Fig. S3.

(A) Concentration–response curves of BI-167107–stimulated βarr1 (WT or ΔFLR) recruitment to the β₂V₂R and βarr1 (WT or ΔFLR)-mediated β₂V₂R internalization. RLU, relative luminescence units. (B) Interaction between βarr1 (WT or ΔFLR) and either rGFP-CAAX (Top, plasma membrane marker) or rGFP-FYVE (Bottom, early endosomal marker) upon agonist stimulation of β₂AR, β₂V₂R, or V₂R. BRET titration curves were obtained using a constant amount of rGFP-CAAX and with increasing amounts of RlucII-βarr1 (WT or ΔFLR). BRET titration curves were obtained using a constant amount of rGFP-FYVE and with increasing amounts of RlucII-βarr1 (WT or ΔFLR). BRET was measured 35 min following addition of agonist or vehicle. To stimulate the GPCRs, 1 μM BI-167107 was applied for the SNAP-β₂AR and SNAP-β₂V₂R, and 100 nM AVP was applied for the SNAP-V₂R. Data are expressed as net BRET absolute values, represent the mean ± SE, and are pooled from six experiments. (C) Two hundred nanomolar 6×His-βarr1 (WT or ΔFLR) was incubated with equal concentrations of GST-Src-3D and with either control buffer or V₂Rpp. The complexes were pulled down using Glutathione Sepharose beads, and the amount of 6×His-βarr1 (WT or ΔFLR) bound to GST-Src-3D was determined by immunoblotting using an anti-His antibody. Data represent the mean ± SE of three experiments. One-way ANOVA was performed to determine statistical differences between basal and V₂Rpp-stimulated states (**P < 0.01, ****P < 0.0001) or V₂Rpp-stimulated states of βarr1 (WT) and βarr1 (ΔFLR) (##P < 0.01).