Biochemical and pharmacological characterization of P-site inhibitors on homodimeric guanylyl cyclase domain from natriuretic peptide receptor-A

Joubert, Simon; McNicoll, N.; Léan, André De

doi:10.1016/j.bcp.2006.12.008

Cited by 11 publications

(24 citation statements)

References 35 publications

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“…Several observations and experiments support the idea that ATP binds to the allosteric site that resides in the catalytic domain of GC-A (35, 36). First, ATP and GTP are structurally similar.…”

Section: Discussionmentioning

confidence: 77%

“…Finally, we investigated whether the catalytic domain of GC-A was a symmetric homodimer, as was suggested by Joubert and colleagues (35), or an asymmetric homodimer as envisioned for soluble GC (sGC) and adenylyl cyclase (AC) (35, 40, 41). On the basis of a symmetric homodimeric catalytic domain model, coexpression of mutant (D849A or N968S) and wild-type enzymes would be expected to produce 25% active homodimers of wild-type enzyme (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Fourth, ATP reduced the Michaelis constant ( K m ; a measure of enzyme-substrate affinity) without increasing the maximal velocity of highly phosphorylated preparations of GC-A and GC-B (32, 34). A related, unexplained phenomenon is that mGCs demonstrate positive cooperativity through an allosteric site in the GC domain when assayed under synthetic conditions (in the presence of Mn 2+ and nonionic detergent) but not under physiologic conditions (in the presence of Mg 2+ and ATP) (32, 35, 36). Here, we connect these seemingly disparate observations into a new unified activation model that illuminates the activation mechanism of this important enzyme family.…”

Section: Introductionmentioning

confidence: 99%

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Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

Robinson

Potter

2012

Sci. Signal.

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It is not known how natriuretic peptides and adenosine triphosphate (ATP) activate guanylyl cyclase A (GC-A) and GC-B, which generate the second messenger cyclic guanosine monophosphate. We determined that natriuretic peptides increased the maximum rate of these enzymes >10-fold in a positive cooperative manner in the absence of ATP. In the absence of natriuretic peptides, ATP shifted substrate-velocity profiles from cooperative to linear but did not increase the affinity of GCs for the substrate guanosine triphosphate (GTP) since the Michaelis constant was unchanged. However, in the presence of natriuretic peptides, ATP competed with GTP for binding to an allosteric site, which enhanced the activation of GCs by decreasing the Michaelis constant. Thus, natriuretic peptide binding was required for communication of the allosteric activation signal to the catalytic site. The ability of ATP to activate GCs decreased and enzyme potency (a measure of sensitivity to stimulation) increased with increasing GTP concentrations. Point mutations in the purine-binding site of the catalytic domain abolished GC activity but not allosteric activation. Coexpression of inactive mutants produced half the activity expectedfor symmetric catalytic dimers. 2′-Deoxy-ATP and 2′-deoxy-GTP were poorallosteric activators, but 2′-deoxy-GTP was an effective substrate, consistent with distinct binding requirements for the allosteric and catalytic sites. We conclude that membrane GC domains are asymmetric homodimers with distinct and reciprocally regulated catalytic and allosteric sites that bind to GTP and ATP, respectively. These data define a new membrane GC activation model and provide evidence of a previously unidentified GC drug interaction site.

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Section: Discussionmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

Robinson

Potter

2012

Sci. Signal.

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“…Each monomer contributes unique residues to the catalytic site. Joubert and colleagues created a mutant form of the catalytic domain of rat GC-A by making single amino acid substitutions on each monomer to inactive only one GTP binding site and observed linear kinetics, consistent with mammalian GCs adopting a similar structure to the cyanobacterial GC [17]. Mutagenesis studies converting human GC-E and rat soluble guanylyl cyclase to adenylyl cyclases are consistent with mammalian guanylyl cyclases adopting similar structures and catalytic mechanisms as adenylyl cyclases [10, 11].…”

Section: Introductionmentioning

confidence: 96%

Guanylyl cyclase structure, function and regulation

Potter

2011

Cellular Signalling

241

195

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Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.

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“…The KHD and the GC domains are connected by a coiled-coil that maintains the catalytic moieties in close contact. The GC domain presents two functional and allosterically regulated catalytic sites whose structure is jointly contributed by both subunits (Joubert et al, 2007).…”

mentioning

confidence: 99%

Role of Extracellular Domain Dimerization in Agonist-Induced Activation of Natriuretic Peptide Receptor A

Parat¹,

McNicoll²,

Wilkes³

et al. 2007

Mol Pharmacol

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Natriuretic peptide receptor (NPR)A is composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region, a kinase homology domain, and a guanylyl cyclase domain. The natural agonists atrial and brain natriuretic peptides (ANP, BNP) bind and activate NPRA, leading to cyclic GMP production, which is responsible for their role in cardiovascular homeostasis. Previous studies suggested that stabilization of a dimeric form of NPRA by agonist is essential for receptor activation. However, ligand specificity and sequential steps of this dimerization process have not been investigated. We used radioligand binding, fluorescence resonance energy transfer homoquenching, and molecular modeling to characterize the interaction of human NPRA-ECD with ANP, BNP, the superagonist (Arg 10 ,Leu 12 ,Ser 17 ,Leu 18 )-rANP-(1-28), the minimized analog mini-ANP and the antagonist (Arg 6 ,␤-cyclohexylAla 8 ,D-Tic 16 ,Arg 17 ,Cys 18 )-rANP-(6 -18)-amide (A71915). ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. All the studied peptides, including A71915 antagonist, induce a dosedependent fluorescence homoquenching, specific to dimerization, with potencies highly correlated with their binding affinities. A71915 induced more quenching than other peptides, suggesting stabilization by the antagonist of ECD dimer in a distinct inactive conformation. In summary, these results indicate that the ligand-induced dimerization process of NPRA is different from that for cytokine receptor model. Agonists or antagonists bind to preformed dimeric ECD, leading to dimer stabilization in an active or inactive conformation, respectively. Furthermore, the highly sensitive fluorescence assay designed to assess dimerization could serve as a powerful tool for further detailing the kinetic steps involved in natriuretic peptide receptor binding and activation.

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Biochemical and pharmacological characterization of P-site inhibitors on homodimeric guanylyl cyclase domain from natriuretic peptide receptor-A

Cited by 11 publications

References 35 publications

Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

Guanylyl cyclase structure, function and regulation

Role of Extracellular Domain Dimerization in Agonist-Induced Activation of Natriuretic Peptide Receptor A

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