Photoaffinity labelling of the renal V2 vasopressin receptor: identification and enrichment of a vasopressin-binding subunit

• Published in: European journal of biochemistry Vol. 152; no. 3; pp. 589 - 595
• Main Authors: FAHRENHOLZ, F, BOER, R, CRAUSE, P, TOTH, V
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 04.11.1985
• Subjects: Adenylyl Cyclases - analysis; Affinity Labels - chemical synthesis; Animals; Biological and medical sciences; Cattle; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Kidney - metabolism; Kidney Medulla - metabolism; Molecular and cellular biology; Photochemistry; Radioligand Assay; Rats; Receptors, Angiotensin - isolation & purification; Receptors, Cell Surface - isolation & purification; Receptors, Vasopressin; Ultraviolet Rays; Vasopressins - chemical synthesis; Enrichment; Rat; Rodentia; Peptide hormone; Identification; Photoaffinity labelling; Subunit; Kidney; Vasopressin; Vertebrata; Mammalia; Artiodactyla; Beef; Ungulata; Hormonal receptor
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1985.tb09236.x

To identify renal vasopressin receptor proteins, analogues of 1-deamino-vasopressin i.e. ([1-(2-mercapto)propionic acid]vasopressin, [Mpa1]VP) with photoreactive aryl-azido groups in position 4 and 8 of the vasopressin sequence were prepared. In the absence of ultraviolet light, these ligands exhibit a high binding affinity for the V2 vasopressin receptor in plasma membranes from bovine and rat kidney medulla (apparent dissociation constants 1.8 X 10(-9) M to 1.7 X 10(-8)M); the photoreactive analogues stimulate the renal vasopressin-sensitive adenylate cyclase. In photoaffinity labelling experiments with tritium-labelled ligands (34-50 Ci/mmol), a membrane protein from bovine kidney or rat kidney medulla with an apparent relative molecular mass (Mr) of 30 000 was preferentially and specifically labelled. The labelling of the 30 000-Mr protein was completely inhibited by a 10-100-fold molar excess of vasopressin; in contrast, angiotensin II, bradykinin or low-affinity analogues of vasopressin did not suppress the incorporation of the reactive ligands into this protein. The highest specific labelling yield and only a low amount of unspecific labelling was obtained with the analogue [Mpa1,Lys(N6-4-azidobenzoyl)8]VP. Preparative sodium dodecyl sulfate gel electrophoresis of bovine kidney membranes photolabelled with this analogue resulted in a 20-30-fold enrichment of the 30 000-Mr vasopressin-binding protein. Our results suggest that this photoreactive analogue of [1-deamino, 8-lysine]vasopressin is a suitable tool for further purification of the renal V2 vasopressin receptor binding subunit.