Direct identification of an extracellular agonist binding site in the renal V2 vasopressin receptor.
To purify the renal V2 receptor and identify domains involved in hormone binding, photoaffinity labeling of the membrane-bound bovine V2 receptor with a tritium-labeled photoreactive vasopressin agonist was performed. The labeled 30,000 M(r) protein was purified to homogeneity by anion-exchange chromatography, isoelectric focusing, gel filtration, gel electrophoresis, and reversed-phase HPLC. N-terminal sequencing showed that the isolated protein which contains the covalently bound hormonal ligand, represents an N-terminal truncated bovine V2 receptor. The purified labeled V2 vasopressin receptor protein was digested with V8 protease, and peptide fragments were isolated. Protein microsequencing and comparison with the cDNA sequence of a cloned PCR product identified two extra- and two intracellular peptides of the bovine V2 receptor. Radioactivity was incorporated into two amino acid residues localized in the second extracellular domain. Our results indicate that this extracellular domain is involved in peptide agonist binding of the V2 receptor.
Kojro
E
E
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Eich
P
P
Gimpl
G
G
Fahrenholz
F
F
D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007679:Kidney Medulla / Q000737:chemistry*; D008236:Lypressin / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D017483:Receptors, Vasopressin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility
D000345:Affinity Labels; D010446:Peptide Fragments; D017483:Receptors, Vasopressin; D008236:Lypressin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase