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

Francis S. Willard, Adam J. Kimple, Christopher A. Johnston, David P. Siderovski

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


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 languageEnglish
Pages (from-to)341-351
Number of pages11
JournalAnalytical Biochemistry
Issue number2
StatePublished - 15 May 2005


  • Fluorescence
  • GAP
  • GTPase accelerating protein
  • Heterotrimeric G-protein
  • RGS
  • Regulator of G-protein signaling


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