Purification and in vitro functional analysis of R7 subfamily RGS proteins in complex with Gbeta5.
The study of purified regulator of G-protein signaling (RGS) proteins in steady-state GTPase assays using reconstituted proteoliposomes is a powerful approach to characterizing the RGS protein-mediated acceleration of intrinsic Galpha subunit GTPase activity in the context of various G-protein and G-protein-coupled receptor (GPCR) combinations. This approach has been applied successfully to the R7 subfamily of RGS proteins, RGS6, -7, -9, and -11, which form heterodimers with Gbeta5 subunits via the G-protein gamma-like domain of R7 proteins. This article describes the purification of heterodimers from Sf9 insect cells following the expression of recombinant R7 protein and histidine-tagged Gbeta5 using affinity and ion-exchange chromatography. The ability of the heterodimers to accelerate the intrinsic GTPase activity of Galpha subunits was assessed in steady-state GTPase assays performed on proteoliposomes consisting of phospholipids, purified G proteins, and purified GPCRs.
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
Shelley B
SB
Hooks
T Kendall
TK
Harden
D000818:Animals; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D011485:Protein Binding; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology
D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; C490610:Gnb5 protein, rat; D008081:Liposomes; D046911:Macromolecular Substances; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D020558:GTP Phosphohydrolases