Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues

Emily E. Jameson, Rebecca A. Roof, Matthew H. Whorton, Henry I. Mosberg, Roger K. Sunahara, Richard R. Neubig, Robert T. Kennedy

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Hydrolysis of fluorescent GTP analogues BODIPY® FL guanosine 5′-O-(thiotriphosphate) (BGTPγS) and BODIPY® FL GTP (BGTP) by Gαi1 and Gαo was characterized using on-line capillary electrophoresis laser-induced fluorescence assays in order that changes in substrate, substrate-enzyme complex, and product could be monitored separately. Apparent kcat values (Vmax/[E]) and Km values were determined from steady-state assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Gαo and Gαi1 were 8.3 ± 1 × 10-3 and 3.0 ± 0.2 × 10-2 s-1, respectively, and K m values were 120 ± 60 and 940 ± 160 nM. Assays with BGTPγS yielded maximum turnover numbers of 1.6 ± 0.1 × 10-4 and 5.5 ± 0.3 × 10-4 s-1 for Gαo and Gαi1; Km values were 14 ± 8 and 87 ± 22 nM. Acceleration of Gα GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY® FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199-778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 μM YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Gα subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPγS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.

Original languageEnglish (US)
Pages (from-to)7712-7719
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number9
DOIs
StatePublished - Mar 4 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Jameson, E. E., Roof, R. A., Whorton, M. H., Mosberg, H. I., Sunahara, R. K., Neubig, R. R., & Kennedy, R. T. (2005). Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues. Journal of Biological Chemistry, 280(9), 7712-7719. https://doi.org/10.1074/jbc.M413810200