A new linear V1A vasopressin antagonist and its use in characterizing receptor/G protein interactions.

We characterized a new iodinated, high affinity, linear V1a vasopressin antagonist, phenylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2. The antagonist bound specifically to the V1a vasopressin receptor in crude rat liver membranes with an apparent Kd value of 0.168 nM. This affinity is approximately 1 order of magnitude greater than that of the natural agonist, vasopressin. The inhibitory activity of the antagonist can be demonstrated by its inability to elicit activation and uncoupling of G proteins from the receptor. Thus, after occupancy of receptor sites in rat liver membranes with labeled antagonist and detergent solubilization, the labeled receptor (approximately 60 kDa) was eluted as a stable 400-kDa complex on size-exclusion chromatography. In contrast, when the receptor sites were occupied by the agonist [3H]vasopressin, the receptor eluted as a 60-kDa peak. Coincubation of membranes with iodinated antagonist and an excess of unlabeled vasopressin caused both reduced antagonist binding and a complete shift from the 400-kDa to the 60-kDa peak. The addition of vasopressin to unliganded 400-kDa fractions resulted in a 75% increase in [35S]guanosine-5'-O-(3-thio)triphosphate binding activity, indicating that the 400-kDa fraction contains complexes between the V1a receptor and G proteins. The vasopressin-elicited increase was inhibited by antagonist. Using specific antibodies 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 effector enzymes PLC-beta1, PLC-gamma2 and PLA-2 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 V1a receptor and the appropriate G proteins and effector enzymes are likely preassociated with each other and do not aggregate after antagonist addition. The association of V1a receptor with the different specific G proteins and effector enzymes is consistent with the multiple actions of vasopressin on liver cells. Antibodies directed against a portion of the carboxyl-terminal domain of the V1a receptor interacted with 60-kDa antagonist-occupied receptor but not with receptor in the 400-kDa complex. These results suggest that the carboxyl-terminal region of the receptor is sterically hindered when coupled to G proteins. The iodinated linear vasopressin antagonist therefore allows stable receptor/G protein complexes and can be an important tool (along with the antisera) for use in the study of factors that control V1a receptor/G protein coupling.