Randall J. Kimple, Miller B. Jones, Adam Shutes, Benjamin R. Yerxa, David P. Siderovski and Francis S. Willard Pages 399 - 407 ( 9 )
Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate Gprotein- mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric Gprotein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in highthroughput screening and drug discovery.
fluorescence-based assays, fret, g-protein alpha subunits, rgs proteins
Department of Pharmacology, CB No.7365, 1106 Mary Ellen Jones Bldg., UNC-Chapel HillSchool of Medicine, Chapel Hill, NC 27599-7365 USA.