Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization.
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.
Romano
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Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA. romano@vision.wustl.edu email
Miller
J K
JK
Hyrc
K
K
Dikranian
S
S
Mennerick
S
S
Takeuchi
Y
Y
Goldberg
M P
MP
O'Malley
K L
KL
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D006801:Humans; D017354:Point Mutation; D016318:Quisqualic Acid / Q000494:pharmacology; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014316:Tritium
D018118:Chloride Channels; D018690:Excitatory Amino Acid Agonists; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D014316:Tritium; D016318:Quisqualic Acid; D003545:Cysteine; D002118:Calcium