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.

By light and electron microscope radioautography in vivo, competitive binding sites for 125I-Arg 101-Tyr 126 atrial natriuretic factor were localized mostly on the "pigmented" epithelium of the rat ciliary process. Further investigation using isolated ciliary processes from rabbits demonstrated the presence of specific receptors for 125I-atrial natriuretic factor. In addition, synthetic atrial natriuretic factor inhibited basal and stimulated adenylate cyclase activity. These results demonstrate for the first time the presence of specific receptors for atrial natriuretic factor in the ciliary processes which are negatively coupled to adenylate cyclase. The possible role of this peptide in the control of intraocular pressure is suggested.

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A portion of the ligand binding domain for atrial natriuretic peptide (ANP) was identified as an affinity cross-linked proteolytic fragment of bovine adrenal natriuretic peptide receptor type-A (NPR-A). Affinity purified NPR-A was UV-cross-linked to the amino terminus of 125I-[Tyr2] rat ANP-(2-27). A chymotryptic fragment of the affinity labeled NPR-A was isolated by chromatography and electrophoresis. This fragment yielded a major microsequence corresponding to a region from Met173 to Phe188 of the receptor extracellular domain and containing one N-glycosylation site at Asn180. Bovine NPR-A receptor was then cross-linked to the carboxy terminus of the highly efficient photoaffinity derivative 125I-[Tyr18,Bpa27] rat ANP(1-27). Proteolysis of the affinity labeled NPR-A with cyanogen bromide and trypsin produced radiolabeled and glycosylated fragments of size 15 and 9 kDa, respectively, which contained the epitope Ile181-Phe188 (CS328) and which were detectable by immunoprecipitation with a monospecific polyclonal antibody against CS328. Proteolysis with cyanogen bromide followed by Glu-C produced a shorter photolabeled 6 kDa fragment which was not immunoprecipitable by anti-CS328 antibody and which was not glycosylated. The results lead to the identification of the short segment Asp191-Arg198 as the site of covalent binding of [Tyr18,Bpa27] rat ANP(1-27). This hydrophilic region is adjacent to the epitope Ile181-Phe188 and to the glycosylation site Asn180. It displays the species variability and the high surface probability expected for a portion of the binding domain of NPR-A in contact with ANP.

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED50 of 12 +/- 2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a Kd of 13 +/- 1 pM, and natriuretic peptides compete for [125I]-CNP binding with a rank order of pCNP > pBNP > rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.