Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

Pattanaik, Priyaranjan; Fromondi, Laura; Ng, Kwok Peng; He, Jiangyan; Akker, F. van den

doi:10.1016/j.biochi.2009.04.011

Cited by 11 publications

(12 citation statements)

References 29 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: 78%

“…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: 78%

Section: Introductionmentioning

confidence: 99%

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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“…On ANP binding, there is no change in the oligomeric state, but apparently a conformational change occurs which activates the cyclase domain [ 11 ]. Two cyclase domains form an active site and the second messenger cGMP is produced [ 11 , 12 ]. cGMP activates different intracellular signalling cascades which ultimately mediate the above-mentioned cardiovascular functions of ANP and BNP.…”

Section: Introductionmentioning

confidence: 99%

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

et al. 2010

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Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.Structured digital abstract•MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

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“…All animal GCs studied to date show higher catalytic activity in the presence of Mn 2+ compared with Mg 2+ (23). GC activity of the affinity-purified recombinant (plant) AtBRI1 protein is increased by both Mn 2+ and Mg 2+ , although activity is higher in the presence of Mg 2+ (13).…”

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confidence: 98%

Ca ² ⁺ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca ²⁺ channels

Verma

Gehring

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

236

240

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A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep-and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca 2+ -dependent manner.calcium signaling | plant innate immunity | flagellin receptor 2 | brassinosteroid associated kinase 1

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Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

Cited by 11 publications

References 29 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

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

Ca ² ⁺ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca ²⁺ channels

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Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

Cited by 11 publications

References 29 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

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

Ca 2 + signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

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Ca ² ⁺ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca ²⁺ channels