Fig. 1.

V₂R forms GDP-resistant agonist-induced complexes with G₁₂ heterotrimers. (A) BRET between V₂R-Rluc8 and Gαβγ-Venus in the presence or absence of AVP (1 μM), and the presence or absence of GDP. When GDP was absent, apyrase (apy) was added to remove residual nucleotides. AVP-induced BRET to G_s (Left) but not G₁₂ (Right) heterotrimers was enhanced when GDP was absent; **P < 0.005; n.s., not significant (P = 0.58); one-way ANOVA (Sidak’s test) compared to GDP+AVP; n = 6. (B) Cartoon representation of two steps of V₂R-G protein coupling: agonist-induced formation of receptor-G protein complexes, and GDP release. (C) Time course of BRET between V₂R-Rluc8 and Gαβγ-Venus in response to injection of 1 μM AVP, followed by injection of 100 μM GTP in permeabilized cells treated with apyrase (mean ± SEM; n = 4–6). (D) BRET between V₂R-Rluc8 and Gαβγ-Venus as a function of AVP concentration in permeabilized cells expressing either G_s (Left) or G₁₂ (Right) heterotrimers treated with apyrase, GDP (100 μM), or GTP (100 μM). The logEC₅₀ for association with G_s was −6.8 ± 0.5 in apyrase-treated cells, and the logEC₅₀s for association with G₁₂ were −7.5 ± 0.3, −7.4 ± 0.4, and −7.5 ± 0.5 in the presence of apyrase, GDP, and GTP, respectively. Data points represent the change in BRET (ΔBRET) in response to AVP (mean ± SEM; n = 3).