Báo cáo Y học: Soluble guanylate cyclase is allosterically inhibited by direct interaction with 2-substituted adenine nucleotides

doi:10.1046/j.1432-1033.2002.02874.x

Eur. J. Biochem. 269, 2186–2193 (2002) (cid:2) FEBS 2002

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

tlswlC yt vrl tpBtvsyvl oyving onMyovnsK LgriBiving nM tTJ Bu bdtpBtvivpvlC gpo,lnviClt Eyt yttnoiyvlC Eivr y Closlytl ig Vmax, 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.

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 rlyvd tvyB,l lgvlsnvnfig ig vrl igvltviglK 2rl msltlgv tvpCu lfyffd iglC vrl lVlovt nM bdtpBtvivpvlC yClgigl gpo,lnviClt ng ospCl ygC mpsi9lC tTJK bd6pBtvivpvlC gpo,lnviClt igriBivlC Byty, ygC DadyoviwyvlC ospCl ygC mpsi9lC tTJh Erlg Ae bR tlswlC yt vrl tpBtvsyvl oyving onMyovnsK 6iffi,ys,uh bdtpBtvid vpvlC yClgigl gpo,lnviClt igriBivlC vrntl lgUufflt Erlg Ag bR h Erior yoviwyvlt tTJ ig y ,ieygCdigClmlgClgv Mytringh

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].

Cyclic GMP (cGMP) is an important signaling molecule functions, that regulates many physiological 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 Mn2+ through ligand-independent mechanisms, and require a divalent cation (Mn2+ or Mg2+) 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

pGCs are allosterically regulated by adenine nucleotides in a complex fashion. When Mg2+ 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 Ki (cid:1) 10)4 M [19]. Allosteric inhibition by those nucleotides was associated with a decrease in Vmax, 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)

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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].

protein were incubated for 5 min at 37 (cid:3)C in 100 lL of 50 mM Tris buffer (pH 7.5), which contained 500 lM isobutylmethylxanthine (IBMX), 15 mM creatine phos- phate, 2.7 U of creatine phosphokinase, MgCl2 or MnCl2 (3 mM 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 (cid:3)C in 100 lL of 50 mM Tris buffer (pH 7.4), 0.5 mgÆmL)1 BSA, 1 mM dithiothreitol, MgCl2 or MnCl2 (3 mM in excess of nucleo- tide unless otherwise stated), and GTP, 50 lM SNP, and 2- substituted adenine nucleotides where indicated. Enzyme reactions were terminated by the addition of 50 mM 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

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.

M A T E R I A L S A N D M E T H O D S

Cell culture

these preparations

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 following SDS/PAGE revealed that > 95% of their composition was a and b subunits (data not shown). These observations are iden-

T84 cells (ATCC, Rockville, MD, USA) were grown at 37 (cid:3)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% CO2 [23].

Preparation of membranes

Confluent cells were washed twice with TED [50 mM Tris/ HCL (pH 7.5) containing 1 mM EDTA, 1 mM dithothre- itol, and 1 mM phenylmethanesulfoxide], collected by scraping into 5 mL of TED, and homogenized on ice in TED using a Wheaton overhead stirrer. Homogenates were centrifuged (4 (cid:3)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 (cid:3)C and 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) of TEDS (20 mM Tris/HCl (pH 7.5) containing, 1 mM EDTA, 1 mM dithiothreitol, and 250 mM sucrose) followed by centrifugation (4 (cid:3)C) at 100 000 g for 60 min. Supernatants were recovered, adjusted to 2 mg pro- teinÆmL)1 with TEDS, stored at )20 (cid:3)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

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.

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tical to those reported previously for purification of this enzyme by immunoaffinity chromatography employing the same antibody [25].

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].

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.

R E S U L T S

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 Ki for inhibition of sGC by those nucleotides was (cid:1) 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 (Ki; 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.

Mn2+ activates sGC and pGCs in a ligand-independent fashion [1,2,5]. 2MeSATP and 2ClATP inhibited GC-C activity when either Mn2+ or Mg2+ was employed as the substrate cation cofactor [19,20,26]. Similarly, those nucleo- tides maximally inhibited purified sGC activity > 80% in a

Previous studies demonstrated that 1 mM 2MeSATP or 2ClATP inhibited basal and ST- and Mn2+-activated GC-C (Fig. 2A) [19,20,26]. Similarly, 1 mM 2MeSATP or 2ClATP inhibited basal and NO- and Mn2+-stimulated crude rat lung sGC (Fig. 2B). These nucleotides inhibited basal sGC (cid:1) 60%, NO-activated enzyme (cid:1) 50%, and Mn2+-activated sGC (cid:2)90%. In addition, 1 mM 2MeSATP or 2ClATP inhibited basal and NO- and Mn2+-stimulated sGC puri- fied to apparent homogeneity (Figs 1 and 2C). Inhibition of crude and purified sGC was comparable suggesting that for mediating 2-substituted adenine factors important

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 lM ST, 50 lM SNP, 3 mM excess Mg2+ or Mn2+, or 1 mM 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 Mg2+ as the sub- strate cation cofactor. Guanylate cyclase activity was measured in the presence of varying concentrations of 2MeSATP (h) or 2ClATP (m) in the absence (upper panels) or presence (lower panels) of 50 lM 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 ± 1.2 pmol cGMP min)1Æmg)1 of protein and 77.3 ± 33.03 nmol cGMP min)1Æmg)1 of protein, respectively. Activities of crude and purified sGC stimulated by SNP were 100 ± 18 pmol cGMP min)1Æmg)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 Ki values presented in Table 1.

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Table 1. Ki 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 mM GTP, and 3 mM excess metal cation. Values ± SEM were determined from nonlinear regression analysis of the sigmoidial plots from three separate experiments. ND, not determined.

Ki ± SEM (lM)

Mg2+ Mg2+ + 50 lM SNP Mn2+ sGC Nucleotide

Crude

concentration-dependent fashion when Mn2+ was utilized as the substrate cofactor (Fig. 4). Interestingly, the potencies of 2-substituted nucleotides to inhibit sGC significantly increased employing Mn2+ as the cation cofactor. Thus, the Ki values of 2MeSATP and 2ClATP decreased greater than ninefold in the presence of Mn2+ compared to Mg2+ (Table 1).

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 lM, consistent with previous observations (Table 3) [26]. At concentrations > 100 lM, GTP inhibited GC-C (data not shown) [26]. In contrast, increasing concentrations of GTP from 10 to

The effects of 2-substituted nucleotides on sGC activity were examined in the presence of increasing concentrations of substrate. Employing Mg2+ as the substrate cofactor, 2MeSATP reduced the Vmax of basal and SNP-stimulated purified sGC activity by 65% and 77%, respectively (Fig. 5, Table 2). 2MeSATP also increased the Km of purified basal and SNP-stimulated sGC threefold and fourfold, respect- ively [Table 2]. Employing Mn2+ as the cation cofactor, 2MeSATP decreased the Vmax of purified sGC by 80% and increased the Km (cid:1) 1.5-fold (Fig. 6, Table 2). These char- acteristics, including a decrease in Vmax and increase in Km, 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].

Purified 2MeSATP 2ClATP 2MeSATP 2ClATP 570 ± 240 457 ± 57 561 ± 166 370 ± 136 767 ± 158 282 ± 66 460 ± 119 99 ± 31 ND ND 53 ± 24 42 ± 22

Fig. 5. Effect of 2MeSATP on the relationship between activity and substrate concentration of (A) basal and (B) SNP-stimulated purified sGC using Mg2+ 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 MgCl2 as the substrate cation cofactor, employing Michaelis (left) and Lineweaver–Burke (right) plots analysis. Open circles, no addition; closed squares, 1 mM 2MeSATP; open triangles, 50 lM SNP; closed diamonds, 50 lM SNP + 1 mM 2MeSATP. Fig. 4. Effect of adenine nucleotides on purified sGC activity using Mn2+ as the substrate cation cofactor. Guanylate cyclase activity was measured in the presence of increasing concentrations of 2MeSATP (h) or 2ClATP (m), 1 mM MnGTP, and 3 mM Mn2+ in excess of nucleotides. Enzyme activities are expressed as fractional response as described in Fig. 3. Basal activity of purified sGC using Mn2+ as the substrate cofactor was 369 ± 47 nmol cGMP min)1Æmg)1 of protein. Nonlinear regression analysis of the sigmoidial plots for each of the nucleotides was used to estimate the Ki values presented in Table 1.

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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 lM)10 mM) or MnGTP (3.9 lM to 1 mM) in the presence or absence of 2MeSATP. The Vmax and Km were determined by nonlinear regression analysis of Michaelis plots. Values ± SEM are representative of three experiments. ND, not determined.

MgGTP MnGTP

a Vmax

a Vmax

Agonist Km (lM) Km (lM)

a Nanomoles of cGMP produced per minÆmg)1 of protein.

Basal 2MeSATP SNP SNP + 2MeSATP 181 ± 55 65 ± 24 2711 ± 1079 630 ± 190 0.50 ± 0.18 1.36 ± 0.33 0.07 ± 0.01 0.28 ± 0.11 311 ± 93 67 ± 0.06 ND ND 13 ± 5.5 17 ± 6.2 ND ND

Table 3. Effect of GTP on the potency of 2-substituted adenine nucleotides to inhibit purified sGC. ND, not determined.

Ki ± SEM

GC-C Purified sGC GTP (lM)

a < 0.05 vs. the Ki value of 2MeSATP at 10 lM GTP for GC-C.

88.2 ± 5.9 ND ND 12.1 ± 2.7 a 10 20 50 100 12.5 ± 3.6 11.0 ± 4.0 16.5 ± 2.5 38.2 ± 9.6

D I S C U S S I O N

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 C1–C2 interface of the catalytic domain of adenylate cyclase, the P site, which mediates allosteric inhibition [28]. P site effectors inhibit forskolin-, Gsa-, or Mn2+-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

100 lM did not increase the potency of 2MeSATP to induce inhibition and concentrations of GTP > 100 lM, did not directly inhibit sGC (Fig. 6, Table 3).

Fig. 6. Effect of 2MeSATP on the relationship between activity and substrate concentration of purified sGC using Mn2+ 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 mM 2MeSATP with Mn2+ as the substrate cation cofactor. Open circles, 1 mM MnGTP; closed squares, 1 mM MnGTP + 1 mM 2MeSATP. (B) Double-reciprocal plot of the data presented in panel (A). Open circles, 1 mM MnGTP; closed squares, 1 mM MnGTP + 1 mM 2MeSATP.

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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 C1–C2 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 C1–C2 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.

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 Mg2+ or Mn2+ as the substrate cation cofactor. Those nucleotides inhibited GC-C in cell-free preparations and in intact cells, in which they blocked the including downstream effects of ST-GC-C interaction, 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 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 AP3A and AP4A, 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 (cid:2)alarmones(cid:3), 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+ (kATP) channel. In this system, ATP directly binds to a subunit of the kATP 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.

The present study demonstrates that, like GC-C, 2-sub- stituted adenine nucleotides allosterically inhibit basal and NO-activated sGC, employing Mg2+ or Mn2+ 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 the catalytic domain [19]. Soluble and particulate of 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

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

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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.

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.

A C K N O W L E D G E M E N T S

(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. These studies were supported by a grant from the NIH (HL59214-01). I. R. S. was supported by a NIH minority supplement (HL59214- 0151).

R E F E R E N C E S

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