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Two G_i1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity

^* To whom correspondence should be addressed. E-mail: dsiderov{at}med.unc.edu.

	Abstract

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate G 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 G 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_i1mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. G_i1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on G_i subunits and not affected by those unable to act on G_i, demonstrating that the G/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of G_i1(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on G_i1(R178M/A326S), the authors developed a Transcreener^®fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining (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 XXXX:xxx-xxx)

First published on October 9, 2009, doi:10.1177/1087057109347473
This version was published on October 12, 2009


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