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

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 35SATPgammaS, 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.