Real-time Detection of Basal and Stimulated G Protein GTPase Activity Using Fluorescent GTP Analogues

Abstract

Hydrolysis of fluorescent GTP analogues BODIPY® FL guanosine 5-O-(thiotriphosphate) (BGTP␥S) and BODIPY® FL GTP (BGTP)by G␣ i1 and G␣ o 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. 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 pseudosingle-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38 -81% by 40 M YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that G␣ subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTP␥S hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe. G proteins1 are involved in many important physiological processes. Ligands binding to cell surface receptors signaling through G protein pathways (like G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs)) induce the G protein to exchange GDP for GTP, activating the protein. Activated G protein can transmit signals to downstream effectors such as adenylyl cyclase, phospholipases, ion channels, or cGMP phosphodiesterase, which ultimately result in cellular responses initiated by the extracellular ligand stimulus. Signaling is ceased by the hydrolysis of GTP to GDP via the intrinsic GTPase activity of the G protein (reviewed in Refs. 1 and 2). G protein deactivation can be accelerated by interactions with GTPase-activating proteins (GAPs) such as regulators of G protein signaling (RGS) (3, 4).Most studies of G protein-mediated signaling have relied on utilization of guanine nucleotide analogues that both behave similarly to the native species and have been modified such that they can be sensitively detected. The most common modification involves insertion of radiolabeled isotopes of phosphorous or sulfur to yield [␥-32 P]GTP and [␥-35 S]GTP␥S, which can then be used in filter-binding assays to measure GTPase activity and the extent of nucleotide binding, respectively. More recently, fluorescent guanine nucleotide derivatives N-methyl-3Ј-O-anthranoyl (MANT) and BODIPY® have been developed (5, 6), allowing for G protein assays with greater sensitivity than previous fluorescence assays that relied on detection of changes in intrinisic tryptophan fluorescence. A number of studies have reported on use of these fluorescent GTP derivatives with low molecular weight, Ras-like G proteins. In general, the binding affinities and rate constants reported for MANT analogues of GTP and GDP are within a factor of 2 of those for unmodified guanine nucleotides (7), rendering these probes useful for studies of basal and GAP-stimulated GTPase activity (8 -13) and interactions with guanine nucleotide exchange factors (14...

Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

“…The data were plotted using Michaelis-Menten models to estimate the K m and V max values with or without Ric-8 proteins present. The calculated K m of G␣ s short for GTP was 385.1 Ϯ 10 nM, which was consistent with previous reports for G␣ subunits (39,40). Ric-8BFL and Ric-8B⌬9 increased the K m dramatically to ϳ42.4 Ϯ 8.1 and ϳ2.6 Ϯ 0.2 M, respectively.…”

High-Throughput Fluorescence Polarization Assay for the Enzymatic Activity of GTPase-Activating Protein of ADP-Ribosylation Factor (ARFGAP)

“…More recently, fluorescent derivatives (n-methyl-3′-o-anthranoyl [ManT] and boDiPy) of guanine nucleotides have been developed 25,26 and used to monitor the loading and hydrolysis of gTP by g-proteins and ras-related gTPases. [27][28][29][30] However, these reagents have not been applied to arf activity assays, and our results (data not shown) suggest that the fluorescent gTP derivatives cannot be effectively loaded to arf1. clearly, a high-throughput arfgaP assay that can be used for development of small-molecule regulators is needed.…”

Structural flexibility of the Gαs α-helical domain in the β 2 -adrenoceptor Gs complex

“…PPi or foscarnet were added together with bodipy-GTPγS-FL and initiated by the addition of G protein (1 mM) in 20 mM Tris-HCl, pH 8.0, 3 mM MgCl 2 , 1 mM DTT in a final volume of 200 mL bodipy-GTPγS-FL binding to heterotrimeric G protein included 0.1% dodecylmaltoside (final). Fluorescence was measured on a short time scale (600 s) to minimize the accumulation of hydrolysis product bodipy-phosphate (21). Hydrolysis, which also appears as an increase in fluorescence, was determined simply by chelating Mg 2+ with 10 mM EDTA following the 600-s incubation.…”