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MOLECULAR PHARMACOLOGY 51:217-224 (1997).

A New Linear V_1A Vasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions

Zuzana Strakova, Amalendra Kumar,¹ A. John Watson,² and Melvyn S. Soloff

Department of Obstetrics and Gynecology (Z.S., M.S.S.) and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1062 (A.K., M.S.S.), Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia (Z.S.), and Division of Biology, California Institute of Technology, Pasadena, California 91125 (A.J.W.)

We characterized a new iodinated, high affinity, linear V_1a vasopressin antagonist, phenylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH₂. The antagonist bound specifically to the V_1a vasopressin receptor in crude rat liver membranes with an apparent K_d value of 0.168 nM. This affinity is ~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 (~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 V_1a 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_q/11, G_i3, and G_s, and effector enzymes PLC-1, PLC-2 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 V_1a 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 V_1a 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 V_1a 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 V_1a receptor/G protein coupling.