LOCALIZATION BY PHOTOAFFINITY-LABELING OF NATRIURETIC PEPTIDE RECEPTOR-A BINDING DOMAIN

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). Affinit y purified NPR-A was UV-cross-linked to the amino terminus of I-125-[T yr(2)] rat ANP(2-27). A chymotryptic fragment of the affinity labeled NPR-A was isolated by chromatography and electrophoresis. This fragmen t yielded a major microsequence corresponding to a region from Met(173 ) to Phe(188) of the receptor extracellular domain and containing one N-glycosylation site at Asn(180). Bovine NPR-A receptor was then cross -linked to the carboxy terminus of the highly efficient photoaffinity derivative I-125-[Tyr(18),Bpa(27)] ray ANP(1-27). Proteolysis of the a ffinity labeled NPR-A with cyanogen bromide and trypsin produced radio labeled and glycosylated fragments of size 15 and 9 kDa, respectively, which contained the epitope Lle(181)-Phe(188) (CS328) and which were detectable by immunoprecipitation with a monospecific polyclonal antib ody against CS328. Proteolysis with cyanogen bromide followed by Glu-C produced a shorter photolabeled 6 kDa fragment which was not immunopr ecipitable by anti-CS328 antibody and which was not glycosylated. The results lead to the identification of the short segment Asp(191)-Arg(1 98) as the site of covalent binding of [Tyr(18),Bpa(27)] rat ANP(1-27) . This hydrophilic region is adjacent to the epitope Ile(181)-Phe(188) and to the glycosylation site Asn(180). It displays the species varia bility and the high surface probability expected for a portion of the binding domain of NPR-A in contact with ANP.