MANT-substituted guanine nucleotides: a novel class of potent adenylyl cyclase inhibitors.
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'-[gamma-thio]triphosphate (MANT-GTPgammaS) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on Galpha(s)- and Galpha(i)-mediated signaling. MANT-GTPgammaS and MANT-GppNHp had lower affinities for Galpha(s) and Galpha(i) than GTPgammaS and GppNHp. In contrast to guanosine 5'-[beta-thio]diphosphate, MANT-GTPgammaS noncompetitively inhibited GTPgammaS-stimulated AC in Galpha(s)-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPgammaS and MANT-GppNHp was not due to Galpha(s) inhibition since it was also observed in Galpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked Galpha(i)-mediated AC inhibition by GTPgammaS and GppNHp in S49 cyc(-) membranes but not AC inhibition by MANT-GTPgammaS and MANT-GppNHp. MANT-GTPgammaS 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.
Gille
Andreas
A
Department of Pharmacology and Toxicology, The University of Kansas, 1251 Wescoe Hall Drive, Malott Hall, Room 5064, Lawrence, KS 66045-7582, USA.
Seifert
Roland
R
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology; D006150:Guanine Nucleotides / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; D006165:Guanylyl Imidodiphosphate; D019205:GTP-Binding Protein alpha Subunits, Gs