Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Yoder, Andrea R.; Stone, Matthew D.; Griffin, Timothy J.; Potter, Lincoln R.

doi:10.1021/bi101700e

Cited by 35 publications

(50 citation statements)

References 28 publications

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“…ATP also reduced the K m of both enzymes sevenfold to ~0.1 mM, which is in the range of cellular concentrations of GTP (39). Together, these data indicate that ATP binds to the receptors in the absence of NPs and that ATP is not required for NP-dependent increases in maximal velocity, as was previously reported for phosphorylated enzyme preparations (22, 23). However, the data also demonstrate that NP binding is required for the ATP-dependent reduction in the K m .…”

Section: Resultssupporting

confidence: 86%

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: Resultssupporting

confidence: 86%

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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“…GC-A and GC-B are phosphorylated on multiple serines and threonines located slightly before and expanding into the N-terminal portion of the kinase homology domain (9). Phosphorylation is required for peptide activation, and prolonged natriuretic peptide exposure or acute exposure to phorbol esters or calcium-elevating agents causes receptor dephosphorylation and inactivation (10).…”

Section: Natriuretic Peptides and Atp Activate And Gö6976 Inhibits Gumentioning

confidence: 99%

ATP Potentiates Competitive Inhibition of Guanylyl Cyclase A and B by the Staurosporine Analog, Gö6976

Robinson

Potter

2011

Journal of Biological Chemistry

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Natriuretic peptides and ATP activate and Gö6976 inhibits guanylyl cyclase (GC)-A and GC-B. Here, the mechanism of inhibition was determined. Gö6976 progressively increased the Michaelis-Menten constant and decreased the Hill coefficient without reducing the maximal velocity of GC-A and GC-B. In the presence of 1 mM ATP, the K i was 1 M for both enzymes. Inhibition of GC-B was minimal in the absence of ATP, and 1 mM ATP increased the inhibition 4-fold. In a reciprocal manner, 10 M Gö6976 increased the potency of ATP for GC-B 4-fold. In contrast to a recent study (Duda, T., Yadav, P., and Sharma, R. K. (2010) FEBS J. 277, 2550 -2553), neither staurosporine nor Gö6976 activated GC-A or GC-B. This is the first study to show that Gö6976 reduces GTP binding and the first demonstration of a competitive inhibitor of a receptor guanylyl cyclase. We conclude that Gö6976 reduces GTP binding to the catalytic site of GC-A and GC-B and that ATP increases the magnitude of the inhibition.

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“…In order for the CNP activation signal to be transmitted to the catalytic domain, the juxtamembrane intracellular region of NPR2 must be phosphorylated on some combination of five serine residues and two threonine residues that have been identified as regulatory (Potter, 1998;Potter and Hunter, 1998;Yoder et al, 2010Yoder et al, , 2012. However, unlike many growth factor receptors, NPR2 phosphorylation is not increased upon binding to its agonist CNP (Potter, 1998).…”

Section: Introductionmentioning

confidence: 99%

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

et al. 2014

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In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

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Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Cited by 35 publications

References 28 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

ATP Potentiates Competitive Inhibition of Guanylyl Cyclase A and B by the Staurosporine Analog, Gö6976

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

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