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 transmembr ane domain, an intracellular regulatory kinase homology domain (KHD), and a guanylyl cyclase catalytic domain. Upon stimulation, NPR-A receptors are a ctivated to produce cyclic guanosine monophosphate (cGMP) and are subsequen tly desensitized through dephosphorylation of residues at their KHD. We use d wild-type rat (r) NPR-A (WT) and a disulfide-bridged mutant (C423S) expre ssed in human embryonic kidney (HEK) 293 cells to study receptor phosphoryl ation. We have previously characterized the C423S receptor as constitutivel y active and desensitized. At basal state, P-32 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 wi th [S-35]ATP gammaS, the mutant dimer receptor displays 3.5% of the thiopho sphate 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 d ephosphorylation. As previously documented, adding ANP globally induces tim e-dependent dephosphorylation of the receptor. However, in pulse-chase expe riments with the WT rNPR-A, adding ANP during the chase does not lead to a significant effect on receptor dephosphorylation. On the other hand, thioph osphorylation of the WT rNPR-A previously desensitized with ANP is reduced to 8.3% of the incorporation for untreated receptor, similar to results fou nd with the rNPR-A(C423S) at basal state. These results demonstrate that AN P-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 clos e relationship between tight dimerization, dephosphorylation, and desensiti zation.