Ligand binding profile of the rat metabotropic glutamate receptor mGluR3 expressed in a transfected cell line

Abstract

A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [3H]glutamate as radioligand. The rank order of affinity was L-glutamate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) > L(+)-2-amino-3-phosphonopropionic acid (L-AP3) > quisqualic acid > L(+)-2-amino-4-phosphonobutyric acid (L-AP4) > ibotenic acid. The active enantiomers of several phenylglycines displayed Ki values of 300 to 400 microM. The nonactive enantiomers and the standard ionotropic glutamate receptor ligands N-methyl-D-aspartic acid (NMDA), (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid only weakly displaced [3H]glutamate. In this cell line, L-glutamate and (2S,3S,4S)-alpha-(Carboxycyclopropyl)-glycine (L-CCG-I) reduced cAMP levels in a dose-dependent manner. The sensitivity of this system and its easy applicability make it feasible to envisage ligand binding assays on cell lines expressing cloned receptors as useful screening tools to discover and characterize new and specific agonists and antagonists.

Characterization of [3H]‐(2S,2′R,3′R)‐2‐(2′,3′‐dicarboxy‐ cyclopropyl)glycine ([3H]‐DCG IV) binding to metabotropic mGlu2 receptor‐transfected cell membranes

“…Furthermore, binding was inhibited by GTP γ S, confirming that, in mGlu 2 receptor‐transfected CHO cells, [ 3 H]‐DCG IV binding is to the G‐protein‐coupled mGlu 2 receptor. In contrast, Laurie et al (1995) failed to obtained an effect of GTP γ S on [ 3 H]‐glutamate binding to membranes from mGlu 3 receptor‐transfected HEK cells, the lack of effect of GTP γ S possibly being due to the non‐selectivity of [ 3 H]‐glutamate binding. As the inhibition evoked by GTP γ S in our study was incomplete, further experiments are necessary in order to determine if GTP γ S causes a shift in affinity of [ 3 H]‐DCG IV.…”

“…In recent years, a number of groups have used [ 3 H]‐glutamate to study the radioligand binding properties of group II mGlu receptors in a variety of preparations. For example, Laurie et al (1995) characterized the binding of [ 3 H]‐glutamate to membranes of mGlu 3 receptor‐transfected HEK cells. In addition, [ 3 H]‐glutamate, in the presence of ionotropic glutamate receptor agonists, has been used to investigate native group II receptors in rat brain membranes (Schoepp & True, 1992; Wright et al , 1994).…”

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor‐stimulated phosphoinositide turnover

“…Although heterologous expression of specific mGluR subtypes can in theory provide almost unlimited supplies of material for pharmacological characterization of putative mGluR agonists/antagonists using radioligand binding methods, few studies have adopted this approach (Thomsen et al, 1993;Laurie et al, 1995) (Schoepp et al, 1991;Manzoni et al, 1992;Jones & Roberts, 1993;Challiss et al, 1994b). In contrast, IR, 3S-ACPD was essentially without effect at concentrations up to 300 gM.…”

“…In parallel with the cloning and functional characterization of the eight mGluRs which presently constitute the mGluR family (Nakanishi, 1994;Pin & Duvoisin, 1995) has been the synthesis of an increasing array of molecules which exhibit selective agonist or antagonist properties at this class of receptor (Hayashi et al, 1992;1994;Schoepp & Conn, 1993;Birse et al, 1994;Pin & Duvoisin, 1995). Although heterologous expression of specific mGluR subtypes can in theory provide almost unlimited supplies of material for pharmacological characterization of putative mGluR agonists/antagonists using radioligand binding methods, few studies have adopted this approach (Thomsen et al, 1993;Laurie et al, 1995), probably because radiolabelled L-[3H]-glutamate is the only ligand available at present. Thus, the vast majority of pharmacological studies have used the activation or inhibition of particular signal transduction pathways as an index of receptor activation or blockade.…”

Stimulatory effects of the putative metabotropic glutamate receptor antagonist L‐AP3 on phosphoinositide turnover in neonatal rat cerebral cortex

“…It should be noted also that in studies where L-[3H]-glutamate binding has been assessed with membranes prepared from clonal cells expressing specific mGluRs, L-AP3 has been shown to displace specfic [3Hl-glutamate binding with Ki values of 298 and 125 giM for mGluRla and mGluR3 (Laurie et al, 1995) respectively, suggesting that L-AP3 interacts directly with the mGluR to affect agonist binding.…”