Covalent and Noncovalent Interactions Mediate Metabotropic Glutamate Receptor mGlu5 Dimerization
- Carmelo Romano1,2,
- Judith Kelvin Miller2,
- Krzysztof Hyrc3,
- Seta Dikranian1,
- Steven Mennerick4,
- Yutaka Takeuchi5,
- Mark P. Goldberg3 and
- Karen L. O'Malley2
- Departments of 1Ophthalmology and Visual Sciences (C.R., S.D.),2Anatomy and Neurobiology (C.R., J.K.M., K.L.O.), 3Neurology (K.H., M.P.G.), and 4Psychiatry (S.M.), Washington University School of Medicine, St. Louis, Missouri; and 5Banyu Tsukaba Research Institute, Tsukaba, Japan (Y.T.)
Abstract
Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.
Footnotes
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Send reprint requests to: Carmelo Romano, Ph.D., Department of Ophthalmology & Visual Sciences, Campus Box 8096, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. E-mail: romano{at}vision.wustl.eduemail
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This work was supported by National Institutes of Health Grants MH57817 and EY02687, an unrestricted grant from Research to Prevent Blindness, and the McDonnell Center for Cellular and Molecular Neuroscience.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- mGluR
- metabotropic glutamate receptor
- HA
- hemagglutinin epitope
- HEK
- human embryonic kidney
- AM
- acetoxymethyl ester
- t
- truncated
- h
- human
- wt
- wild-type
- DTT
- dithiothreitol
- mGABA
- metabotropic γ-aminobutryic acid
- CaR
- Ca2+-sensing receptor
- PBP
- periplasmic binding protein
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- Received March 29, 2000.
- Accepted September 25, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
