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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.
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