Copurification of brain G-protein β5 with RGS6 and RGS7

A structurally divergent G-protein beta subunit expressed in brain and retina, G beta(5), exhibits functional specialization in its protein-protein interactions in vitro. In retina, Gb5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of G beta(5) in brain are unknown. To identify tightly bound proteins associated with G beta(5) in the brain, it was immunoaffinity-purified from a nonionic detergent extract of washed mouse brain membranes using an antibody directed against its N terminus. Elution with cognate peptide revealed a broad band of 55 kDa that coeluted with G beta(5) on SDS-PAGE. The copurifying 55 kDa band was identified as a similar to 1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. G beta(5) and RGS7 could be reciprocally coimmunoprecipitated from unfractionated brain membrane extracts confirming the tight association of native proteins. In contrast, immunoblotting of the peptide eluate revealed no copurifying G alpha q/11, G alpha i1/2, G gamma 2, G gamma 3, or G gamma 7. These findings implicate RGS6 and RGS7 in the function of G beta(5) in the brain and suggest that a large fraction of membrane-targeted G beta 5 has no associated G gamma subunit and therefore functions outside the canonical framework of G beta gamma interactions.