Figure 6.

HEK293A cells transiently transfected with B2R^(Ø)mutant (lacking AltB2R) show no difference in receptor internalization kinetics but show decreased G protein coupling efficiencies following agonist stimulation.A–C, G protein coupling efficiency after stimulation with NG291 in HEK293A cells transfected with B2R and B2R^(Ø), by following BRET proximity of Gα_q-RLuc2 and GFP10-Gγ₁ (A), Gα_i2-RLuc2 and GFP10-Gγ₁ (B), or Gα_i3-RLuc2 and GFP10-Gγ₁ (C). Data represent mean ± SD, n = 3, 4, 4, unpaired t-test for EC₅₀ (not significant) and E_max, ^†p < 0.05, ^††p < 0.01. D, β-arrestin2 recruitment at the plasma membrane after stimulation with NG291 in HEK293A cells transfected with B2R and B2R^(Ø) by following BRET proximity of β-arr2-RLuc2 and rGFP-CAAX. Data represent mean ± SD, n = 3, unpaired t-test for EC₅₀ and E_max, not significant. E and F, internalization kinetics of B2R by cell surface ELISA after stimulation with 1 μM RMP-7 (E) or NG291 (F) in HEK293A cells transfected with ^FlagB2R or ^FlagB2R^(Ø), respectively. Note. B2R-HEK293A and B2R(Ø)-HEK293A cells gave equivalent signals at time zero (unstimulated) (not shown). Data represent mean ± SD, n = 3, 3, multiple comparison versus corresponding time point using two-way ANOVA with Sidak's correction, unpaired t-test for I_max (maximal internalization) and half-life, not significant.