Cys-140 Is Critical for Metabotropic Glutamate Receptor-1 Dimerization

Kausik Ray,Benjamin C Hauschild

doi:10.1074/jbc.m005581200

Abstract

Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1.

Highlights

Tors (T1Rs) [7], and GABAB receptors [8]
MGluR1 Is a Cell Surface-expressed Disulfide-linked Dimer—To determine the domain(s) involved in Metabotropic glutamate receptor 1 (mGluR1) dimerization, we sought to construct a truncated form of the mGluR1 lacking the extracellular amino-terminal domain (ECD)
To confirm observations derived from study of mGluRs [10, 11] that suggested that the dimerization domain is localized to the ECD, we expressed a truncation mutant receptor (RhodCR1) that contained the seven transmembrane domains and carboxyl-terminal tail of the mGluR1 but lacked the ECD

Summary

Introduction

Tors (T1Rs) [7], and GABAB receptors [8]. The eight members of the mGluR family (mGluR1– 8) of receptors have been divided into three subgroups based on sequence homology, signal transduction properties, and pharmacological profile [1]. The rat mGluR subtype 1␣ (described as mGluR1 on) ECD contains a total of 19 cysteines [16] of which several are highly conserved in other mGluRs, Ca2ϩ receptor, V2Rs, and T1Rs. Proteolysis of the mGluR5 receptor localized cysteine(s) critical for dimer formation to the first 17 kDa of the ECD [17]. Proteolysis of the mGluR5 receptor localized cysteine(s) critical for dimer formation to the first 17 kDa of the ECD [17] This region contains three cysteines conserved in all mGluRs, and we mutated these cysteines of the mGluR1 and investigated the role of these cysteine mutants in receptor dimerization and function. This study led to the identification of a conserved Cys-140 as critical for disulfide-linked dimerization of mGluR1, and functional study indicated that, like the wild type receptor, C140S mutant receptor is capable of intracellular signaling via Gq-phospholipase C pathway

Methods

Results

Conclusion