Cation and structural requirements for P site-mediated inhibition of adenylate cyclase

RA Johnson, SM Yeung, D Stubner, M Bushfield and I Shoshani

Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.

The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovine brain. Sensitivity of adenylate cyclase to P site-mediated inhibition was enhanced by reversible and irreversible activators of the enzyme. The most effective sensitization of the platelet and brain adenylate cyclases was observed with Mn2+ and upon proteolysis with inhibin in the presence of guanosine 5'-O-(3-thiotriphosphate). These resulted in IC50 values for (2',5'dideoxy-adenosine (2',5'-dd-Ado) and 2'-deoxy-3'-AMP of approximately 1-2 microM. The data were consistent with the ideas that P site-mediated inhibition of adenylate cyclase is dependent on divalent cation and is a function of enzyme activity. A number of nucleosides and nucleotides were synthesized and used to define structural requirements for P site-mediated inhibition of a detergent- solubilized adenylate cyclase from rat brain. The data suggest a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety. 2'-Deoxy-and especially 2',5'-dideoxy-ribosyl moieties enhanced sensitivity and a strong preference for phosphate at the 3'-position was exhibited. Substitutions at the 5'-ribose position impaired sensitivity. The order of potency and IC50 values of the more potent adenosine analogs were 2',5'-dideoxy-3'-AMP (congruent to 0.1 microM) greater than 2'-deoxy-3'-AMP (congruent to 1 microM) greater than 2',5'-dd-Ado (congruent to 3 microM) greater than 3'-AMP (congruent to 9 microM) greater than 2'-deoxy-adenosine (congruent to 15 microM) greater than adenosine (congruent to 80 microM). Large substitutions at the 3'-ribose position were tolerated, e.g., dApdN di- and dAp(dN)4 penta-nucleotides and succinyl- and p-fluoro-sulfonyl- benzoyl- moieties. The purified adenylate cyclase from bovine brain was inhibited by P site agonists with IC50 values of 34 and 45 microM for 2'-deoxy-3'-AMP and 2',5'-dd-Ado, respectively. The data imply, first, that the locus of the P site is the catalytic subunit of adenylate cyclase and, second, that the increased sensitivity observed with Mn2+ is due to an effect of the cation on the catalytic subunit. In contrast with adenylate cyclases from other mammalian tissues, the enzyme from bovine sperm exhibited only weak sensitivity to P site agonists; 2'- deoxy-3'-AMP congruent to 2',5'-dd-Ado greater than adenosine, each with IC50 values greater than 1000 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 35, Issue 5, pp. 681-688, 05/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				A. M. Stessin, J. H. Zippin, M. Kamenetsky, K. C. Hess, J. Buck, and L. R. Levin Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1 J. Biol. Chem., June 23, 2006; 281(25): 17253 - 17258. [Abstract] [Full Text] [PDF]

				T.-C. Mou, A. Gille, D. A. Fancy, R. Seifert, and S. R. Sprang Structural Basis for the Inhibition of Mammalian Membrane Adenylyl Cyclase by 2 '(3')-O-(N-Methylanthraniloyl)-guanosine 5 '-Triphosphate J. Biol. Chem., February 25, 2005; 280(8): 7253 - 7261. [Abstract] [Full Text] [PDF]

				B. D. Spiegelberg, J. dela Cruz, T.-H. Law, and J. D. York Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism J. Biol. Chem., February 18, 2005; 280(7): 5400 - 5405. [Abstract] [Full Text] [PDF]

				M. Spehr, K. Schwane, J. A. Riffell, J. Barbour, R. K. Zimmer, E. M. Neuhaus, and H. Hatt Particulate Adenylate Cyclase Plays a Key Role in Human Sperm Olfactory Receptor-mediated Chemotaxis J. Biol. Chem., September 17, 2004; 279(38): 40194 - 40203. [Abstract] [Full Text] [PDF]

				A. Gille, G. H. Lushington, T.-C. Mou, M. B. Doughty, R. A. Johnson, and R. Seifert Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides J. Biol. Chem., May 7, 2004; 279(19): 19955 - 19969. [Abstract] [Full Text] [PDF]

				W. H. G. Laux, P. Pande, I. Shoshani, J. Gao, V. Boudou-Vivet, G. Gosselin, and R. A. Johnson Pro-nucleotide Inhibitors of Adenylyl Cyclases in Intact Cells J. Biol. Chem., April 2, 2004; 279(14): 13317 - 13332. [Abstract] [Full Text] [PDF]

				A. Gille and R. Seifert 2'(3')-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors J. Biol. Chem., April 4, 2003; 278(15): 12672 - 12679. [Abstract] [Full Text] [PDF]

				J. U. Linder, T. Hoffmann, U. Kurz, and J. E. Schultz A Guanylyl Cyclase from Paramecium with 22 Transmembrane Spans. EXPRESSION OF THE CATALYTIC DOMAINS AND FORMATION OF CHIMERAS WITH THE CATALYTIC DOMAINS OF MAMMALIAN ADENYLYL CYCLASES J. Biol. Chem., April 6, 2000; 275(15): 11235 - 11240. [Abstract] [Full Text] [PDF]

				I. Shoshani, V. Boudou, C. Pierra, G. Gosselin, and R. A. Johnson Enzymatic Synthesis of Unlabeled and beta -32P-labeled beta -L-2',3'-Dideoxyadenosine-5'-triphosphate as a Potent Inhibitor of Adenylyl Cyclases and Its Use as Reversible Binding Ligand J. Biol. Chem., December 3, 1999; 274(49): 34735 - 34741. [Abstract] [Full Text] [PDF]

				I. Shoshani, W. H. G. Laux, C. Perigaud, G. Gosselin, and R. A. Johnson Inhibition of Adenylyl Cyclase by Acyclic Nucleoside Phosphonate Antiviral Agents J. Biol. Chem., December 3, 1999; 274(49): 34742 - 34744. [Abstract] [Full Text] [PDF]

				S. Doronin, L. Murray, C. W. Dessauer, and R. A. Johnson Covalent Labeling of Adenylyl Cyclase Cytosolic Domains with gamma -Methylimidazole-2',5'-dideoxy-[gamma -32P]3'-ATP and the Mechanism for P-site-mediated Inhibition J. Biol. Chem., December 3, 1999; 274(49): 34745 - 34750. [Abstract] [Full Text] [PDF]

				D. B. Bylund and D. M. Chacko Characterization of {alpha}2 Adrenergic Receptor Subtypes in Human Ocular Tissue Homogenates Invest. Ophthalmol. Vis. Sci., September 1, 1999; 40(10): 2299 - 2306. [Abstract] [Full Text] [PDF]

				A. Ibrahimi, N. Abumrad, H. Maghareie, M. Golia, I. Shoshani, L. Desaubry, and R. A. Johnson Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes Am J Physiol Cell Physiol, February 1, 1999; 276(2): C487 - C496. [Abstract] [Full Text] [PDF]

				S. Doronin, C. Dessauer, and R. A. Johnson Direct Photoaffinity Labeling of Individual Cytosolic Domains of Adenylyl Cyclase by [32P]2'-deoxy-3'-AMP and [alpha -32P]5'-ATP J. Biol. Chem., December 4, 1998; 273(49): 32416 - 32420. [Abstract] [Full Text] [PDF]

				L. Desaubry and R. A. Johnson Adenine Nucleoside 3'-Tetraphosphates Are Novel and Potent Inhibitors of Adenylyl Cyclases J. Biol. Chem., September 18, 1998; 273(38): 24972 - 24977. [Abstract] [Full Text] [PDF]

				W.-J. Tang and J. H. Hurley Catalytic Mechanism and Regulation of Mammalian Adenylyl Cyclases Mol. Pharmacol., August 1, 1998; 54(2): 231 - 240. [Full Text]

				C. W. Dessauer and A. G. Gilman The Catalytic Mechanism of Mammalian Adenylyl Cyclase. EQUILIBRIUM BINDING AND KINETIC ANALYSIS OF P-SITE INHIBITION J. Biol. Chem., October 31, 1997; 272(44): 27787 - 27795. [Abstract] [Full Text] [PDF]

				D. B. Bylund, L. J. Iversen, W. J. Matulka, and D. M. Chacko J. Pharmacol. Exp. Ther., June 1, 1997; 281(3): 1171 - 1177. [Abstract] [Full Text]

				R. A. Johnson, L. Desaubry, G. Bianchi, I. Shoshani, E. Lyons Jr., R. Taussig, P. A. Watson, J. J. Cali, J. Krupinski, J. P. Pieroni, et al. Isozyme-dependent Sensitivity of Adenylyl Cyclases to P-site-mediated Inhibition by Adenine Nucleosides and Nucleoside 3'-Polyphosphates J. Biol. Chem., April 4, 1997; 272(14): 8962 - 8966. [Abstract] [Full Text] [PDF]

				C. W. Dessauer and A. G. Gilman Purification and Characterization of a Soluble Form of Mammalian Adenylyl Cyclase J. Biol. Chem., July 12, 1996; 271(28): 16967 - 16974. [Abstract] [Full Text] [PDF]

				L. Desaubry, I. Shoshani, and R. A. Johnson Inhibition of Adenylyl Cyclase by a Family of Newly Synthesized Adenine Nucleoside 3'-Polyphosphates J. Biol. Chem., June 14, 1996; 271(24): 14028 - 14034. [Abstract] [Full Text] [PDF]

				L. Désaubry, I. Shoshani, and R. A. Johnson 2`,5`-Dideoxyadenosine 3`-Polyphosphates Are Potent Inhibitors of Adenylyl Cyclases J. Biol. Chem., February 2, 1996; 271(5): 2380 - 2382. [Abstract] [Full Text] [PDF]