Reduced Activity of the NPR-A Kinase Triggers Dephosphorylation and Homologous Desensitization of the Receptor

Abstract

NPR-A, the receptor for the atrial natriuretic peptide (ANP), is a 130-kDa protein presenting an extracellular ANP-binding domain, a single transmembrane domain, an intracellular regulatory kinase homology domain (KHD), and a guanylyl cyclase catalytic domain. Upon stimulation, NPR-A receptors are activated to produce cyclic guanosine monophosphate (cGMP) and are subsequently desensitized through dephosphorylation of residues at their KHD. We used wild-type rat (r) NPR-A (WT) and a disulfide-bridged mutant (C423S) expressed in human embryonic kidney (HEK) 293 cells to study receptor phosphorylation. We have previously characterized the C423S receptor as constitutively active and desensitized. At basal state, 32P incorporation in the rNPR-A(C423S) covalent dimer is about 24 times less efficient than incorporation in the WT rNPR-A. When membranes from WT and rNPR-A(C423S) are incubated with [35S]ATPgammaS, the mutant dimer receptor displays 3.5% of the thiophosphate incorporation found for WT rNPR-A. Since the rNPR-A(C423S) dimer is already extensively dephosphorylated, we then used the WT rNPR-A to study dephosphorylation. As previously documented, adding ANP globally induces time-dependent dephosphorylation of the receptor. However, in pulse-chase experiments with the WT rNPR-A, adding ANP during the chase does not lead to a significant effect on receptor dephosphorylation. On the other hand, thiophosphorylation of the WT rNPR-A previously desensitized with ANP is reduced to 8.3% of the incorporation for untreated receptor, similar to results found with the rNPR-A(C423S) at basal state. These results demonstrate that ANP-induced rNPR-A desensitization is modulated by a significant reduction in the activity or affinity of the rNPR-A kinase that contributes to the low phosphorylation level after induction. Moreover, we further document a close relationship between tight dimerization, dephosphorylation, and desensitization.

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

“…Recently, Joubert et al (28) reported that ANP-dependent dephosphorylation of NPR-A in whole transfected 293 cells results primarily from the inactivation of the NPR-A kinase, with little contribution from increased phosphatase activity. These data are completely consistent with our findings in the 293-NPR-A model system.…”

“…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).…”

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

“…ATP increases the activity of both receptors in broken cell preparations by serving as a phosphate donor (11)(12)(13). ATP is also an allosteric activator that reduces the Michaelis-Menten constant without affecting maximal velocities (14).…”

Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

“…Each receptor consists of an extracellular ligand-binding domain, single membrane-spanning domain, and an intracellular region made up of a kinase homology domain-regulatory, hinge-dimerization, and guanylyl cyclase-catalytic domains. Phosphorylation of the kinase homology domains of NPR-A and NPR-B is required for natriuretic peptide-dependent activation (30), and receptor dephosphorylation is associated with desensitization (21,22,30,32,33). Although all particulate guanylyl cyclases contain a domain with limited homology to protein kinases, only GC-E has been reported to possess intrinsic phosphotransferase activity (4).…”