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Methods Enzymol. 2004;390:163-77.

Purification and in vitro functional analysis of R7 subfamily RGS proteins in complex with Gbeta5.

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Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.


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.

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