A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Abstract

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 5Ј-(␤,␥-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.

The Atrial Natriuretic Peptide Receptor (NPR-A/GC-A) Is Dephosphorylated by Distinct Microcystin-sensitive and Magnesium-dependent Protein Phosphatases

“…Microcystin prevents the desensitization of NPR-A in membrane fractions (38), and experiments with the constitutively phosphorylated receptor variant (NPR-A-6E) indicated that the target of the microcystin-sensitive phosphatase is NPR-A itself (31). In this study, we provide evidence for a second distinct NPR-A phosphatase that has characteristics in common with PP2C.…”

“…This process is called homologous desensitization and is mediated, at least in part, by dephosphorylation of all six NPR-A phosphorylation sites, a process termed global dephosphorylation (27)(28)(29)(30). Consistent with this model of desensitization, a "constitutively phosphorylated" version of NPR-A containing glutamate residues at all six phosphorylation sites (NPR-A-6E) to mimic the negative charge of phosphate is hormonally responsive and resistant to homologous desensitization (31). NPR-A is also desensitized in the absence of ANP or BNP by pressor hormones that antagonize the physiologic consequences of natriuretic peptides (32,33).…”

“…The latter receptor is responsive to stimulation by ANP and ATP but cannot be dephosphorylated (31). Hence, if dephosphorylation contributes to the desensitization of NPR-A in this assay, then one would expect that membranes containing NPR-A-6E would have a diminished desensitization response, i.e.…”

“…We also used another stable cell 293 line called 293-NPR-A-6E in these studies. We made this cell line by transfecting the pCMV3-GC-A-6E construct, which encodes the rat cDNA for NPR-A that has all six of its phosphorylation sites mutated to glutamate (31), into the same parental cell line as described above. Cells were grown to 40 -50% confluence in 10-cm dishes that had been precoated with 50 g/ml poly-L-lysine.…”

Regulation of Guanylate Cyclase by ATP and Dithiothreitol in Rat Lung Membrane: Involvement of an Insensitive and a Sensitive State to ATP / Dithiothreitol-Stimulation

“…However, the identity of the responsible protein kinase and PP is not known. From the inhibitory effects of microcystin and okadaic acid on desensitization in cells overexpressing GC-A, the responsible PP was reported to be a microcystin-and okadaic acid-sensitive phosphatase species (19,20). The relevant protein kinase has not yet been clarified.…”