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 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.
D000262:Adenylyl Cyclases / Q000032:analysis; D000345:Affinity Labels / Q000138:chemical synthesis; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007668:Kidney / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism; D010777:Photochemistry; D011869:Radioligand Assay; D051381:Rats; D011945:Receptors, Angiotensin / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D017483:Receptors, Vasopressin; D014466:Ultraviolet Rays; D014667:Vasopressins / Q000138:chemical synthesis
D000345:Affinity Labels; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D017483:Receptors, Vasopressin; D014667:Vasopressins; D000262:Adenylyl Cyclases