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

Abstract

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G s. 2′(3′)- O-( N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G i/G o-proteins. We studied the effects of MANT-guanosine 5′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-guanosine 5′-[β,γ-imido]triphosphate (MANT-GppNHp) on Gα s- and Gα i-mediated signaling. MANT-GTPγS and MANT-GppNHp had lower affinities for Gα s and Gα i than GTPγS and GppNHp. In contrast to guanosine 5′-[β-thio]diphosphate, MANT-GTPγS noncompetitively inhibited GTPγS-stimulated AC in Gα s-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPγS and MANT-GppNHp was not due to Gα s inhibition since it was also observed in Gα s-deficient S49 cyc − lymphoma cell membranes. Mn 2+ blocked Gα i-mediated AC inhibition by GTPγS and GppNHp in S49 cyc − membranes but not AC inhibition by MANT-GTPγS and MANT-GppNHp. MANT-GTPγS and MANT-GppNHp competitively inhibited forskolin/Mn 2+-stimulated AC in S49 cyc − membranes with K i values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.