[³H]-LY341495 as a novel antagonist radioligand for group II metabotropic glutamate (mGlu) receptors: characterization of binding to membranes of mGlu receptor subtype expressing cells

Abstract

Metabotropic glutamate (mGlu) receptors are a family of eight known subtypes termed mGlu1-8. Currently, few ligands are available to study the pharmacology of mGlu receptor subtypes. In functional assays, we previously described LY341495 as a highly potent and selective mGlu2 and mGlu3 receptor antagonist. In this study, radiolabeled [³H]-LY341495 was used to investigate the characteristics of receptor binding to membranes from cells expressing human mGlu receptor subtypes. Using membranes from cells expressing human mGlu2 and mGlu3 receptors, [³H]-LY341495 (1 nM) specific binding was>90% of total binding. At an approximate K_D concentration for [³H]-LY341495 binding to human mGlu2 and mGlu3 receptors (1 nM), no appreciable specific binding of [³H-]LY341495 was found in membranes of cells expressing human mGlu1a, mGlu5a, mGlu4a, mGlu6, or mGlu7a receptors. However, modest (∼20% of mGlu2/3) specific [³H]-LY341495 (1 nM) binding was observed in human mGlu8 expressing cells. [³H]-LY341495 bound to membranes expressing human mGlu2 and mGlu3 receptors in a reversible and saturable manner with relatively high affinities (B_max 20.5±5.4 and 32.0±7.0 pmol/mg protein; and K_D=1.67±0.20 and 0.75±0.43 nM, respectively). The pharmacology of [³H]-LY341495 binding in mGlu2 and mGlu3 expressing cells was consistent with that previously described for LY341495 in functional assays. [³H]-LY341495 binding provides a useful way to further investigate regulation of receptor expression and pharmacological properties of mGlu2 and mGlu3 receptor subtypes in recombinant systems.

Introduction

Glutamate binds to two major types of receptor proteins termed ionotropic glutamate (iGlu) receptors and metabotropic glutamate (mGlu) receptors (Nakanishi, 1992). Ionotropic glutamate receptors are homomeric or heteromeric ligand-gated cation-specific ion channels (Hollman and Heinemann, 1994). Ionotropic receptors are defined by their pharmacology and molecular properties, and are selectively activated by the agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoazolepropionic acid (AMPA), and kainate. In contrast, mGlu receptor subtypes are coupled by G-proteins to effector systems to generate second messengers within the cell. Initially, mGlu receptors were pharmacologically distinguished by their insensitivity to NMDA, AMPA or kainate, but their activation by (±)trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (or its 1S,3R-isomer), the first agonist shown to be selective for mGlu receptors as a class (Palmer et al., 1989, Desai and Conn, 1990, Schoepp et al., 1991). The metabotropic glutamate receptor family has been separated into three groups based on sequence homology, second messenger transduction mechanisms, and pharmacological properties of each subtype (Nakanishi, 1992). The discovery of multiple cloned mGlu receptors has prompted the search for newer compounds with higher potency and selectivity than earlier compounds (such as 1S,3R-ACPD) which are only μM potent and/or act across multiple mGlu receptor subtypes (Schoepp and Conn, 1993, Pin and Duvoisin, 1995).

Group I mGlu receptors (including mGlu1a and mGlu5a receptors and their splice variants) are positively coupled via Gq to the phosphoinositide hydrolysis signal transduction pathway and are selectively activated by quisqualate and 3,5-dihydroxyphenyl glycine (DHPG) (Schoepp et al., 1994, Gereau and Conn, 1995). Both group II and group III mGlu receptors are negatively coupled to adenyl cyclase via G_i, thereby reducing stimulated cAMP levels in non-neuronal cell lines expressing these receptors. Group III mGlu receptors include mGlu4, mGlu6, mGlu7 and mGlu8 receptor subtypes and are all activated by l-2-amino-4-phosphonobutyrate (l-AP4). In contrast, group II mGlu receptors (mGlu2 and mGlu3 receptor subtypes) are relatively insensitive to l-AP4, but are potently and selectively activated by compounds such as is (2S,1′R, 2′R, 3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine, (DCG-IV), (Ohfune et al., 1993, Hayashi et al., 1993), 2R,4R-aminopyrrolidine-2,4-dicarboxylate, (2R,4R-APDC), (Monn et al., 1996, Schoepp et al., 1996), and LY354740 (Monn et al., 1997, Schoepp et al., 1997).

Until very recently, the lack of potent and selective mGlu receptor agonists and antagonists have made it difficult to devise receptor binding assays for studying mGlu receptor expression and pharmacology. l-[³H]Glutamate has been reported to label metabotropic glutamate receptors using autoradiographic techniques in the rat brain (Cha et al., 1990), rat forebrain homogenates by centrifugation techniques (Schoepp and True, 1992, Wright et al., 1994), and the rat mGlu3 receptor subtype expressed in a transfected cell line (Laurie et al., 1995). However, glutamate is a non-subtype selective receptor agonist for both ionotropic and metabotropic receptors, thus in hetereogenous receptor systems specific binding to mGlu receptors has to be defined by the amount of [³H]glutamate binding which was displaced by mGlu receptor selective compounds such as 1S,3R-ACPD. Furthermore, the relatively low affinity of glutamate makes it difficult to use rapid filtration techniques for separation of bound and free ligand.

Recent reports have described the binding of nM potent and selective mGlu2/3 agonists. [³H]-LY354740 was reported to bind with high affinity to rat brain membranes (Schaffhauser et al., 1998) and [³H]-DCG-IV binds to mGlu2 receptor transfected CHO cells (Cartmell et al., 1998). Nevertheless, there is a still a lack of mGlu receptor ligands, particularly high affinity subtype selective antagonists, and this has hampered investigation of mGlu receptor functions in situ. Recently, we reported that 2S-2-amino-2-(1S,2S-2-carboxycyclopropy-1-yl)-3-(xanth-9-yl)propionic acid, (LY341495) is a nM potent competitive antagonist of functional mGlu receptor agonist responses in cells expressing human mGlu2 and mGlu3 receptors (Kingston et al., 1998). This study investigated the [³H]-LY341495 binding properties and pharmacology in membranes of non-neuronal cells expressing cloned human mGlu receptor subtypes.