A NEW LINEAR V-1A VASOPRESSIN ANTAGONIST AND ITS USE IN CHARACTERIZING RECEPTOR G-PROTEIN INTERACTIONS

We characterized a new iodinated, high affinity, linear V-1a vasopress in antagonist, enylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2 The antagonist bound specifically to the V-1a vasopressin receptor in crud e rat liver membranes with an apparent K-d value of 0.168 nM. This aff inity is similar to 1 order of magnitude greater than that of the natu ral agonist, vasopressin. The inhibitory activity of the antagonist ca n be demonstrated by its inability to elicit activation and uncoupling of G proteins from the receptor. Thus, after occupancy of receptor si tes in rat liver membranes with labeled antagonist and detergent solub ilization, the labeled receptor (similar to 60 kDa) was eluted as a st able 400-kDa complex on size-exclusion chromatography. In contrast, wh en the receptor sites were occupied by the agonist [H-3]vasopressin, t he receptor eluted as a 60-kDa peak. Coincubation of membranes with io dinated antagonist and an excess of unlabeled vasopressin caused both reduced antagonist binding and a complete shift from the 400-kDa to th e 60-kDa peak. The addition of vasopressin to unliganded 400-kDa fract ions resulted in a 75% increase in [S-35]guanosine-5'-O-(3-thio)tripho sphate binding activity, indicating that the 400-kDa fraction contains complexes between the V-1a receptor and G proteins. The vasopressin-e licited increase was inhibited by antagonist. Using specific antibodie s and immunoadsorption to protein A/Sepharose columns, we found that G protein isotypes G(alpha q/11), G(alpha i3), and G(alpha S), and effe ctor enzymes PLC-beta 1, PLC-gamma 2 and FLAP were associated with the antagonist-labeled receptor in the 400-kDa fraction. Because the 400- kDa complex was found in the absence of ligand, the V-1a receptor and the appropriate G proteins and effector enzymes are likely preassociat ed with each other and do not aggregate after antagonist addition. The association of V-1a receptor with the different specific G proteins a nd effector enzymes is consistent with the multiple actions of vasopre ssin on liver cells. Antibodies directed against a portion of the carb oxyl-terminal domain of the V-1a receptor interacted with 60-kDa antag onist-occupied receptor but not with receptor in the 400-kDa complex. These results suggest that the carboxyl-terminal region of the recepto r is sterically hindered when coupled to G proteins. The iodinated lin ear vasopressin antagonist therefore allows stable receptor/G protein complexes and can be an important tool (along with the antisera) for u se in the study of factors that control V-1a receptor/G protein coupli ng.