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

Andreas Gille,Roland Seifert

doi:10.1074/jbc.m211292200

Abstract

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

Highlights

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]
In contrast to the observations made with Gi/Goproteins, 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]
We examined the interactions of MANT-GTP␥S/MANTGppNHp with GTP, GTP␥S, GppNHp, GDP␤S and AlF4Ϫ, i.e. substances that all bind to the nucleotide-binding pocket of G␣

Summary

Introduction

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]. MANT-GTP␥S and MANT-GppNHp (Fig. 1) bind to purified Go-proteins with higher affinity than to Gi-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 Gi/Go-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]. In contrast to the observations made with Gi/Goproteins, 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 Gs-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 Gs- and Gi-protein-mediated signaling. We found that MANT-GTP␥S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors

Objectives

Results

Conclusion