A direct fluorescence-based assay for RGS domain GTPase accelerating activity

Abstract

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 Gα subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Gα 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 Gα o. BODIPYFL-GTP bound to Gα o 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 Gα o. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Gα o and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds.