Z. Strakova et al., A NEW LINEAR V-1A VASOPRESSIN ANTAGONIST AND ITS USE IN CHARACTERIZING RECEPTOR G-PROTEIN INTERACTIONS, Molecular pharmacology, 51(2), 1997, pp. 217-224
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.