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). 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.
McNicoll
N
N
Department of Pharmacology, Faculty of Medicine, University of Montreal, Canada.
Gagnon
J
J
Rondeau
J J
JJ
Ong
H
H
De Léan
A
A
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D045283:Natriuretic Agents / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D015384:Zona Glomerulosa
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D008024:Ligands; D045283:Natriuretic Agents; D010446:Peptide Fragments; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D003488:Cyanogen Bromide