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.