Research reportCharacterization of mGluR5R, a novel, metabotropic glutamate receptor 5-related gene
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.
References (45)
- et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Molecular determinants of metabotropic glutamate receptor 1B trafficking
Mol. Cell. Neurosci.
(2001) - et al.
Metabotropic glutamate 1α and adenosine A1 receptors assemble into functionally interacting complexes
J. Biol. Chem.
(2001) Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking
Trends Pharmacol. Sci.
(2001)- et al.
Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons
J. Biol. Chem.
(2001) - et al.
Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor
J. Biol. Chem.
(1997) - et al.
Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor
J. Biol. Chem.
(1999) - et al.
Cloning and functional expression of alternative spliced variants of the human metabotropic glutamate receptor 8
Brain Res. Mol. Brain Res.
(1999) - et al.
A variant of metabotropic glutamate receptor subtype 5: an evolutionally conserved insertion with no termination codon
Biochem. Biophys. Res. Commun.
(1993) - et al.
The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins
Neuron
(1993)
Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1
J. Biol. Chem.
Metabotropic glutamate receptors: a structural view point
Pharm. Acta Helv.
Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors
Brain Res. Mol. Brain Res.
The metabotropic glutamate receptors: structure and functions
Neuropharmacology
Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction
J. Biol. Chem.
Cys-140 is critical for metabotropic glutamate receptor-1 dimerization
J. Biol. Chem.
Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca2+ receptor critical for dimerization. Implications for function of monomeric Ca2+ receptor
J. Biol. Chem.
Homer 1b regulates the trafficking of group I metabotropic glutamate receptors
J. Biol. Chem.
Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers
J. Biol. Chem.
Metabotropic glutamate receptor 5 is a disulfide-linked dimer
J. Biol. Chem.
Characterization of γ-aminobutyric acid receptor GABAB(1e), a GABAB(1) splice variant encoding a truncated receptor
J. Biol. Chem.
Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination
J. Biol. Chem.
Cited by (5)
Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor
2004, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Thus, the pattern for the site of truncation for secretion and ligand binding appears to be similar for Group II and Group III receptors. In contrast, it has been reported that with the Group I mGluRs, mGluR1 and mGluR5, and the calcium-sensing receptor, truncation at or near the junction of the extracellular domain and the first transmembrane domain does result in secreted receptors [20–23]. In the case of mGluR1 and mGluR5, the secreted receptors are capable of binding the Group I agonist [3H]quisqualic acid.
Gene structure of the human metabotropic glutamate receptor 5 and functional analysis of its multiple promoters in neuroblastoma and astroglioma cells
2003, Journal of Biological ChemistryCitation Excerpt :Only a very weak signal was detected in spinal cord and corpus callosum (Fig. 2). An additional band of ∼2.5 kbp was observed in most of the brain areas analyzed, which corresponds to a novel metabotropic glutamate receptor 5-related gene (27). The genomic structures of rat and mouse Grm5 were also mapped (data not shown) by using publicly available mRNA and genomic sequences, as well as the 5′-end organization of the rat gene reported previously (10).
Adenosine 2A receptor: A crucial neuromodulator with bidirectional effect in neuroinflammation and brain injury
2011, Reviews in the NeurosciencesVenus fly trap domain of mGluR1 functions as a dominant negative against group I mGluR signaling
2010, Journal of NeurophysiologyNeurochemical aspects of excitotoxicity
2008, Neurochemical Aspects of Excitotoxicity