Cys-140 Is Critical for Metabotropic Glutamate Receptor-1 Dimerization*
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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Laboratory of Signal Transduction, National Institute on Deafness and Other Communication Disorders, 5 Research Ct., Rockville, MD 20850. Tel.: 301-496-9168; Fax: 301-480-8019; E-mail: Kray@helix.nih.gov.
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Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M005581200
- Abbreviations:
- mGluR
-
metabotropic glutamate receptor
- GPCR
-
G-protein-coupled receptor
- PhI
-
phosphoinositide
- V2R
-
vomeronasal organ receptor (Type 2)
- T1R
-
taste receptor (Type 1)
- ECD
-
extracellular domain
- HEK-293 cells
-
human embryonic kidney 293 cells
- PAGE
-
polyacrylamide gel electrophoresis
- PNGase-F
-
peptide N-glycosidase F
- Endo-H
-
endo-β-N-acetylglucosaminidase H
- Biotin-7-NHS
-
d-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester
- PIPES
-
1,4-piperazinediethanesulfonic acid
- PCR
-
polymerase chain reaction
- POD
-
peroxidase-conjugated
- GABA
-
γ-aminobutyric acid
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- Received June 26, 2000.
- Revision received August 2, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
