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Abstract

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.