Figure 7.

Effect of intrabodies on βarr1 recruitment, V₂R endocytosis, and endosomal localization of βarr1. A, intrabodies do not significantly alter agonist-induced βarr1 recruitment to V₂R as assessed in intermolecular BRET assay. HEK-293 cells expressing V₂R–venus, βarr1–RlucII, and the indicated intrabodies were stimulated with varying doses of AVP, and the levels of BRET signal were recorded using a plate reader. The data represent means ± S.E.M. from three independent experiments, each performed in triplicate. B, Ib30 co-localizes with internalized V₂R and βarr1 upon agonist stimulation as visualized using confocal microscopy of HEK-293 cells expressing FLAG–V₂R, βarr1-YFP, and Ib30–HA. The merged image shows co-localization of all three protein upon receptor internalization. The cells were “fed” anti-FLAG M2 antibody prior to agonist stimulation (AVP 100 nm, 12 min) and were subsequently fixed, permeabilized, treated with HA antibody, and imaged (PCC of V₂R and βarr1 in unstimulated cells = 0.38 ± 0.03 and in stimulated cells = 0.88 ± 0.03, Ib30 and V₂R in unstimulated cells = 0.29 ± 0.04 and in stimulated cells = 0.83 ± 0.01, and βarr1 with Ib30 in unstimulated cells = 0.43 ± 0.08 and in stimulated cells = 0.63 ± 0.04, no. of cells = 3). A representative image of n = 3 cells/condition is shown here. Scale bar, 5 μm. C, Ib30 does not significantly alter agonist-induced internalization of V₂R as assessed by confocal microscopy. Comparative analysis of V₂R co-localization with two early endosomal markers, EEA1 and APPL1, upon agonist stimulation was performed in the presence of either Ib–CTL or Ib30. Cells expressing FLAG–V₂R and Ib30–HA were treated with anti-FLAG antibody prior to agonist stimulation (AVP, 100 nm, 3–12 min) followed by fixation, permeabilization, and staining for endosomal markers APPL1 or EEA1 (Pearson's coefficient of V₂R and EEA1 in Ib–CTL cells = 0.70 ± 0.01 and in Ib30 cells = 0.42 ± 0.01, no. of cells = 4, and Pearson's coefficient of V₂R and APPL1 in Ib–CTL cells = 0.69 ± 0.07 and in Ib30 cells = 0.30 ± 0.07, no. of cells = 4). D, co-localization was also measured by manual counting of punctae in confocal images, and quantified data representing means ± S.E. from four different cells per condition are presented. E, an intermolecular BRET assay to measure the effect of intrabodies on the endosomal localization of βarr1 upon agonist stimulation. HEK-293 cells expressing V₂R, βarr1–RlucII, rGFP-FYVE, and the indicated intrabodies were stimulated with varying doses of AVP, and the levels of BRET signal were recorded using a plate reader. The data represent means ± S.E.M. from four independent experiments, each performed in triplicate. F, agonist-induced change in BRET signal (i.e. the difference in BRET signal between the highest and the lowest AVP doses) as measured in panel E is presented as ΔBRET and analyzed using one-way ANOVA. **, p < 0.01; ***, p < 0.001.