Soluble guanylate cyclase is allosterically inhibited by direct interaction with 2-substituted adenine nucleotides Inez Ruiz-Stewart, Shiva Kazerounian, Giovanni M. Pitari, Stephanie Schulz and Scott A. Waldman Division of Clinical Pharmacology, Departments of Medicine and Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA, USA Nitric oxide (NO), the principal endogenous ligand for sol- uble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, inclu- ding 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat- stable enterotoxin in the intestine. The present study exam- ined the effects of 2-substituted adenine nucleotides on crude and purified sGC. 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg 2+ served as the substrate cation cofactor. Similarly, 2-substi- tuted adenine nucleotides inhibited those enzymes when Mn 2+ , which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in V max , consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mech- anism. These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site. Keywords: soluble guanylate cyclase; adenine nucleotide. Cyclic GMP (cGMP) is an important signaling molecule that regulates many physiological functions, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, cellular proliferation, and photo- transduction (reviewed in [1]). The family of enzymes that synthesize cGMP from GTP, the guanylate cyclases, are expressed by most tissues in the cytoplasmic (soluble) and membrane (particulate) compartments [2–4]. These enzymes can be activated by specific ligands or by free Mn 2+ through ligand-independent mechanisms, and require a divalent cation (Mn 2+ or Mg 2+ ) as an essential cofactor for catalytic activity [5]. Particulate guanylate cyclases (pGCs) are multidomain homo-oligomers and each monomer contains an extracellu- lar ligand-binding domain, a single transmembrane domain, an intracellular kinase homology domain (KHD) and a catalytic domain (reviewed in [1]). Soluble guanylate cyclases (sGCs) are heterodimers composed of a and b subunits and each monomer contains a heme binding domain, a dimeri- zation domain, and a catalytic domain [1,6]. The primary structure of the catalytic domains of sGC and pGC are homologous, reflecting their similarity of function [7,8]. pGCs are allosterically regulated by adenine nucleotides in a complex fashion. When Mg 2+ serves as the cation cofactor, ATP potentiates ligand activation of pGCs presumably by binding to the KHD. The working hypothesis suggests that the KHD is intrinsically inhibitory and ligand–receptor interaction permits association of that domain with ATP resulting in derepression of the catalytic domain [9–11]. It remains unclear whether ATP binding to the KHD dere- presses the enzyme or an intrinsic kinase activity mediates derepression [12]. In addition, ligand activation of pGCs is dependent upon the phosphorylation state of serine and threonine residues within the KHD, which, in turn, is dependent upon ATP [13,14]. Indeed, one mechanism by which desensitization of pGCs may be mediated is ligand- dependent dephosphorylation of those residues [15–17]. Recently, a novel allosteric mechanism mediating inhibi- tion of pGC by adenine nucleotides was identified. Thus, adenine nucleotides substituted in the 2-position of the purine ring inhibited the isoform of pGC expressed in intestinal epithelial cells, GC-C, the receptor for ST that is a major cause of diarrhea in animals and humans [18]. Indeed, 2ClATP and 2MeSATP inhibited basal and ST-stimulated GC-C in a concentration-dependent manner with a K i  10 )4 M [19]. Allosteric inhibition by those nucleotides was associated with a decrease in V max , characteristic of a noncompetitive mechanism and was mediated by the intracellular domains of GC-C [19]. Furthermore, inhibition Correspondence to I. Ruiz-Stewart, Division of Clinical Pharmacology, Thomas Jefferson University, 1100 Walnut Street, MOB 810, Philadelphia, PA 19107, USA. Fax: +1 215 955 7006, Tel.: +1 215 955 0054, E-mail: iar001@jefferson.edu Abbreviations: cGMP, cyclic GMP; 2ClAdo, 2-chloroadenosine; 2ClATP, 2-chloroadenosine triphosphate; GCA, guanylate cyclase A; GC-C, guanylate cyclase C; GTPcS, guanosine 5¢O-(3-triphosphate); IBMX, isobutylmethylxanthine; KHD, kinase homology domain 2MeSATP, 2-methylthioadenosine triphosphate; NO, nitric oxide; pGC, particulate guanylate cyclase; sGC, soluble guanylate cyclase; SNP, sodium nitroprusside; ST, Escherichia coli heat-stable entero- toxin. (Received 3 December 2001, revised 4 March 2002, accepted 11 March 2002) Eur. J. Biochem. 269, 2186–2193 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02874.x of GC-C by 2-substituted nucleotides was guanine nucleo- tide-dependent, suggesting a role for a guanine nucleotide- binding protein in the mechanism mediating allosteric inhibition of GC-C [19]. Incubation of intestinal epithelial cells in vitro with 2ClAdo, which undergoes intracellular transformation to 2ClATP, prevented ST-induced [cGMP] i accumulation and electrolyte transport [20]. While ligand activation by pGCs is regulated in a complex fashion by adenine nucleotides, there appears to be a less well-defined role for those nucleotides in the regulation of NO-activation of sGC. ATP does not activate basal sGC nor is it required to potentiate activation of sGC by NO. Indeed, sGC lacks the KHD present in all known mammalian pGCs that presumably mediates allosteric activation of those enzymes. Previous studies have demon- strated that phosphorylation of sGC by cAMP-dependent protein kinase and protein kinase C increases the respon- siveness of that enzyme to NO [21,22]. Although adenine nucleotides do not appear to be absolutely required for ligand activation, their ability to allosterically inhibit sGC remains unclear. In this study, we examine the allosteric regulation of crude and purified basal, and NO-activated sGC by 2-substituted adenine nucleotides. MATERIALS AND METHODS Cell culture T84 cells (ATCC, Rockville, MD, USA) were grown at 37 °C in Dulbecco’s modified Eagle’s medium/F12 (Mediatech, Herndon, VA, USA), 10% fetal bovine serum (Mediatech, Herndon, VA, USA), and 1% penicillin/ streptomycin (Gibco, Grand Island, NY, USA) in a humi- dified atmosphere of 5% CO 2 [23]. Preparation of membranes Confluent cells were washed twice with TED [50 m M Tris/ HCL (pH 7.5) containing 1 m M EDTA, 1 m M dithothre- itol, and 1 m M phenylmethanesulfoxide], collected by scraping into 5 mL of TED, and homogenized on ice in TED using a Wheaton overhead stirrer. Homogenates were centrifuged (4 °C) at 100 000 g for 60 min to produce a pellet, which was then resuspended in TED at 2 mg proteinÆmL )1 . Membranes were stored at )20 °Cand frozen-thawed once only for analyses. Preparation of crude sGC Rat lungs (Pelfreeze, Rogers, AR, USA) were washed in ice- cold 0.9% NaCl to remove residual blood. Lungs were homogenized on ice with a Wheaton overhead stirrer in 9 vol.(w/v)ofTEDS(20 m M Tris/HCl (pH 7.5) containing, 1m M EDTA, 1 m M dithiothreitol, and 250 m M sucrose) followed by centrifugation (4 °C) at 100 000 g for 60 min. Supernatants were recovered, adjusted to 2 mg pro- teinÆmL )1 with TEDS, stored at )20 °C and frozen-thawed once only for analyses. Guanylate cyclase activity Guanylate cyclase activity was quantified as described previously [24]. Briefly, 20 lg of supernatant or membrane protein were incubated for 5 min at 37 °Cin100lLof 50 m M Tris buffer (pH 7.5), which contained 500 l M isobutylmethylxanthine (IBMX), 15 m M creatine phos- phate, 2.7 U of creatine phosphokinase, MgCl 2 or MnCl 2 (3 m M in excess of nucleotide), and GTP, activating ligand, and 2-substituted adenine nucleotide as indicated in the figure legend. For sGC purified from bovine lung (Alexis Biochemical Corporation, San Diego, CA, USA), 5 ng of protein was incubated for 5 min at 37 °C in 100 lLof 50 m M Tris buffer (pH 7.4), 0.5 mgÆmL )1 BSA, 1 m M dithiothreitol, MgCl 2 or MnCl 2 (3 m M in excess of nucleo- tide unless otherwise stated), and GTP, 50 l M SNP, and 2- substituted adenine nucleotides where indicated. Enzyme reactions were terminated by the addition of 50 m M sodium acetate (pH 4.0) followed by boiling for 3 min. Samples were acetylated and cGMP production quantified by radioimmunoassay [20]. All enzyme reactions were per- formed in duplicate and radioimmunoassays were per- formed in triplicate. Results reflect enzyme activities that were linear with respect to time and protein concentrations. Purified sGC sGC (1.25 lg), purified by immunoaffinity chromatography employing an antibody to the C-terminus of the b1 subunit [25], was analyzed by SDS/PAGE on a precast 8 · 10 cm 12.5% polyacrylamide gel (Owl, Portsmouth, NH, USA) as described previously [25]. The gel, stained with Gelcode Blue (Pierce, Rockford, IL, USA), demonstrated that these preparations were composed of 73- and 70-kDa proteins (the a and b subunits, respectively) (Fig. 1). Densitometric analysis of these preparations following SDS/PAGE revealed that > 95% of their composition was a and b subunits (data not shown). These observations are iden- Fig. 1. SDS/PAGE analysis of sGC immunopurified from bovine lung. sGC (1.25 lg) immunopurified from bovine lung was subjected to SDS/PAGE on a 12.5% polyacrylamide gel and stained with Gelcode Blue, as described in Materials and methods. Ó FEBS 2002 Adenine nucleotides directly inhibit sGC (Eur. J. Biochem. 269) 2187 tical to those reported previously for purification of this enzyme by immunoaffinity chromatography employing the same antibody [25]. Miscellaneous All results are representative of three experiments. 2-subti- tuted adenine nucleotides, EDTA, dithiothreitol, phenyl- methanesulfoxide, sodium nitroprusside (SNP), GTP, IBMX, creatine phosphate, and creatine phosphokinase were obtained from Sigma (St Louis, MO, USA). Protein concentration was determined according to the Bradford method (Bio-Rad, Hercules, CA, USA). Statistical signifi- cance was analyzed employing Student’s t-test. RESULTS Previous studies demonstrated that 1 m M 2MeSATP or 2ClATP inhibited basal and ST- and Mn 2+ -activated GC-C (Fig. 2A) [19,20,26]. Similarly, 1 m M 2MeSATP or 2ClATP inhibitedbasalandNO-andMn 2+ -stimulated crude rat lung sGC (Fig. 2B). These nucleotides inhibited basal sGC  60%, NO-activated enzyme  50%, and Mn 2+ -activated sGC 90%. In addition, 1 m M 2MeSATP or 2ClATP inhibited basal and NO- and Mn 2+ -stimulated sGC puri- fied to apparent homogeneity (Figs 1 and 2C). Inhibition of crude and purified sGC was comparable suggesting that factors important for mediating 2-substituted adenine nucleotide inhibition were not removed during immunopu- rification. This is the first demonstration that 2-substituted nucleotides inhibit guanylate cyclase by directly interacting with the purified enzyme, without a requirement for an intermediate cofactor [26]. 2MeSATP and 2ClATP inhibited basal and NO-activa- ted crude and purified sGC in a concentration-dependent and saturable fashion (Fig. 3). These preparations were maximally inhibited ‡ 80% by those nucleotides. The K i for inhibition of sGC by those nucleotides was  10 )4 M and there were no significant differences in their potency (Table 1). The potencies of adenine nucleotides to inhibit crude and purified sGC (K i ; Table 1) are comparable to those reported for inhibition of GC-C [19]. That the pharmacological characteristics of inhibition by 2-substi- tuted nucleotides were virtually identical for crude and purified sGC supports the suggestion that this inhibition is mediated by direct interaction of those nucleotides with sGC. Mn 2+ activates sGC and pGCs in a ligand-independent fashion [1,2,5]. 2MeSATP and 2ClATP inhibited GC-C activity when either Mn 2+ or Mg 2+ was employed as the substrate cation cofactor [19,20,26]. Similarly, those nucleo- tides maximally inhibited purified sGC activity > 80% in a Fig. 2. Effect of 2-substituted adenine nucleotides on GC-C and sGC. GC-C and sGC activities were determined as described in Materials and methods. Incubations contained 1 l M ST, 50 l M SNP, 3 m M excess Mg 2+ or Mn 2+ ,or1m M 2ClATP or 2MeSATP, where indi- cated. (A) GC-C in T84 cell membranes; (B) crude sGC extracted from rat lung; (C) sGC purified from bovine lung. Fig. 3. Concentration-dependence of inhibition of crude and purified sGC by 2-substituted adenine nucleotides employing Mg 2+ as the sub- strate cation cofactor. Guanylate cyclase activity was measured in the presence of varying concentrations of 2MeSATP (h)or2ClATP(m) in the absence (upper panels) or presence (lower panels) of 50 l M SNP. Enzyme activities are expressed as the ratio of [(enzyme activity in the presence of nucleotide)/(enzyme activity in the absence of nucleotide)] (fractional response). Basal activities of crude and purified sGC were 13.7±1.2pmol cGMPmin )1 Æmg )1 of protein and 77.3 ± 33.03 nmol cGMP min )1 Æmg )1 of protein, respectively. Activities of crude and purified sGC stimulated by SNP were 100 ± 18 pmol cGMP min )1 Æmg )1 of protein and 1.8 ± 0.5 lmol of cGMP per minÆmg )1 of protein, respectively. Nonlinear regression analysis of the sigmoidial plots for each of the nucleotides was used to estimate the K i values presented in Table 1. 2188 I. Ruiz-Stewart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 concentration-dependent fashion when Mn 2+ was utilized as the substrate cofactor (Fig. 4). Interestingly, the potencies of 2-substituted nucleotides to inhibit sGC significantly increased employing Mn 2+ as the cation cofactor. Thus, the K i values of 2MeSATP and 2ClATP decreased greater than ninefold in the presence of Mn 2+ compared to Mg 2+ (Table 1) . The effects of 2-substituted nucleotides on sGC activity were examined in the presence of increasing concentrations of substrate. Employing Mg 2+ as the substrate cofactor, 2MeSATP reduced the V max of basal and SNP-stimulated purified sGC activity by 65% and 77%, respectively (Fig. 5, Table 2). 2MeSATP also increased the K m of purified basal and SNP-stimulated sGC threefold and fourfold, respect- ively [Table 2]. Employing Mn 2+ as the cation cofactor, 2MeSATP decreased the V max of purified sGC by 80% and increased the K m  1.5-fold (Fig. 6, Table 2). These char- acteristics, including a decrease in V max and increase in K m , suggest that 2-substituted adenine nucleotides inhibit puri- fied sGC by a mixed noncompetitive mechanism, consistent with allosteric regulation. These results are nearly identical to those obtained examining the regulation of GC-C by 2-substituted nucleotides [19]. Regulation of GC-C by 2-substituted adenine nucleotides is guanine-nucleotide dependent, and increasing concentra- tions of GTP increase the potency of 2MeSATP and 2ClATP to inhibit GC-C [26]. Thus, the effect of guanine nucleotides on the inhibition of purified sGC by 2-substi- tuted nucleotides was examined. 2MeSATP inhibited GC-C in T84 human colon carcinoma cells (Fig. 2A) and the potency of that nucleotide to induce inhibition was increased nearly eightfold by increasing concentrations of guanine nucleotide from 10 to 100 l M , consistent with previous observations (Table 3) [26]. At concentrations >100 l M , GTP inhibited GC-C (data not shown) [26]. In contrast, increasing concentrations of GTP from 10 to Table 1. K i values for 2MeSATP and 2ClATP inhibition of crude and purified sGC. Guanylate cyclase was assayed in the presence of increasing concentrations of the indicated nucleotide, 1 m M GTP, and 3 m M excess metal cation. Values ± SEM were determined from nonlinear regression analysis of the sigmoidial plots from three separate experiments. ND, not determined. sGC Nucleotide K i ± SEM (l M ) Mg 2+ Mg 2+ +50l M SNP Mn 2+ Crude 2MeSATP 570 ± 240 767 ± 158 ND 2ClATP 457 ± 57 282 ± 66 ND Purified 2MeSATP 561 ± 166 460 ± 119 53 ± 24 2ClATP 370 ± 136 99 ± 31 42 ± 22 Fig. 4. Effect of adenine nucleotides on purified sGC activity using Mn 2+ as the substrate cation cofactor. Guanylate cyclase activity was measured in the presence of increasing concentrations of 2MeSATP (h)or2ClATP(m), 1 m M MnGTP, and 3 m M Mn 2+ in excess of nucleotides. Enzyme activities are expressed as fractional response as described in Fig. 3. Basal activity of purified sGC using Mn 2+ as the substrate cofactor was 369 ± 47 nmol cGMP min )1 Æmg )1 of protein. Nonlinear regression analysis of the sigmoidial plots for each of the nucleotides was used to estimate the K i values presented in Table 1. Fig. 5. Effect of 2MeSATP on the relationship between activity and substrate concentration of (A) basal and (B) SNP-stimulated purified sGC using Mg 2+ as the substrate cation cofactor. Purified sGC activity was quantified, using a range of substrate concentrations in the pres- ence or absence of 2MeSATP with MgCl 2 as the substrate cation cofactor, employing Michaelis (left) and Lineweaver–Burke (right) plots analysis. Open circles, no addition; closed squares, 1 m M 2MeSATP; open triangles, 50 l M SNP; closed diamonds, 50 l M SNP + 1 m M 2MeSATP. Ó FEBS 2002 Adenine nucleotides directly inhibit sGC (Eur. J. Biochem. 269) 2189 100 l M did not increase the potency of 2MeSATP to induce inhibition and concentrations of GTP > 100 l M ,didnot directly inhibit sGC (Fig. 6, Table 3). DISCUSSION Regulation of receptor–effector coupling and effector response by purine nucleotides is a general mechanism regulating transmembrane signaling by nucleotide cyclases. Seven-transmembrane-domain receptors are coupled to adenylate cyclase and cAMP production by heterotrimeric guanine nucleotide-binding (G) proteins. In this system, ligand–receptor interaction induces exchange of GDP for GTP by G proteins which activate their coupling function, permitting receptor-coupled regulation of adenylate cyclase and accumulation of [cAMP] i . In addition, the catalytic domains of adenylate cyclases are allosterically regulated by adenine nucleotides. Thus, adenine nucleotides, including 2¢,5¢-dideoxy-3¢ATP and 2¢,5¢-dideoxy-3¢ADP, inhibit crude and purified adenylate cyclases by a noncompetitive or uncompetitive mechanism [27–30]. These nucleotides are thought to bind directly to the C 1 –C 2 interface of the catalytic domain of adenylate cyclase, the P site, which mediates allosteric inhibition [28]. P site effectors inhibit forskolin-, G sa -, or Mn 2+ -stimulated adenylate cyclase [31–34]. Although 2¢,5¢-dideoxy-3¢ATP and 2¢,5¢-dideoxy- 3¢ADP are not natural products of cellular metabolism, recent studies suggest that 2¢-deoxyadenosine 3¢-polyphos- phates might be the natural allosteric effectors for P site regulation of adenylate cyclases [27]. Regulation of guanylate cyclases by purine nucleotides also is complex. Coupling between the ligand binding and catalytic domains of pGCs is mediated by the KHD in the cytoplasm, which serves as a constitutive repressor of the catalytic domain. This domain contains the 11 subdomains characteristic of protein kinases, but lacks the critical aspartate residue in subdomain VI required for phospho- transferase activity [35]. Ligand–receptor interaction induces Fig. 6. Effect of 2MeSATP on the relationship between activity and substrate concentration of purified sGC using Mn 2+ as the substrate cation cofactor. (A) Michaelis plot of purified sGC in the presence of 2MeSATP. Guanylate cyclase activity was quantified using a range of substrate concentrations in the presence or absence of 1 m M 2MeSATP with Mn 2+ as the substrate cation cofactor. Open circles, 1m M MnGTP; closed squares, 1 m M MnGTP + 1 m M 2MeSATP. (B) Double-reciprocal plot of the data presented in panel (A). Open circles, 1 m M MnGTP; closed squares, 1 m M MnGTP + 1 m M 2MeSATP. Table 3. Effect of GTP on the potency of 2-substituted adenine nucleotides to inhibit purified sGC. ND, not determined. GTP (l M ) K i ± SEM GC-C Purified sGC 10 88.2 ± 5.9 12.5 ± 3.6 20 ND 11.0 ± 4.0 50 ND 16.5 ± 2.5 100 12.1 ± 2.7 a 38.2 ± 9.6 a < 0.05 vs. the K i value of 2MeSATP at 10 l M GTP for GC-C. Table 2. Effect of 2-substituted adenine nucleotides on the kinetic parameters of purified sGC. Guanylate cyclase was assayed in the presence of increasing concentrations of MgGTP (10 l M )10 m M )orMnGTP(3.9l M to 1 m M ) in the presence or absence of 2MeSATP. The V max and K m were determined by nonlinear regression analysis of Michaelis plots. Values ± SEM are representative of three experiments. ND, not determined. Agonist MgGTP MnGTP V max a K m (l M ) V max a K m (l M ) Basal 181 ± 55 0.50 ± 0.18 311 ± 93 13 ± 5.5 2MeSATP 65 ± 24 1.36 ± 0.33 67 ± 0.06 17 ± 6.2 SNP 2711 ± 1079 0.07 ± 0.01 ND ND SNP + 2MeSATP 630 ± 190 0.28 ± 0.11 ND ND a Nanomoles of cGMP produced per minÆmg )1 of protein. 2190 I. Ruiz-Stewart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 association of ATP with the KHD which derepresses the catalytic domain, resulting in accumulation of [cGMP] i [9–11,36,37]. Also, the KHD of GCA contains six critical serine and threonine residues whose phosphorylation by ATP is required for coupling natriuretic peptide–receptor interaction with guanylate cyclase activation [13]. Indeed, one working hypothesis suggests that termination of ligand- induced signaling by GC-A is mediated, in part, by ligand- induced dephosphorylation of the KHD, resulting in desensitization [17]. In addition, GC-C possesses a serine residue (Ser1029) in the C-terminal, the phosphorylation of which by ATP and protein kinase C potentiates stimulation of that enzyme by ST [38,39]. Soluble guanylate cyclase does not possess a KHD and ATP does not allosterically potentiate its activation by NO. However, activation of sGC by NO is regulated by phosphorylation by cAMP- dependent protein kinase and protein kinase C [21,22]. Recently, a novel mechanism by which adenine nucleo- tides allosterically inhibit guanylate cyclases was identified that is analogous to P site inhibition of adenylate cyclases. Thus, the 2-substituted adenine nucleotides, 2MeSATP and 2ClATP, inhibit GC-C by a noncompetitive mechanism [19]. Inhibition was mediated by intracellular domains of GC-C other than the ATP-binding KHD region, which regulates pGCs in a positive allosteric fashion [19]. 2-Sub- stituted nucleotides inhibited basal and ST-stimulated GC-C, employing either Mg 2+ or Mn 2+ as the substrate cation cofactor. Those nucleotides inhibited GC-C in cell-free preparations and in intact cells, in which they blocked the downstream effects of ST-GC-C interaction, including accumulation of [cGMP] i , chloride transport by the cystic fibrosis transmembrane conductance regulator, and vecto- rial water transport [20]. Interestingly, the potency of 2-substituted nucleotides to inhibit GC-C was increased in a concentration-dependent fashion by GTP and the hydro- lysis-resistant analogue GTPcS [26]. These data suggest that allosteric inhibition of GC-C by 2-substituted nucleotides is mediated by a region in the catalytic domain of that enzyme outside the substrate-binding site and may involve a guanine nucleotide-dependent accessory protein [26]. The present study demonstrates that, like GC-C, 2-sub- stituted adenine nucleotides allosterically inhibit basal and NO-activated sGC, employing Mg 2+ or Mn 2+ as the substrate cation cofactor. These data support the suggestion that allosteric inhibition by adenine nucleotides is a generalized mechanism regulating particulate and soluble guanylate cyclases. Also, 2-substituted nucleotides inhibited crude and purified sGC, demonstrating that those nucleo- tides inhibit guanylate cyclases by interacting directly with the enzyme, rather than through a separate coupling protein. Indeed, inhibition of purified sGC by 2-substituted nucleotides was not regulated by guanine nucleotides, supporting a model in which direct interaction of adenine nucleotides with guanylate cyclases mediates allosteric inhibition. Previous studies demonstrated that adenine nucleotide inhibition of GC-C was mediated by an intra- cellular domain outside the KHD or substrate-binding site of the catalytic domain [19]. Soluble and particulate guanylate cyclases exhibit the highest homology in their catalytic domains, which share the common function of converting GTP into cGMP [7]. That 2-substituted adenine nucleotides inhibit sGC and pGC by a noncompetitive mechanism mediated by intracellular domains other than the KHD, and that those enzymes display significant homology only in their catalytic domains supports the suggestion that their allosteric regulation by 2-substituted nucleotides is mediated by regions of those domains outside the substrate binding site. The P site that binds adenine nucleotides and mediates allosteric inhibition resides in the C 1 –C 2 interface of the catalytic domain of adenylate cyclase and deletion or mutation of that site eliminates the ability of those nucleotides to inhibit that enzyme. However, although adenine nucleotide inhibition of adenylate and guanylate cyclases is analogous, the two basic residues important for binding and stabilization of P site inhibitors in the C 1 –C 2 interface of the catalytic domain of adenylate cyclase do not exist in guanylate cyclases. Thus, the site of adenine nucleotide binding and allosteric regulation in guanylate cyclases remains undefined. The physiological effectors and role for adenine nucleo- tide inhibition of nucleotide cyclases remain unclear. P site regulation of adenylate cyclase appears to be mediated by adenosine and 2-deoxyadenosine. This suggests a working hypothesis in which adenosine and 2¢-deoxyadenosine 3¢-polyphosphates are potentially important intracellular regulatory nucleotides of the adenylate cyclase transmem- brane signaling system [27]. 2-Substituted adenine nucleo- tides are not natural products of cellular metabolism, making it unlikely that they are the physiological regulators of guanylate cyclases. However, previous studies demon- strated that diadenosine polyphosphates inhibit sGC [40]. Indeed, preliminary studies suggest that diadenosine poly- phosphates, particularly AP 3 AandAP 4 A, inhibit sGCs and pGCs with kinetic characteristics that are similar to those of 2-substituted nucleotides (I. Ruiz-Stewart & S. A. Waldman, unpublished observations). Diadenosine polyphosphates, also termed ÔalarmonesÕ, are nucleotides produced under pathophysiological conditions, including heat and oxidative stress, that participate in modulating cellular responses to stress [41,42]. These data suggest a working hypothesis in which sGCs, pGCs and [cGMP] i are coordi- nately regulated by diadenosine polyphosphates as part of the integrated stress response of cells. The pharmacological properties of diadenosine polyphosphate regulation of guanylate cyclases will be described in a separate study. In addition to soluble guanylate cyclase, adenine nucle- otides allosterically regulate other proteins and cellular processes. For example, these nucleotides inhibit glycogen synthase, the rate-limiting enzyme in glycogen synthesis, and the uncoupling protein involved in fatty-acid-induced proton transport [43,44]. Also, adenine nucleotides, inclu- ding ATP and ADP, allosterically regulate the ATP- sensitive K + (k ATP ) channel. In this system, ATP directly binds to a subunit of the k ATP channel and mediates channel inhibition [45]. Taken together, these observations highlight the regulatory role of adenine nucleotides in controlling cellular processes and signal transduction, in addition to their more classical role in cellular energy metabolism. In summary, the present study demonstrates that 2-sub- stituted adenine nucleotides inhibit sGC, suggesting that allosteric regulation by those nucleotides is a generalized characteristic of the family of guanylate cyclases. Allosteric inhibition by 2-substituted nucleotides is mediated by their direct interaction with purified sGC, rather than by an inter- mediate coupling protein. Structural homology between sGCs and pGCs suggest that the catalytic domain at a Ó FEBS 2002 Adenine nucleotides directly inhibit sGC (Eur. J. Biochem. 269) 2191 region outside the substrate-binding site mediates inhibition by adenine nucleotides. The endogenous effectors of this allosteric inhibitory mechanism regulating guanylate cyclases remain undefined and their identification is the focus of ongoing studies in this laboratory. ACKNOWLEDGEMENTS These studies were supported by a grant from the NIH (HL59214-01). I. R. S. was supported by a NIH minority supplement (HL59214- 0151). REFERENCES 1. Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S., Chepenik, K.P. & Waldman, S.A. (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol. Rev. 52, 375–414. 2. Hardman, J.G. & Sutherland, E.W. (1969) Guanyl cyclase, an enzyme catalyzing the formation of guanosine 3¢,5¢-monophos- phate from guanosine trihosphate. J. Biol. Chem. 244, 6363–6370. 3. Ishikawa, E., Ishikawa, S., Davis, J.W. & Sutherland, E.W. (1969) Determination of guanosine 3¢,5¢-monophosphate in tissues and of guanyl cyclase in rat intestine. J. Biol. Chem. 244, 6371–6376. 4. Schultz, G., Bohme, E. & Munske, K. (1969) Guanyl cyclase. Determination of enzyme activity. Life Sci. 8, 1323–1332. 5. Waldman, S.A. & Murad, F. (1987) Cyclic GMP synthesis and function. Pharmacol. Rev. 39, 163–196. 6. Kamisaki, Y., Saheki, S., Nakane, M., Palmieri, J.A., Kuno, T., Chang, B.Y., Waldman, S.A. & Murad, F. (1986) Soluble gua- nylate cyclase from rat lung exists as a heterodimer. J. Biol. Chem. 261, 7236–7241. 7. Thorpe, D.S. & Garbers, D.L. (1989) The membrane form of guanylate cyclase. Homology with a subunit of the cytoplasmic form of the enzyme. J. Biol. Chem. 264, 6545–6549. 8. Thorpe, D.S. & Morkin, E. (1990) The carboxyl region contains the catalytic domain of the membrane form of guanylate cyclase. J. Biol. Chem. 265, 14717–14720. 9. Chinkers, M. & Garbers, D.L. (1989) The protein kinase domain of the ANP receptor is required for signaling. Science 245, 1392– 1394. 10. Marala, R.B., Sitaramayya, A. & Sharma, R.K. (1991) Dual regulation of atrial natriuretic factor-dependent guanylate cyclase activity by ATP. FEBS Lett. 281, 73–76. 11. Chinkers, M., Singh, S. & Garbers, D.L. (1991) Adenine nucleo- tides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system. J. Biol. Chem. 266, 4088–4093. 12. Foster, D.C. & Garbers, D.L. (1998) Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor, guanylyl cyclase-A. J. Biol. Chem. 273, 16311–16318. 13. Potter, L.R. & Hunter, T. (1998) Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor. Mol. Cell. Biol. 18, 2164–2172. 14. Potter, L.R. & Hunter, T. (1998) Identification and characteriza- tion of the major phosphorylation sites of the B-type natriuretic peptide receptor. J. Biol. Chem. 273, 15533–15539. 15. Potter, L.R. (1998) Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptor B: dephos- phorylation is a mechanism of desensitization. Biochemistry 37, 2422–2429. 16. Potter, L.R. & Garbers, D.L. (1992) Dephosphorylation of the guanylyl cyclase-A receptor causes desensitization. J. Biol. Chem. 267, 14531–14534. 17. Potter, L.R. & Hunter, T. (1999) A constitutively ÔphosphorylatedÕ guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization. Mol. Biol. Cell 10, 1811–1820. 18. Schulz, S., Green, C.K., Yuen, P.S. & Garbers, D.L. (1990) Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell 63, 941–948. 19. Parkinson, S.J., Carrithers, S.L. & Waldman, S.A. (1994) Opposing adenine nucleotide-dependent pathways regulate gua- nylyl cyclase C in rat intestine. J. Biol. Chem. 269, 22683–22690. 20. Parkinson, S.J., Alekseev, A.E., Gomez, L.A., Wagner, F., Terzic, A. & Waldman, S.A. (1997) Interruption of Escherichia coli heat-stable enterotoxin-induced guanylyl cyclase signaling and associated chloride current in human intestinal cells by 2-chloro- adenosine. J. Biol. Chem. 272, 754–758. 21. Zwiller, J., Revel, M.O. & Basset, P. (1981) Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP- dependent protein kinase. Biochem. Biophys. Res. Comm. 101, 1381–1387. 22. Zwiller, J., Revel, M.O. & Malviya, A.N. (1985) Protein kinase C catalyzes phosphorylation of guanylate cyclase in vitro. J. Biol. Chem. 260, 1350–1353. 23. Cohen, M.B., Jensen, N.J., Hawkins, J.A., Mann, E.A., Thompson, M.R., Lentze, M.J. & Giannella, R.A. (1993) Receptors for Escherichia coli heat stable enterotoxin in human intestine andinahumanintestinalcellline(Caco-2).J. Cell. Physiol. 156, 138–144. 24. Waldman, S.A., O’Hanley, P., Falkow, S., Schoolnik, G. & Murad, F. (1984) A simple, sensitive, and specific assay for the heat-stable enterotoxin of Escherichia coli. J. Infect. Dis. 149, 83–89. 25. Humbert, P., Niroomand, F., Fischer, G., Mayer, B., Koesling, D.,Hinsch,K.D.,Gausepohl,H.,Frank,R.,Schultz,G.& Bo ¨ hme, E. (1990) Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method. Eur.J.Biochem.190, 273–278. 26. Parkinson, S.J. & Waldman, S.A. (1996) An intracellular adenine nucleotide binding site inhibits guanyly cyclase C by a guanine nucleotide-dependent mechanism. Biochemistry 35, 3213–3221. 27. Desaubry, L., Shoshani, I. & Johnson, R.A. (1996) Inhibition of adenylyl cyclase by a family of newly synthesized adenine nucleoside 3¢-polyphosphates. J. Biol. Chem. 271, 14028–14034. 28. Dessauer, C.W. & Gilman, A.G. (1997) The catalytic mecha- nism of mammalian adenylyl cyclase. Equilibrium binding and kinetic analysis of P-site inhibition. J. Biol. Chem. 272, 27787– 27795. 29. Wolff, J., Londos, C. & Cooper, D.M. (1981) Adenosine receptors and the regulation of adenylate cyclase. Adv. Cyc. Nucl. Res. 14, 199–214. 30. Johnson, R.A., Saur, W. & Jakobs, K.H. (1979) Effects of pros- taglandin E1 and adenosine on metal and metal-ATP kinetics of platelet adenylate cyclase. J. Biol. Chem. 254, 1094–1101. 31. Londos,C.&Wolff,J.(1977)Twodistinctadenosine-sensitive sites on adenylate cyclase. Proc. Natl Acad. Sci. USA 74, 5482– 5486. 32. Londos, C. & Preston, M.S. (1977) Activation of the hepatic adenylate cyclase system by divalent cations. J. Biol. Chem. 252, 5957–5961. 33. Johnson, R.A., Desaubry, L., Bianchi, G., Shoshani, I., Lyons, E.J., Taussig, R., Watson, P.A., Cali, J.J., Krupinski, J., Pieroni, J.P. & Iyengar, R. (1997) Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3¢-polyphosphates. J. Biol. Chem. 272, 8962–8966. 34. Johnson, R.A. & Shoshani, I. (1990) Kinetics of ‘‘P’’-site-mediated inhibition of adenylyl cyclase and the requirements for substrate. J. Biol. Chem. 265, 11595–11600. 35. Chinkers, M., Garbers, D.L., Chang, M.S., Lowe, D.G., Chin, H.M.,Goeddel,D.V.&Schulz,S.(1989)Amembraneformof guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338, 78–83. 2192 I. Ruiz-Stewart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 36. Larose,L.,McNicoll,N.,Ong,H.&DeLe ´ an, A. (1991) Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions. Biochemistry 30, 8990–8995. 37. Wong, S.K., Ma, C.P., Foster, D.C., Chen, A.Y. & Garbers, D.L. (1995) The guanylyl cyclase-A receptor transduces an atrial natriuretic peptide/ATP activation signal in the absence of other proteins. J. Biol. Chem. 270, 30818–30822. 38. Wada, A., Hasegawa, M., Matsumoto, K., Niidome, T., Kawano, Y., Hidaka, Y., Padilla, P.I., Kurazono, H., Shimonishi, Y. & Hirayama, T. (1996) The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate. FEBS Lett. 384, 75–77. 39. Crane, J.K. & Shanks, K.L. (1996) Phosphorylation and activa- tion of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C. Mol. Cell. Biochem. 165, 111–120. 40. Gu, J.G. & Geiger, J.D. (1994) Effects of diadenosine poly- phosphates on sodium nitroprusside-induced soluble guanylate cyclase activity in rat cerebellum. Neurosci. Lett. 169, 185–187. 41. Baker, J.C. & Jacobson, M.K. (1986) Alteration of adenyl dinu- cleotide metabolism by environmental stress. Proc. Natl Acad. Sci. USA 83, 2350–2352. 42. Johnstone, D.B. & Farr, S.B. (1991) AppppA binds to several proteins in Escherichia coli, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40. EMBO J. 10, 3897–3904. 43. Nuttall, F.Q. & Gannon, M.C. (1993) Allosteric regulation of glycogen synthase in liver. A physiological dilemma. J. Biol. Chem. 268, 13286–13290. 44. Modriansky, M., Murdza Inglis, D.L., Patel, H.V., Freeman, K.B. & Garlid, K.D. (1997) Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nu- cleotide binding and inhibition. J. Biol. Chem. 272, 24759–24762. 45. Zingman, L.V., Alekseev, A.E., Bienengraeber, M., Hodgson, D., Karger, A.B., Dzeja, P.P. & Terzic, A. (2001) Signaling in channel/ enzyme multimers: ATPase transitions in SUR module gate ATP- sensitive K + conductance. Neuron 31, 233–245. Ó FEBS 2002 Adenine nucleotides directly inhibit sGC (Eur. J. Biochem. 269) 2193 . Soluble guanylate cyclase is allosterically inhibited by direct interaction with 2-substituted adenine nucleotides Inez Ruiz-Stewart,. characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at