Guanosine 5′‐<i>O</i>‐(2‐thiodiphosphate) as a competitive inhibitor of GTP in hormone or cholera toxin‐stimulated pancreatic adenylate cyclase

2(3)-O-(N-MethylanthraniloylG-proteins are heterotrimeric (␣␤␥-structure) and serve as signal transducers between agonist-occupied GPCRs 1 and effector systems (1, 2). GPCR promotes GDP dissociation from G␣. GDP dissociation is the rate-limiting step of the G-protein cycle. Agonist-occupied GPCR then forms a ternary complex with guanine nucleotide-free G-protein. Thereafter, GPCR catalyzes GTP binding to G␣. G␣ GTP dissociates from GPCR, thereby disrupting the ternary complex. In addition, G␣ GTP and ␤␥ dissociate from each other, and both G␣ GTP and ␤␥ regulate the activity of effector systems. G-proteins are deactivated by the GTPase of G␣ that cleaves GTP into GDP and P i . The GTP hydrolysis-resistant GTP analogs GTP␥S and GppNHp ( Fig. 1) induce persistent G-protein activation as does AlF 4 Ϫ , the latter mimicking the transition state of GTP hydrolysis as GDP-AlF 4 Ϫ complex (1, 3, 4). The hydrolysisresistant GDP analog GDP␤S (Fig. 1) is a partial G-protein activator (5, 6).Nucleotides substituted with a MANT group at the 2Ј(3Ј)-Oposition of the ribosyl residue are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins (7). However, only few studies with MANT-nucleotides and G-proteins have been conducted so far, and the data are controversial. MANT-GTP␥S and MANT-GppNHp (Fig. 1) bind to purified G o -proteins with higher affinity than to G i -proteins, and the MANT group does not have an effect on the affinity of GTP␥S for purified G␣ i1 (8, 9). The maximum fluorescence of G i /G o -proteins induced by MANT-GTP␥S is higher than the maximum fluorescence induced by MANT-GppNHp, suggesting that the two nucleotides stabilize different conformations in G-proteins (8, 10). Moreover, like GTP␥S/GppNHp, MANT-GTP␥S/MANT-GppNHp confer protease protection to G i /G oproteins (8). In contrast to the observations made with G i /G oproteins, the MANT group substantially reduces the affinity of GTP for the retinal G-protein transducin, and MANT-GTP is ineffective at activating the effector of transducin, cGMP-degrading phosphodiesterase (11). To the best of our knowledge, the effects of MANT-nucleotides on G s -proteins have not yet been studied.The goal of our study was to learn more about the functional effects of MANT-GTP␥S/MANT-GppNHp on G s -and G i -protein-mediated signaling. As models we used fusion proteins and co-expression systems of the ␤ 2 AR with the G␣ s -proteins, G␣ sL , G␣ sS , or G␣ olf (12-14), fusion proteins, and co-expression systems of the FPR with the G␣ i -proteins, G␣ i1 , G␣ i2 , or G␣ i3 * This work was supported by Army Research Office Grant DAAD19-00-1-0069, The J. R. and Inez Jay Biomedical Research Award of the University of Kansas (to R. S.), and a predoctoral fellowship from the Studienstiftung des Deutschen Volkes (to A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To...

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“…5B). These data confirm the competitive interaction of guanine nucleotides at the nucleotide-binding site of G␣ s (31).…”

Section: Mant-gtp␥s/mant-gppnhp Are Potent Inhibitors Of G␣ Sstimulatsupporting

confidence: 76%

2′(3′)-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors

Gille

Seifert

2003

Journal of Biological Chemistry

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“…2B). These data are indicative for competitive interaction of GDPhS and GTPgS at the guanine nucleotide binding site of Ga s (Eckstein et al, 1979;Svoboda et al, 1980). In contrast to GDPhS, MANT-GTPgS inhibited AC activation by GTPgS noncompetitively, i.e.…”

Section: Unmasking Ac Inhibition By Mant-gtp;s and Mant-gppnhpmentioning

confidence: 80%

MANT-substituted guanine nucleotides: A novel class of potent adenylyl cyclase inhibitors

Gille

Seifert

2003

Life Sciences

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“…Control membranes underwent the complète procédure without choiera toxin. This method, first developed by others for turkey erythrocyte membranes [31], is described elsewhere [23],…”

Section: Expérimentalmentioning

confidence: 99%

“…The terms used in the présent study are defined in the appendix. I EFFECT OF CHOLERA TOXIN PRETREATMENT ON THE EFFICACY OF FIVE ACTIVATORS ON RAT PAN-CREATIC ADENYLATE CYCLASE ACTIVITY AT EQUILIBRIUM Membranes were pretreated in human erythrocyte cytosol, in the absence or présence of choiera toxin at 0.5 wg/ml or 30 yiig/ml, as previously described [23]. Membrane proteins (5-10 Mg) were incubated for 7 min at 37°C in 60 fi\ of an adenylate cyclase assay médium emiched with saturating concentrations of the listed activators.…”

Section: Expérimentalmentioning

confidence: 99%

“…In the présent study, the values of the kinetic constants of activation and deactivation by guanine nucleotides were determined experimentally in rat pancreatic plasma membranes, either native or pretreated with choiera toxin, and in the présence or absence of cholecystokinin (tested as CCK-8). We took advantage of GDP/3S (guanosine 5'-0-(2-thiodiphosphate) and of dibutyryl cycHc GMP as spécifie inhibitors of, respectively, GTP [21][22][23] and CCK-8 [24]. The altérations of kinetic parameters were correlated with the steady-state concentration of activated adenylate cyclase and the activity of spécifie GTPase observed at equilibrium.…”

Section: Introductionmentioning

confidence: 99%

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Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

Svoboda

Lambert

Christophe

1981

Biochimica et Biophysica Acta (BBA) - General Subjects

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(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTP gamma S as nucleotide activator, cholecystokinin as peptide hormone, and GDP beta S and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant kappa+1), and a deactivation process (rate constant kappa off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant kappa 2) and/or the dissociation of the intact nucleotide (rate constant kappa-1), so that EA/ETOT = kappa+1/(kappa+1 + kappa 2 + kappa-1). (2) The hormone CCK-8 increased the value of kappa+1 with GTP dose-dependently, from 0.2 to 10.9 min-1. The value of kappa-1 increased 0.01 to 0.3 min-1 but the value of kappa 2 was unaltered at 7 min-1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 micrograms/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the kappa off value of the GTP-activated enzyme (from 7 min-1 to 0.5 min-1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of kappa+1 (from 0.2 to 0.8 min-1) and the occurrence of a significant 0.3 min-1 value for kappa-1.

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Guanosine 5′‐O‐(2‐thiodiphosphate) as a competitive inhibitor of GTP in hormone or cholera toxin‐stimulated pancreatic adenylate cyclase

Cited by 19 publications

References 20 publications

2′(3′)-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors

2′(3′)-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors

MANT-substituted guanine nucleotides: A novel class of potent adenylyl cyclase inhibitors

Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

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