A direct fluorescence-based assay for RGS domain GTPase accelerating activity.
Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Galpha subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Galpha subunits. In this article, we describe the use of the ribose-conjugated fluorescent guanine nucleotide analog BODIPYFL-GTP as a spectroscopic probe to measure intrinsic and RGS protein-catalyzed nucleotide hydrolysis by Galphao. BODIPYFL-GTP bound to Galphao exhibits a 200% increase in fluorescence quantum yield. Hydrolysis of BODIPYFL-GTP to BODIPYFL-GDP reduces the quantum yield to 27% above its unbound value. We demonstrate that BODIPYFL-GTP can be used as a rapid real-time probe for measuring RGS domain-catalyzed GTP hydrolysis by Galphao. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Galphao and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds.
Willard
Francis S
FS
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. fwillard@med.unc.edu
Kimple
Adam J
AJ
Johnston
Christopher A
CA
Siderovski
David P
DP
D000595:Amino Acid Sequence; D001896:Boron Compounds / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods; D020349:Surface Plasmon Resonance
C503506:BODIPY-FL-GTP; D001896:Boron Compounds; D005456:Fluorescent Dyes; D044385:GTP-Binding Protein alpha Subunits; D020690:GTPase-Activating Proteins; D020710:RGS Proteins; D006160:Guanosine Triphosphate