Soluble guanylate cyclase is allosterically inhibited by directinteraction with 2-substituted adenine nucleotides Inez Ruiz-Stewart, Shiva Kazerounian, Giovanni M.. Previous studies demo
Trang 1Soluble 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 Mg2+
served as the substrate cation cofactor Similarly,
2-substi-tuted adenine nucleotides inhibited those enzymes when
Mn2+, 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 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
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 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
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 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
10)4M[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 toI 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)
Trang 2of 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
M A T E R I A L S A N D M E T H O D S
Cell culture
T84 cells (ATCC, Rockville, MD, USA) were grown
at 37C 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 mMTris/
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 (4C) 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 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 mMTris/HCl (pH 7.5) containing,
1 mM EDTA, 1 mM dithiothreitol, and 250 mM sucrose)
followed by centrifugation (4C) at 100 000 g for 60 min
Supernatants were recovered, adjusted to 2 mg
pro-teinÆmL)1with 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 37C 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, MgCl2or MnCl2 (3 mMin 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 37C in 100 lL of
50 mM Tris buffer (pH 7.4), 0.5 mgÆmL)1 BSA, 1 mM dithiothreitol, MgCl2or MnCl2(3 mMin excess of nucleo-tide unless otherwise stated), and GTP, 50 lMSNP, and 2-substituted adenine nucleotides where indicated Enzyme reactions were terminated by the addition of 50 mMsodium acetate (pH 4.0) followed by boiling for 3 min Samples were acetylated and cGMP production quantified by radioimmunoassay [20] All enzyme reactions were 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.
Trang 3tical 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
R E S U L T S
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 mM2MeSATP or 2ClATP
inhibited basal and NO- and Mn2+-stimulated crude rat
lung sGC (Fig 2B) These nucleotides inhibited basal sGC
60%, NO-activated enzyme 50%, and Mn2+-activated
sGC 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
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 Kifor inhibition of sGC by those nucleotides was 10)4Mand 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
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 Mg2+or Mn2+, or 1 m 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 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 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
w ere 13.7 ± 1.2 pmol cGMP min)1Æmg)1 of protein and 77.3 ± 33.03 nmol cGMP min)1Æmg)1of 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)1of 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.
Trang 4concentration-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).
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 Vmaxof basal and SNP-stimulated
purified sGC activity by 65% and 77%, respectively (Fig 5,
Table 2) 2MeSATP also increased the Kmof purified basal
and SNP-stimulated sGC threefold and fourfold,
respect-ively [Table 2] Employing Mn2+as the cation cofactor,
2MeSATP decreased the Vmaxof purified sGC by 80% and
increased the Km 1.5-fold (Fig 6, Table 2) These
char-acteristics, including a decrease in Vmaxand 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]
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
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.
K i ± SEM (l M )
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 m M MnGTP, and 3 m M Mn2+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)1Æmg)1of protein.
Nonlinear regression analysis of the sigmoidial plots for each of the
nucleotides was used to estimate the K 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 abspres-ence 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 2MeSATP.
Trang 5100 lMdid not increase the potency of 2MeSATP to induce
inhibition and concentrations of GTP > 100 lM, did not
directly inhibit sGC (Fig 6, Table 3)
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 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 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 m M
2MeSATP with Mn 2+ as the substrate cation cofactor Open circles,
1 m 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
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 ) or MnGTP (3.9 l 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
a Nanomoles of cGMP produced per minÆmg)1of protein.
Trang 6association 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
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
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 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
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
C1–C2interface 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
The physiological effectors and role for adenine nucleo-tide inhibition of nucleonucleo-tide 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 alarmones, 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]iare 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 kATPchannel 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
Trang 7region 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
A C K N O W L E D G E M E N T S
These studies were supported by a grant from the NIH (HL59214-01).
I R S was supported by a NIH minority supplement
(HL59214-0151).
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