Isozyme-dependent Sensitivity of Adenylyl Cyclases to P-site-mediated Inhibition by Adenine Nucleosides and Nucleoside 3′-Polyphosphates

Abstract

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Dé saubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028 -14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2,5-dideoxyadenosine (2,5-ddAdo) > ␤-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3-polyphosphates inhibitory potency followed the order Ado < 2-dAdo < 2,5-ddAdo and 3-mono-< 3-di-< 3-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2,5-dd-3-ATP with IC 50 values of 40 -300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact purine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4 -6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.P-site-mediated inhibition has been characterized pharmacologically (1, 2, 4, 12-16). Inhibition requires an intact adenine moiety, and potency of inhibition is increased substantially for deoxyribose and especially 3Ј-phosphorylribose adenine nucleosides. Inhibitory potency follows the order: 3Ј-mono-Ͻ 3Ј-di Ͻ 3Ј-triphosphate and adenosine (Ado) Ͻ 2Ј-deoxy (d) 1 -Ado Ͻ 2Ј,5Ј-ddAdo, with 2Ј,5Ј-dd-3Ј-ATP being the most potent ligand and exhibiting an IC 50 ϳ40 nM (15). In addition, tolerance for large substitutions at the 3Ј-position and for other ribose modifications has been demonstrated (1, 2, 4).We reported previously that levels of 2Ј-d-3Ј-AMP and 3Ј-AMP varied considerably in different tissues and were dependent on the metabolic state of the animal (17). Moreover, sensitivity o...

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…A similar observation was made for P-site inhibitors (28,29). In general, the structure/activity relationships of inhibitors for ACs 1, 2, 5, and 6 were similar, but BODIPY-FL-GMPPNP (compound 14) exhibited ϳ15-fold selectivity for AC5 relative to AC2.…”

“…The IC 50 value of 2Ј,5Ј-dd-3Ј-ATP was 38 nM for C1⅐C2, 37 nM for AC1, 220 nM for AC2, and 37 nM for AC5. A lower sensitivity of AC2 relative to other AC isoforms toward P-site inhibitors was also noted in previous studies (28,29). Based on the data presently available, it is clear that AC2 is less sensitive to competitive and noncompetitive inhibitors than other membranous AC isoforms.…”

“…Inhibitor Analysis of Rat and Bovine Brain ACs-The dispersed rat brain AC and purified bovine brain AC have served as standard systems for the analysis of AC inhibitors for many years (4,23,25,28,30). Therefore, we wished to calibrate some of the inhibitors analyzed in this study with those standard systems.…”

“…The experiments for the determination of the potencies of 2Ј,5Ј-dd-3Ј-ATP at various AC preparations shown in Fig. 5 were conducted in the presence of 1.0 -1.5 Ci of [␣-32 P]ATP, 100 M unlabeled ATP, 5 mM MnCl 2 , and 100 M forskolin to allow direct comparison with literature data (22,24,25,28). All AC assays with purified proteins contained 0.2% (mass/volume) bovine serum albumin to prevent adsorption of proteins to reaction tubes.…”

“…The most potent P-site inhibitors presently available are 2Ј,5Ј-dd-3Ј-ATP (IC 50 for rat brain AC, 40 nM) and 2Ј,5Ј-dd-3Ј-A 4 P (IC 50 for rat brain AC, 7.4 nM) (24,25). P-site inhibitors exhibit a moderate degree of specificity for mammalian AC isoforms (28,29), and EF is insensitive to P-site inhibitors (4). The most potent competitive AC inhibitor presently available is ␤-L-2Ј,3Ј-dd-5Ј-ATP (K i for rat brain AC, 16 nM) (30).…”