Mapping peptide-binding domains of the human V1a vasopressin receptor with a photoactivatable linear peptide antagonist.
The study of antagonist-binding domains of the human V1a 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 85-90-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 NH2 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 NH2 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 V1a receptor with the photoreactive antagonist occurs in a region including transmembrane domain VII (residues Asn327-Lys370).
Phalipou
S
S
U469 INSERM, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.
Cotte
N
N
Carnazzi
E
E
Seyer
R
R
Mahe
E
E
Jard
S
S
Barberis
C
C
Mouillac
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010455:Peptides / Q000037:antagonists & inhibitors*; D019920:Photoaffinity Labels; D011233:Precipitin Tests; D011485:Protein Binding; D017483:Receptors, Vasopressin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D019920:Photoaffinity Labels; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D010450:Endopeptidases