Photoreactive analogues of vasopressin as tools in the isolation of the renal V2 vasopressin receptor

Eich, Peter; Kojro, Elżbieta; Morgat, Jean-Louis; Fahrenholz, Falk

doi:10.1007/978-94-011-1470-7_322

Cited by 1 publication

(1 citation statement)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…From the amount of radioactivity specifically incorporated into the M, 30 000 protein, it was estimated that the yield of the covalent reaction between the photoreactive aryl azido analogue of 1-deamino [8-lysine] vasopressin and the vasopressin receptor protein was approximately from 3 to 5%. To obtain a higher yield of the V2 receptor labeling, a radioactive analogue of l-deamino[8-lysine] vasopressin with a photoreactive benzoylbenzoyl group in position 8 and a high binding affinity (Ad = 0.4 nM) has been synthesized (Eich et al, 1993). The photolabile benzoylaryl group possesses high reactivity for C-H bonds but a low reactivity toward water (Breslow, 1980;Helene, 1972); photoaffinity labeling of the substance P (Boyd et al, 1991) and cholecystokinin B receptors (Thiele & Fahrenholz, 1993) with a ligand containing this photoreactive group proceeded with a yield of 70%.…”

Section: Discussionmentioning

confidence: 99%

Direct identification of an extracellular agonist binding site in the renal V2 vasopressin receptor

Kojro¹,

Eich²,

Gimpl³

et al. 1993

Biochemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract