Fluorescence-based assays for RGS box function.
Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride.
Willard
Francis S
FS
Department of Pharmacology, The University of North Carolina at Chapel Hill, 27599-7365, USA.
Kimple
Randall J
RJ
Kimple
Adam J
AJ
Johnston
Christopher A
CA
Siderovski
David P
DP
D019943:Amino Acid Substitution; D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020555:GTP-Binding Protein Regulators / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017354:Point Mutation; D020710:RGS Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D020555:GTP-Binding Protein Regulators; D020690:GTPase-Activating Proteins; D020710:RGS Proteins; D012694:Serine; D020558:GTP Phosphohydrolases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins