Photoaffinity labelling of the renal V2 vasopressin receptor. Identification and enrichment of a vasopressin-binding subunit.

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 × 10−9 M to 1.7 × 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 30000 was preferentially and specifically labelled. The labelling of the 30000-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 30000-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.