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.
Original language | English |
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Pages (from-to) | 341-351 |
Number of pages | 11 |
Journal | Analytical Biochemistry |
Volume | 340 |
Issue number | 2 |
DOIs | |
State | Published - 15 May 2005 |
Keywords
- BODIPY
- Fluorescence
- GAP
- GTPase accelerating protein
- Heterotrimeric G-protein
- RGS
- Regulator of G-protein signaling