Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9744934 | Analytical Biochemistry | 2005 | 11 Pages |
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 Gαo. BODIPYFL-GTP bound to Gαo 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 Gαo. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Gαo and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Francis S. Willard, Adam J. Kimple, Christopher A. Johnston, David P. Siderovski,