Research report
Characterization of mGluR5R, a novel, metabotropic glutamate receptor 5-related gene

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Abstract

We report here the isolation of a novel gene termed mGluR5R (mGluR5-related). The N-terminus of mGluR5R is highly similar to the extracellular domain of metabotropic glutamate receptor 5 (mGluR5) whereas the C-terminus bears similarity to the testis-specific gene, RNF18. mGluR5R is expressed in the human CNS in a coordinate fashion with mGluR5. Although the sequence suggests that mGluR5R may be a secreted glutamate binding protein, we found that when expressed in HEK293 cells it was membrane associated and not secreted. Furthermore, mGluR5R was incapable of binding the metabotropic glutamate receptor class I selective agonist, quisqualate. Although mGluR5R could not form disulfide-mediated covalent homodimers, it was able to form a homomeric complex, presumably through noncovalent interactions. mGluR5R also formed noncovalent heteromeric associations with an engineered construct of the extracellular domain of mGluR5 as well as with full-length mGluR5 and mGluR1α. The ability of mGluR5R to associate with mGluR1α and mGluR5 suggests that it may be a modulator of class I metabotropic glutamate receptor function.

Introduction

Glutamate, the major excitatory neurotransmitter in the brain, acts through multiple independent receptors. These receptors fall into two classes, the ligand gated ionotropic receptors, and the seven-transmembrane G protein coupled metabotropic receptors (GPCRs). The metabotropic receptors, of which eight independent genes have been identified, are further subdivided into three distinct classes based on sequence similarity, pharmacology, and effector systems to which they are coupled [25]. Metabotropic glutamate receptors (mGluRs) themselves are part of a larger family of GPCRs termed Family 3 [4]. Family 3 GPCRs, along with the metabotropic glutamate receptors, include the γ-aminobutyric acid type B (GABAB), calcium sensing (CaR), and putative pheromone receptors, and are distinguished by large extracellular domains (ECDs) through which they bind their cognate ligands. These ECDs bear structural homology to the soluble, amino acid binding, periplasmic binding proteins of bacteria [21].

Recently, several splice variants and engineered constructs of Family 3 GPCRs have been described that encode truncated receptors [13], [18], [22], [24], [25], [35], [39], [40], [41], [45]. These truncated versions are generally predicted to be secreted proteins that may, and in some cases do, bind cognate ligand. Secreted receptor ligand binding domains have been shown to have important functional consequences in other systems [11], [37]. However, the function of secreted family 3 GPCR ligand binding domains remains to be determined.

In the case of GABAB1, the truncated receptor, GABAB(1e), physically associates with GABAB(2) and, in so doing, inhibits the formation of GABAB(2)–GABAB(1a) heterodimers [35]. An engineered construct of the ECD of mGluR5 has also been shown to heterodimerize with its full-length version [33], [34] whereas ECDs of mGluR1 and mGluR4 form homodimers, respectively [13], [22]. These associations are presumably mediated through covalent interactions between cysteine residues but also may occur through noncovalent interactions [13], [22], [33].

Homo- and heteromultimerization of GPCRs has received considerable attention in recent years. These associations have, in some instances, been shown to affect receptor pharmacology and function [2], [8], [12], [14], [15], [31], [32]. For example, physical association of CaR with mGluR1α and mGluR5 was found to affect receptor trafficking and internalization [10], whereas angiotensin II (ATII) and bradykinin receptor heterodimers were associated with preeclampsia in pregnancy [1].

Here we report the isolation of a novel clone encoding a 369-amino-acid protein highly homologous to the N-terminal region of mGluR5 that we have termed mGluR5R (mGluR5-Related). mGluR5 and mGluR5R are 98% identical within their N-terminal 303 amino acids after which they are completely divergent. mGluR5R was expressed in the human central nervous system in a pattern coincident with mGluR5. Although it was not competent to bind the mGluR class I selective ligand, quisqualate, when expressed in a heterologous system, mGluR5R protein did form noncovalent homomers and heteromers with mGluR1α and mGluR5.

Section snippets

Clone isolation and analysis of genomic structure

A cDNA library was constructed from human whole brain polyA+ RNA using the Superscript plasmid system for cDNA Synthesis and Plasmid Cloning Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The plasmid library was mass-plated and individual clones were randomly chosen and sequenced. The 7TM database of GPCR sequences was obtained from the web (http://www.gpcr.org/7tm) and curated. A database of sequences of all clones isolated from the library was searched for GPCRs by

Isolation of mGluR5R

GPCRs of neurological interest were identified by screening randomly sequenced, human brain-derived, cDNA clones for homology to known GPCRs by tblastn [3] analysis (see Materials and methods). One clone isolated shared significant homology to mGluR5 and was designated mGluR5R (mGluR5-related). The first 914 nucleotides of mGluR5R were 98% identical to nucleotides 148–1061 of human mGluR5 (GenBank accession no. D28538; Fig. 1A) [9]. The mGluR5R cDNA was predicted to encode a 369-amino-acid

Discussion

We report here the isolation of a novel mGluR5-related gene product we termed mGluR5R. Recent reports have demonstrated that splice variants of family 3 GPCRs encoding only the ECDs are not uncommon [18], [25], [35], [39], [40], [41], [45]. A general feature of these variants is that they are identical to their full-length receptors up to the point where they possess a novel exon resulting in a frame shift and premature termination of the transcript. In contrast, there are nine amino acid

Acknowledgments

The authors wish to thank John Dunlop, Paul Yaworsky and Christopher Miller for critical reading of the manuscript and Karen Marquis, Dianne Kowal, Mark Pausch and Richard Konz for technical assistance, reagents, and invaluable advice.

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