Covalent and Noncovalent Interactions Mediate Metabotropic Glutamate Receptor mGlu₅Dimerization

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.

Ontogeny of postsynaptic density proteins at glutamatergic synapses

“…In immunoblots (Fig. 1), multiple bands represent monomers and dimers as noted previously (Romano et al, 2001(Romano et al, , 2002. The peak in labeling intensity seen at P10 is consistent with previous findings (e.g., Romano et al, 2002;Di Giorgi Gerevini et al, 2004).…”

The activation mechanism of class-C G-protein coupled receptors

“…Of interest, in all class-C GPCRs with a VFTM, except the GABA B receptor, the two subunits are linked by a disulfide bridge at the level of their VFTM (lobe-I) (Romano et al, 1996;Ray et al, 1999), indicating such dimers are likely constitutive, and unlikely able to dissociate. Mutation of the cysteines involved in the disulfide bridge does not prevent the receptors from dimerizing Romano et al, 2001), indicating other regions are involved in the formation of the dimer and that the disulfide bridge simply firmly stabilizes the dimer.…”

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

“…In a recent preliminary study of mGluR5 by Romano and co-workers (32), the cysteine residues Cys in mGluR5 did not impair the pharmacology of the receptor significantly (20,32,33). In contrast, mutation of Cys 240 in mGluR5 resulted in a nonfunctional receptor (32).…”