Localization by Photoaffinity Labeling of Natriuretic Peptide Receptor-A Binding Domain
| Autor: | Huy Ong, Jean Gagnon, J J Rondeau, De Léan A, N. McNicoll |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Glycosylation
medicine.drug_class Blotting Western Molecular Sequence Data Ligands Biochemistry Natriuretic peptide receptor A Atrial natriuretic peptide Natriuretic peptide medicine Animals Chymotrypsin Humans Trypsin Amino Acid Sequence Cyanogen Bromide Receptor Binding Sites Photoaffinity labeling Chemistry Serine Endopeptidases Affinity Labels NPR1 NPR2 Peptide Fragments Rats Cross-Linking Reagents Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Guanylate Cyclase cardiovascular system Cattle Electrophoresis Polyacrylamide Gel Zona Glomerulosa Natriuretic Agents Receptors Atrial Natriuretic Factor Protein Binding Binding domain |
| Zdroj: | Biochemistry. 35:12950-12956 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi960818q |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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