Two Galpha(i1) rate-modifying mutations act in concert to allow receptor-independent, steady-state measurements of RGS protein activity.

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate G alpha subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Ga and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a G alpha(i1) mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. G alpha(i1)(R178M/A326S) GTPase activity was stimulated 6-to 12-fold by RGS proteins known to act on G alpha(i) subunits and not affected by those unable to act on G alpha(i), demonstrating that the G alpha/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of G alpha(i1)(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on G alpha(i1)(R178M/A326S), the authors developed a Transcreener(R) fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining G alpha(i1)(R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. (Journal of Biomolecular Screening 2009:1195-1206)