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Vol. 10, Issue 6, 1811-1820, June 1999
Molecular Biology and Virology Laboratory, The Salk Institute for
Biological Studies, La Jolla, California 92037
Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is
hypothesized to mediate its desensitization in response to atrial
natriuretic peptide (ANP) binding. Recently, we identified six
phosphorylation sites within the kinase homology domain of NPR-A and
determined that the conversion of these residues to alanine abolished
the ability of the receptor to be phosphorylated or to be activated by
ANP and ATP. In an attempt to generate a form of NPR-A that mimics a
fully phosphorylated receptor but that is resistant to
dephosphorylation, we engineered a receptor variant (NPR-A-6E)
containing glutamate substitutions at all six phosphorylation sites.
Consistent with the known ability of negatively charged glutamate
residues to substitute functionally, in some cases, for phosphorylated
residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP.
As determined by guanylyl cyclase assays, the hormone-stimulated
activity of the wild-type receptor declined over time in membrane
preparations in vitro, and this loss was blocked by the
serine/threonine protein phosphatase inhibitor microcystin. In
contrast, the activity of NPR-A-6E was more linear with time and was
unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine
5'-(
,
-imino)-triphosphate was half as effective as ATP
in stimulating the wild-type receptor but was equally as potent in
stimulating NPR-A-6E, suggesting that ATP is required to keep the
wild-type but not 6E variant phosphorylated. Finally, the
desensitization of NPR-A-6E in whole cells was markedly blunted
compared with that of the wild-type receptor, consistent with its
inability to shed the negative charge from its kinase homology
domain via dephosphorylation. These data provide the first
direct test of the requirement for dephosphorylation in guanylyl
cyclase desensitization and they indicate that it is an essential
component of this process.
This article has been cited by other articles:
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  L. K. Antos and L. R. Potter Adenine nucleotides decrease the apparent Km of endogenous natriuretic peptide receptors for GTP Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1756 - E1763. [Abstract] [Full Text] [PDF]
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 L. R. Potter, S. Abbey-Hosch, and D. M. Dickey Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions Endocr. Rev., February 1, 2006; 27(1): 47 - 72. [Abstract] [Full Text] [PDF]
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 D. Fan, P. M. Bryan, L. K. Antos, R. J. Potthast, and L. R. Potter Down-Regulation Does Not Mediate Natriuretic Peptide-Dependent Desensitization of Natriuretic Peptide Receptor (NPR)-A or NPR-B: Guanylyl Cyclase-Linked Natriuretic Peptide Receptors Do Not Internalize Mol. Pharmacol., January 1, 2005; 67(1): 174 - 183. [Abstract] [Full Text] [PDF]
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 N. Airhart, Y.-F. Yang, C. T. Roberts Jr., and M. Silberbach Atrial Natriuretic Peptide Induces Natriuretic Peptide Receptor-cGMP-dependent Protein Kinase Interaction J. Biol. Chem., October 3, 2003; 278(40): 38693 - 38698. [Abstract] [Full Text] [PDF]
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S. E. Abbey and L. R. Potter Vasopressin-dependent Inhibition of the C-type Natriuretic Peptide Receptor, NPR-B/GC-B, Requires Elevated Intracellular Calcium Concentrations J. Biol. Chem., November 1, 2002; 277(45): 42423 - 42430. [Abstract] [Full Text] [PDF]
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P. M. Bryan and L. R. Potter The Atrial Natriuretic Peptide Receptor (NPR-A/GC-A) Is Dephosphorylated by Distinct Microcystin-sensitive and Magnesium-dependent Protein Phosphatases J. Biol. Chem., May 3, 2002; 277(18): 16041 - 16047. [Abstract] [Full Text] [PDF]
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 D. Muller, R. Middendorff, J. Olcese, and A. K. Mukhopadhyay Central Nervous System-Specific Glycosylation of the Type A Natriuretic Peptide Receptor Endocrinology, January 1, 2002; 143(1): 23 - 29. [Abstract] [Full Text] [PDF]
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 M. B. Hussain, R. J. MacAllister, and A. J. Hobbs Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1151 - H1159. [Abstract] [Full Text] [PDF]
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 A. S. Gukovskaya, S. Gukovsky, and S. J. Pandol Endoplasmic reticulum Ca2+-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells Am J Physiol Cell Physiol, February 1, 2000; 278(2): C363 - C371. [Abstract] [Full Text] [PDF]
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L. R. Potter and T. Hunter Activation of Protein Kinase C Stimulates the Dephosphorylation of Natriuretic Peptide Receptor-B at a Single Serine Residue. A POSSIBLE MECHANISM OF HETEROLOGOUS DESENSITIZATION J. Biol. Chem., September 29, 2000; 275(40): 31099 - 31106. [Abstract] [Full Text] [PDF]
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L. R. Potter and T. Hunter Guanylyl Cyclase-linked Natriuretic Peptide Receptors: Structure and Regulation J. Biol. Chem., February 23, 2001; 276(9): 6057 - 6060. [Full Text] [PDF]
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