Identification and Molecular Characterization of m3 Muscarinic Receptor Dimers*
- From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
Abstract
Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3′) as a model system. When membrane lysates prepared from m3′ receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent,N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3′ receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3′ receptor constructs. In addition, these studies showed that m3′ receptors were also able to form non-covalently associated receptor dimers and that m3′ receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3′ receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3′ receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization.
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: NIDDKD, Laboratory of Bioorganic Chemistry, Bldg. 8A, Rm. B1A-05,National Institutes of Health, Bethesda, MD 20892. Tel.: 301-402-3589; Fax: 301-402-4182; E-mail: jwess{at}helix.nih.gov.
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↵2 F.-Y. Zeng, A. Soldner, and J. Wess, manuscript in preparation.
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↵3 F.-Y. Zeng and J. Wess, unpublished data.
- Abbreviations:
- GPCRs
-
G protein-coupled receptors
- ABT
-
3-(2′-aminobenzylhydroxy)-tropane
- BSA
-
bovine serum albumin
- 4-DAMP
-
4-diphenylacetoxy-N-methylpiperidine methiodide
- DTT
-
dithiothreitol
- ECL
-
enhanced chemiluminescence
- HA tag
-
hemagglutinin epitope tag
- i3 loop
-
the third intracellular loop of GPCRs
- mGluR
-
metabotropic glutamate receptor
- NEM
-
N-ethylmaleimide
- NMS
-
N-methylscopolamine
- PBS
-
phosphate-buffered saline
- PI
-
phosphatidylinositol
- PAGE
-
polyacrylamide gel electrophoresis
- TM
-
transmembrane domains
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- Received November 17, 1998.
- Revision received March 12, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
