Membrane Topology of a Metabotropic Glutamate Receptor

Abstract

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsinlike GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide stong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.Glutamate is the primary excitatory neurotransmitter of the central nervous system and activates ligand-gated ion channels or ionotropic glutamate receptors (iGluRs) and G-protein-coupled metabotropic glutamate receptors (mGluRs). 1The mGluRs modulate synaptic transmission and neuronal excitability throughout the peripheral and central nervous system. They have been classified into three groups based on sequence homology, pharmacology, and signal transduction coupling. Group I mGluRs, consisting of mGluR1 and mGluR5, uniformly couple to phospholipase C, while group II and III mGluRs, composed of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8, inhibit adenylyl cyclase activity in heterologous expression systems (1, 2).Since their cloning, the structure-function relationships of mGluRs have garnered extensive interest. Relatively recent classification of G-protein-coupled receptors (GPCRs) has categorized mGluRs as family C GPCRs characterized structurally by a large N-terminal extracellular domain and consisting of mGluRs, GABA B receptors, Ca 2ϩ -sensing receptors, and certain pheromone receptors. The large N-terminal extracellular domain of mGluRs exhibits distant homology to bacterial amino acid periplasmic binding proteins, suggesting the domain is responsible for ligand binding (3). Structural modeling of mGluRs based on this homology accurately predicts important residues for ligand binding (4). Exchange of this domain among mGluRs also endows specific ligand binding properties (5-7). Recently, crystal structure determination of the N-terminal domain with bound ligand has definitively confirmed its role in ligand binding and conformational changes of the domain with binding have made several predictions for potential mechanisms for receptor activation (8).While the structure of the ligand binding N-terminal domain has been clearly resolved, little data exists on the structure and membrane topology of the subsequent transmembrane domains. The complete structure of the canonical GPCR, rhodopsin, has recently been solved and confirms a long line of studies delineating seven membrane spanning ␣-helices (9 -12). Based...

Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization

“…Specifically, N-linked glycosylation would be sensitive to Endo H digestion, whereas Endo H resistance suggests a protein has been trafficked at least to the cis/medialGolgi where PNGase F can remove all N-linked glycans. mGluR5 has six putative N-glycosylation sites, although only one (Asn-444) appears to be utilized (49). By treating plasma membrane preparations with both enzymes and comparing the resulting immunoblots with non-treated protein, we observed an mGluR5 mobility shift akin to what was previously reported following PNGase F but not Endo H treatment (Fig.…”

Night blindness-associated mutations in the ligand-binding, cysteine-rich, and intracellular domains of the metabotropic glutamate receptor 6 abolish protein trafficking

“…All mGluR family members possess a large, bilobed, extracellular N-terminal domain containing the glutamate binding site, which is linked via an extracellular cysteine-rich region to a typical G protein-coupled receptor (GPCR) transmembrane heptahelical domain that mediates G protein activation [Bhave et al, 2003;Pin et al, 2003].…”

Interaction of Novel Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 with the Negative Allosteric Antagonist Site Is Required for Potentiation of Receptor Responses

“…Although mGluRs have a seven transmembrane (7TM)-spanning domain similar to other GPCRs (Conn and Pin, 1997;Bhave et al, 2003), glutamate binds these receptors on a large N-terminal extracellular glutamate binding domain that is composed of two globular domains and a hinge region (O'Hara et al, 1993;Jingami et al, 2003). As expected for a region involved in binding a common endogenous agonist, the glutamate binding sites share high homology across the mGluR subtypes relative to other regions of the receptor (Conn and Pin, 1997).…”