IDENTIFICATION OF THE V1-VASOPRESSIN RECEPTOR BY CHEMICAL CROSS-LINKING AND LIGAND AFFINITY BLOTTING

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with I-125 and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of M(r) 60000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of M(r) 60000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of I-125-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with I-125-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of M(r) 60000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5[Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin Il. Incubation of nitrocellulose containing membrane proteins from pig liver with I-125-LVP showed the labeling of a band of M(r) 58 000 that is inhibited by an excess of unlabeled LVP. This band of M(r) 58 000 seems to correspond with the protein of M(r) 60 000 revealed by the cross-linking experiment. This protein appears not to have internal disulfide bonds since the electrophoretic pattern did not change in the absence or presence of reductant in the polyacrylamide gel electrophoresis. Our results suggest that the V1 vasopressin receptor is a monomeric protein of M(r) 60 000.