Elsevier

Analytical Biochemistry

Volume 340, Issue 2, 15 May 2005, Pages 341-351
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2005.02.015Get rights and content

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.

Section snippets

Materials

BODIPYFL-GTP and guanosine 5′-O-(3-thiotriphosphate), BODIPYFL thioester (BODIPYFL-GTPγS) were obtained from Molecular Probes (Eugene, OR, USA). Unless otherwise specified, all other chemicals were of the highest purity obtainable from Sigma (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).

Cloning

A prokaryotic expression construct encoding an N-terminal GST fusion of the extended RGS domain of rat RGS14 (aa 57–235) was created using the pGEX4TEV2 vector modified from pGEX4T2 (Amersham

Results

We examined the suitability of the ribose-conjugated GTP analog BODIPYFL-GTP (Fig. 1A) as a fluorescent spectroscopic probe for GTP binding and hydrolysis by heterotrimeric G protein α subunits. As shown previously [18], BODIPYFL-GTP undergoes time-dependent changes in fluorescence on interaction with Gαo (Fig. 2A). This response is biphasic, with an initial increase in fluorescence corresponding to GTP binding followed by decay in fluorescence corresponding to GTP hydrolysis by Gαo (for a

Discussion

We have described a direct real-time assay for Gαo GTPase activity with specific applications to the measurement of RGS protein GAP activity. BODIPYFL-GTP undergoes a 200% increase in fluorescence quantum yield when bound to Gαo, and hydrolysis of this nucleotide to its GDP form decreases its fluorescence to 27% above its unbound value. Thus, BODIPYFL-GTP represents a useful fluorescence biosensor for GTPase activity and therefore G-protein activation state.

Among the series of described

Acknowledgments

The authors thank Melinda Hains and Christopher McCudden for critical appraisal of the manuscript. We also thank Randall J. Kimple and Breann L. Wolfe (UNC Pharmacology) for assistance with RGS14 vector construction and GST-RGS14 purification, respectively. Francis Willard is an American Heart Association postdoctoral fellow. This research was supported by National Institutes of Health grants (R01 GM062338 and P01 GM065533).

References (38)

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