Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity

• Published in: SLAS discovery Vol. 14; no. 10; pp. 1195 - 1206
• Main Authors: Zielinski, Thomas, Kimple, Adam J., Hutsell, Stephanie Q., Koeff, Mark D., Siderovski, David P., Lowery, Robert G.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.12.2009
• Subjects: fluorescence polarization; GDP detection; regulators of G-protein signaling; surface plasmon resonance; fluorescence polarization; regulators of G-protein signaling; GDP detection; surface plasmon resonance
• ISSN: 2472-5552; 2472-5560
• DOI: 10.1177/1087057109347473

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα 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 Gα 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 Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Gαi subunits and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Gαi1(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Gαi1(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 Gαi1(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.