Copurification of brain G-protein beta5 with RGS6 and RGS7.
A structurally divergent G-protein beta subunit expressed in brain and retina, Gbeta5, exhibits functional specialization in its protein-protein interactions in vitro. In retina, Gbeta5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of Gbeta5 in brain are unknown. To identify tightly bound proteins associated with Gbeta5 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 Gbeta5 on SDS-PAGE. The copurifying 55 kDa band was identified as an approximately 1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. Gbeta5 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 Galphaq/11, Galphai1/2, Ggamma2, Ggamma3, or Ggamma7. These findings implicate RGS6 and RGS7 in the function of Gbeta5 in the brain and suggest that a large fraction of membrane-targeted Gbeta5 has no associated G subunit and therefore functions outside the canonical framework of G(beta)(gamma) interactions.
Zhang
J H
JH
Metabolic Diseases Branch, National Institute of Diabetes and Digestiveand Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Simonds
W F
WF
D000818:Animals; D001921:Brain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015151:Immunoblotting; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D011233:Precipitin Tests; D020710:RGS Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014020:Tissue Extracts
C490611:GNB5 protein, human; D044387:GTP-Binding Protein beta Subunits; C094668:Gnb5 protein, mouse; C490610:Gnb5 protein, rat; D020710:RGS Proteins; C492039:RGS7 protein, human; C485692:Rgs6 protein, mouse; C485691:Rgs6 protein, rat; C492038:Rgs7 protein, mouse; D014020:Tissue Extracts; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins