Mapping peptide-binding domains of the human V1a vasopressin receptor with a photoactivatable linear peptide antagonist

被引:46
作者
Phalipou, S
Cotte, N
Carnazzi, E
Seyer, R
Mahe, E
Jard, S
Barberis, C
Mouillac, B
机构
[1] CNRS, INSERM, U469, CCIPE, F-34094 MONTPELLIER 05, FRANCE
[2] CNRS, CCIPE, UPR 9023, F-34094 MONTPELLIER 05, FRANCE
关键词
D O I
10.1074/jbc.272.42.26536
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The study of antagonist binding domains of the hu man Via vasopressin receptor was performed using a radioiodinated photoreactive peptide antagonist, This ligand displayed a high affinity for the receptor expressed in Chinese hamster ovary cell membranes, and specifically labeled two protein bands with apparent molecular mass at 85-90 and 46 kDa. Our results clearly show that the V1a receptor is degraded during incubation with the ligand and that the 46-kDa. species is probably the result of the 65-90-kDa species proteolytic cleavage, Truncation of the receptor was then confirmed by deglycosylation with N-glycosidase F, A monoclonal antibody directed against a c-Myc epitope added at the receptor NH,terminus allowed. immunoprecipitation of the 85-90-kDa photolabeled species, The 46 kDa photolabeled protein never immunoprecipitated, indicating that the truncated form of the receptor lacks the NN, terminus region. To localize photolabeled domains of the receptor, the 46 kDa protein was cleaved with V8 and/or Lys-C endoproteinases, The identity of the smallest photolabeled fragment, observed at approximately 6 kDa, was then confirmed by mutation of the potential V8 cleavage sites, Our results indicate that covalent labeling of the vasopressin Via receptor with the photoreactive antagonist: occurs in a region including transmembrane domain VII (residues Asn(327)-Lys(370)).
引用
收藏
页码:26536 / 26544
页数:9
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