Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins.
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 G-protein-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 G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.
Kimple
Randall J
RJ
Department of Pharmacology, Lineberger Comprehensive Cancer Center, and UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA. dsiderov@med.unc.edu
Jones
Miller B
MB
Shutes
Adam
A
Yerxa
Benjamin R
BR
Siderovski
David P
DP
Willard
Francis S
FS
D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D021122:Protein Subunits; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D021122:Protein Subunits; D020710:RGS Proteins; D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins