THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 5, Issue of February 2, pp. 2380 –2382, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Communication 2*,5*-Dideoxyadenosine 3*-Polyphosphates Are Potent Inhibitors of Adenylyl Cyclases* (Received for publication, October 16, 1995, and in revised form, December 8, 1995) Laurent Désaubry, Ilana Shoshani, and Roger A. Johnson‡ From the Department of Physiology and Biophysics, State University of New York, Health Sciences Center, Stony Brook, New York 11794-8661 Adenylyl cyclase is a family of membrane-bound enzymes that catalyze the formation of 39:59-cAMP from 59-ATP. In mammalian systems, the 10 known adenylyl cyclase isozymes are regulated by numerous hormones and neurotransmitters via cell surface receptors linked via stimulatory (Gs) and inhibitory (Gi) guanine nucleotide-dependent regulatory proteins (G-proteins), as well as by numerous other agents also of physiological and biochemical interest. These include agents or enzymes that act on hormone receptors, specific bacterial toxins that act on Gs and Gi, and agents that act directly on adenylyl cyclase. Direct activation can be caused by forskolin with all but one isozyme and by Ca21/calmodulin with four isozymes. The enzyme is also inhibited directly by certain adenosine derivatives which act via a distinct domain, referred to as the “P”-site from a requirement for a purine moiety (1– 8). Of the mammalian adenylyl cyclases that have been tested, all save the enzyme from sperm have been found to be susceptible to P-site-mediated inhibition (5, 9 –12). Although P-site ligands act directly on adenylyl cyclase, enzyme stimulated by hormones (via Gsa) or Mn21 are most sensitive to inhibition, and P-site ligands may be viewed as attenuating the enzyme’s susceptibility to such activation (5, 8, 13–18). Although the three-dimensional structure of adenylyl cyclase is not known, the deduced primary sequence suggests a membrane topology exhibiting a repeated structure of six membrane spanning regions followed by a large cytosolic domain, giving * This work was supported by National Institutes of Health Research Grant DK38828 (to R. A. J.) and a Philippe Foundation award (to L. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 516-444-3040; Fax: 516-444-3432; E-mail: rjohnson@ccmail.sunysb.edu. EXPERIMENTAL PROCEDURES Preparation and Assay of Adenylyl Cyclase—Detergent-solubilized preparations of adenylyl cyclase from rat and bovine brains were prepared and assayed as described previously (5, 14, 15). Bovine brain adenylyl cyclase was purified as described by Pfeuffer et al. (25). Inhibition kinetics were determined on enzyme assayed with concentrations of divalent cation fixed in excess of the ATP concentration as described previously (26). Inhibition was determined on enzyme that was activated in the presence of Mn21 and forskolin. Estimation of Bound Phosphate—Bound phosphate was determined on ashed samples by the malachite green method of Stull and Buss (23). Adenine was estimated by uv-absorption at 259 nm following purification on high performance liquid chromatography. Materials—29,59-dd-39-ADP and 29,59-dd-39-ATP were prepared as described previously (24). Lubrol PX (from Sigma) was filtered through alumina (Neutral, AG7, from Bio-Rad Laboratories) to remove peroxides. Recombinant Type I enzyme was generously supplied by Drs. R. Taussig and A. G. Gilman. Enzyme was purified by forskolin affinity chromatography from extracts of fall army worm ovarian (Sf9) cells infected with a Type I adenylyl cyclase encoding baculovirus (27). Other reagents were of the highest quality from commercial sources. RESULTS AND DISCUSSION 29,59-Dideoxyadenosine 39-phosphates are a family of inhibitors of adenylyl cyclase in which potency increased with the number of 39-phosphates (Fig. 1). IC50 values for inhibition of 1 I. Shoshani, R. Taussig, and R. A. Johnson, submitted for publication. 2 The abbreviations used are: 29,59-dd-39-AMP, 29,59-dideoxyadenosine 39-monophosphate; Ado, adenosine; 29-dAdo, 29-deoxyadenosine; 29,59-dd-39-ADP, 29,59-dideoxyadenosine 39-diphosphate; 29,59-dd-39ATP, 29,59-dideoxyadenosine 39-triphosphate; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid. 2380 Downloaded from http://www.jbc.org/ by guest on March 2, 2017 2*,5*-Dideoxyadenosine 3*-di- and triphosphates were tested as inhibitors of brain adenylyl cyclases. With an IC50 ;40 nM, 2*,5*-dideoxy-3*-ATP is the most potent nonprotein synthetic regulator of adenylyl cyclases thus far described. Neither 2*,5*-dideoxy-3*-ADP nor 2*,5*dideoxy-3*-ATP inhibited activity by competition with substrate, and the linear noncompetitive inhibition observed was consistent with interaction via a distinct domain. The availability of this ligand will permit the development of a variety of probes that will be extremely useful in investigating adenylyl cyclase structure and the role(s) that this class of compound may play in physiologically regulating cell function. twelve membrane spanning regions and two cytosolic domains (19). The two cytosolic domains (C1 and C2) are homologous with each other and with the established catalytic domain of guanylyl cyclases (20), supporting the idea that each contains a nucleotide binding region. However, it is not known whether C1, C2, or both form the catalytic site, nor whether one or both domains participate in inhibition by P-site ligands. Inhibition kinetics and irreversible inactivation studies with P-site-selective covalent affinity probes are consistent with inhibition occurring at a site that is distinct from the catalytic site (13–15).1 Shared key structural requirements for substrate and for P-site ligands include a requirement for adenine, enhanced efficacy with 29-deoxyadenosine derivatives compared with those of adenosine, and a requirement (catalysis) or preference (inhibition) for phosphate (1–5). Given the shared requirements of these ligands, some similarities in the binding domains for each may be expected. Lacking have been high affinity labeled ligands suitable for binding to either or both domains. The most potent inhibitory ligands have been 29,59-dideoxyadenosine 39-monophosphate (29,59-dd-39-AMP)2 and the naturally occurring 39-AMP and 29-d-39-AMP, with IC50 values in the micromolar range (5, 22). In the synthesis of 29,59-dd-39AMP, referred to in a previous study (5), a small amount of a more potent inhibitory peak also was detected in HPLC eluates. Upon ashing, this unexpected inhibitor was found to contain two phosphates per adenine (23) and the most likely product was 29,59-dd-39-ADP. Subsequently, we developed a synthesis for the 39-polyphosphorylated derivatives of 29,59ddAdo (24). Reported here are the effects of these agents on adenylyl cyclase and the mode of their inhibition. 2381 Adenylyl Cyclase Inhibition by 29,59-Dideoxy-39-ATP TABLE I IC50 values for inhibition of rat brain adenylyl cyclase by adenine nucleosides and adenine 39-nucleotides Activities were determined as described for Fig. 1. Nucleoside IC50 Adenosine 59-Deoxyadenosine 29-Deoxyadenosine 39-AMP 29-d-39-AMP 29,59-Dideoxyadenosine 29,59-dd-39-AMP 29,59-dd-39-ADP 29,59-dd-39-ATP 82a 17a 15a 8.9a 1.2a 2.7a 0.46 0.10 0.04 mM a TABLE II IC50 values for inhibition of different adenylyl cyclases by 29,59-dideoxyadenosine 39-polyphosphates FIG. 1. Inhibition of rat brain adenylyl cyclase by 2*,5*dideoxyadenosine and its 3*-phosphorylated derivatives. Activities were determined in the presence of 5 mM MnCl2, 100 mM 59-ATP, 1 mM 3-isobutyl-1-methylxanthine, 100 mM forskolin, 1 mg of bovine serum albumin per ml, 3 mM dithiothreitol, 0.1% (w/v) Lubrol PX, 50 mM MOPS buffer, and an ATP-regenerating system including 4 mM phosphoenolpyruvate and 100 mg of pyruvate kinase per ml. Enzyme source 29,59-dd-39- 29,59-dd-39- 29,59-dd-39AMP ADP ATP nM Rat brain, detergent extracta Bovine brain, purifiedb Type I, purified recombinantd 460 NDc 1990 100 560 250 40 120 170 a Values from Table I. Forskolin eluates from forskolin-affinity column as per Ref. 25. ND, not determined. d Forskolin eluate as per Ref. 27. b c FIG. 2. Kinetics of inhibition of purified bovine brain adenylyl cyclase by 2*,5*-dd-3*-ATP. Activities were determined in the presence of 5 mM MnCl2 in excess of the ATP concentration, 100 mM forskolin, and an ATP-regenerating system including 2 mM creatine phosphate and 100 mg of creatine kinase per ml. and intercepts were also linear with inhibitor concentration. This linear noncompetitive behavior was also seen with the crude, detergent-extracted adenylyl cyclase from rat brain (not shown) and also with 29,59-dd-39-ADP (not shown). This behavior argues strongly that inhibition occurred at a site distinct from catalysis and is fully consistent with inhibition occurring at the P-site. P-site-mediated inhibition of adenylyl cyclases is characteristically noncompetitive with respect to substrate, whether MnzATP or MgzATP (1, 3, 7, 8, 13, 14, 16, 28). Thus, the evidence supports the conclusion that adenylyl cyclases contain distinct and interacting adenine nucleotide binding domains for catalysis (59-ATP) and inhibition (29,59-dd-39-ATP). The straightforward and linear kinetic behavior was somewhat surprising with the cruder detergent extracts as we had expected that phosphohydrolases might have contributed to Downloaded from http://www.jbc.org/ by guest on March 2, 2017 the enzyme by these nucleotides are presented in Table I and are compared with those of the parent nucleoside and with known ligands. 29,59-dd-39-ADP and 29,59-dd-39-ATP exhibited IC50 values of 0.1 mM and 0.04 mM, respectively. Potency of the 39-monophosphate (29,59-dd-39-AMP, IC50 ;0.5 mM) was enhanced approximately 5-fold by the addition of the second phosphate at the 39-position (29,59-dd-39-ADP) and an additional two-plus-fold by the addition of the third phosphate (29,59-dd-39-ATP). Thus, the most effective inhibitory ligands of adenylyl cyclase are effected by the removal of both 29- and 59-hydroxyl groups from adenosine and the addition of polyphosphate at the 39-position (Table I). The rank order noted here was maintained with the purified native bovine type I and with the recombinant wild type type I adenylyl cyclases, although with these enzymes each of the 39-nucleotides was noticeably less potent than with the enzyme in the cruder detergent-dispersed rat brain preparation (Table II). The reason for the loss of inhibitory potency of these 39-nucleotides upon purification of adenylyl cyclase is unknown but is consistent with previously noted observations with 29-d-39-AMP and 29,59-ddAdo with the bovine brain enzyme (5). The reduced sensitivity may be due simply to the changes in structure of the enzyme upon isolation and removal of the native phospholipid environment. 29,59-dd-39-ATP (IC50 ;40 nM) is almost two orders of magnitude more potent than previous ligands in inhibiting native adenylyl cyclase. It the most potent non-protein regulator of adenylyl cyclases thus far described. Since inhibition was also observed with purified adenylyl cyclase, it is clear that 29,59dd-39-ATP acts directly on the enzyme, exerting inhibition independent of either hormone receptor, stimulatory or inhibitory G-protein, or G-protein subunit. It approaches the potency of the stimulatory effect of rGsa on the type I adenylyl cyclase and the stimulatory and inhibitory effects of bg on as-activated types II and I, respectively (27). Structurally, 29,59-dd-39-ATP and 29,59-dd-39-ADP share some properties with 59-ATP. To ascertain whether either 39nucleotide might inhibit activity through simple competition with 59-ATP at the catalytic site, inhibition kinetics were evaluated (Fig. 2). In this experiment, inhibition by 29,59-dd-39-ATP of the forskolin affinity-purified enzyme from bovine brain was found to be noncompetitive with respect to substrate. Moreover, inhibiton was linear (Fig. 2, inset) in that replots of slopes Values from Ref. 5. 2382 Adenylyl Cyclase Inhibition by 29,59-Dideoxy-39-ATP significant breakdown of the 39-polyphosphates (21). However, we noted no meaningful breakdown of either nucleotide during assay incubations either with purified or with the crude Lubrol PX-extracted adenylyl cyclase. The inhibitory potency of 29,59-dd-39-ATP suggests that derivatives of it may be useful as a labeled ligand to probe the P-site binding domain on adenylyl cyclase. This could circumvent one of the major constraints in the identification of amino acids involved in the binding of P-site ligands. Presently one must rely on inhibition of catalysis, even for evaluating efficacy of site-directed mutations of expressed protein. With inhibition as end point one is by definition not directly quantifying a single domain on this enzyme, but is rather measuring a downstream event. Changes in catalysis could occur independent of changes in the binding of inhibitory ligand, simply through modification of a domain that influences coupling between inhibitory and catalytic domains. The affinity of 29,59-dd-39-ATP is sufficient to allow a binding assay to be developed, and this could significantly aid studies in this direction. REFERENCES 1. Weinryb, I., and Michel, I. M. (1974) Biochim. Biophys. Acta 334, 218 –225 2. Londos, C., and Wolff, J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5482–5486 3. Haslam, R. J., Davidson, M. M. L., and Desjardins, J. V. 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Chem. 1996, 271:2380-2382. doi: 10.1074/jbc.271.5.2380 Access the most updated version of this article at http://www.jbc.org/content/271/5/2380 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Downloaded from http://www.jbc.org/ by guest on March 2, 2017 This article cites 28 references, 19 of which can be accessed free at http://www.jbc.org/content/271/5/2380.full.html#ref-list-1