (mGluRs) has been hindered by the lack of such probes. The mGluR family of receptors has been divided into three subgroups based on sequence homology, signal transduction properties, and pharmacological profiles (Pin and Duvoisin, 1995, Conn and Pin, 1997). A problem with many of the radiolabeled and unlabeled mGluR ligands is that in most cases the complete pharmacological profiles with all eight cloned mGluRs have not been determined. Thus to obtain a comprehensive picture of receptor selectivity it has become increasingly imperative to assess ligands across the spectrum of mGluR subtypes.

Recently, several radiolabeled ligands have become available for studying mGluRs (see Pin et al., 1999, Schoepp et al., 1999 for reviews). [³H]Quisqualic acid has been used to examine the pharmacological properties of the group I mGluR, mGluR1 (Okamoto et al., 1998). The agonists [³H]DCG-IV (Cartmell et al., 1998) and [³H]LY354740 (Schaffhauser et al., 1998), and the antagonist [³H]LY341495 (Johnson et al., 1999) were used to label the group II mGluRs which include mGluR2 and mGluR3. For the group III mGluRs (mGluR4, 6, 7, and 8), [³H]L-AP4 has been utilized to assess the pharmacological properties of mGluR4 (Eriksen and Thomsen, 1995, Han and Hampson, 1999, Hampson et al., 1999) and mGluR8 (Peltekova et al., 2000). This radiolabeled agonist has relatively high affinity for both mGluR4 and mGluR8 (K_D=400–800 nM) but its ability to label other mGluRs has not been reported.

The organic synthesis of a series of phenylglycine derivatives has generated a variety of more selective mGluRs agonists and antagonists. Three phenylglycine derivatives, including the competitive antagonists (RS)-α-methyl-4-phosphonophenylglycine (MPPG, Jane et al., 1995, Bushell et al., 1996) and (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG, Toms et al., 1996), and the agonist (R,S)-4-phosphonophenylglycine ((RS)-PPG) appear to be particularly potent at group III mGluRs. (RS)-PPG has been reported to have antiepileptic properties and is neuroprotective against quinolinic acid-induced excitotoxicity (Gasparini et al., 1999, Henrich-Noack et al., 2000). In the present study, we have investigated the binding properties of [³H]L-AP4 and a new radiolabeled phenylglycine antagonist, [³H]CPPG at all eight mGluR subtypes. Our results demonstrate that, at the concentrations used, the two compounds label different but overlapping subsets of group III mGluRs.

Section snippets

Materials

[³H]L-AP4 (specific activity=52 Ci/mmol) and [³H]CPPG (specific activity=16 Ci/mmol) and all amino acids and amino acid analogues were from Tocris Cookson Inc.

cDNA expression constructs

All expression constructs for the rat mGluRs were assembled in the pcDNA3 or pcDNA3.1 expression vectors (InVitrogen Inc). The cDNAs were purified using the Qiagen miniprep kit. The cDNAs coding for rat mGluRs 1a, 2, 3, 4a, 5a, 6, 7a, and 8a were transiently transfected into human embryonic kidney (HEK)-293-TSA-201 cells as described

Expression of recombinant mGluRs in HEK-293 cells

Membranes prepared from HEK-293 cells transfected with different mGluR subtypes were subjected to immunoblotting using receptor-specific antibodies. For each receptor, the appropriate molecular weight bands were detected in mGluR-transfected cells but not in mock-transfected cells (Fig. 1). Specific recognition of each receptor was observed with carboxy terminal domain antibodies to mGluR1a, mGluR5, mGluR6, and mGluR7a, and as expected, the carboxyl terminal anti-mGluR2/3 antibody labeled both

Discussion

In previous studies, we showed that [³H]L-AP4 bound with high affinity (K_D=400–800 nM) to mGluR4a and mGluR8a (Han and Hampson, 1999, Hampson et al., 1999, Peltekova et al., 2000). This analysis has now been extended to other mGluRs. Because of the selectivity of L-AP4 to the group III mGluRs, we did not expect to see appreciable binding of this radioligand in membranes from cells expressing group I or group II mGluRs. Moreover, based on the reported low affinity of agonist activation of mGluR7

Acknowledgements

We thank Dr M.C. Kemp for helpful suggestions, Geoffrey Hornby, Xi-Ping Huang, and Dr V. Peltekova for subcloning the cDNAs. We appreciate the generosity of Drs S. Nakanishi and C. Romano for providing the cDNAs, and Drs P.J. Conn, S. Risso-Bradley, and R. Shigemoto for providing antibodies. This work was supported by grants from the Canadian Institutes of Heath Research and the Heart and Stroke Foundation of Ontario.

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Pharmacological profiles of the metabotropic glutamate receptor ligands [3H]L-AP4 and [3H]CPPG

Abstract

Introduction

Section snippets

Materials

cDNA expression constructs

Expression of recombinant mGluRs in HEK-293 cells

Discussion

Acknowledgements

Neuropharmacology

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Neuropharmcology

Neuropharmacology

Neuropharmacology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Brain Res.

Eur. J. Pharmacol.

Pharmacological profiles of the metabotropic glutamate receptor ligands [³H]L-AP4 and [³H]CPPG