Cys-140 is critical for metabotropic glutamate receptor-1 dimerization.
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.
Ray
K
K
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. Kray@helix.nih.gov
Hauschild
B C
BC
D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D018094:Receptors, Metabotropic Glutamate; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine