Fig. 5: Impaired formation of GPCR–βarr complex and PtdIns(4,5)P2 domain by NC-site-mutant βarr.
From: Membrane-domain compartmentalization of active GPCRs by β-arrestins through PtdIns(4,5)P2 binding
a–c, CI of the three particle pairs under basal conditions (pre) and after 15 min of AVP stimulation (post) in both AVP-treated and vehicle-treated conditions: βarr2–PLCδ-PH (a), V2R–βarr2 (b) and V2R–PLCδ-PH (c). WT, C-3Q and NC-4Q are represented in black, blue and red, respectively. d, Merged images of βarr2 (yellow), V2R (cyan) and PLCδ-PH (magenta) particle trajectories. Trajectories of these three molecules were projected on the colocalized coordinates (round markers). Scale bars, 1 µm. e, Enlarged views of the area enclosed by the white dotted liners of d. Colocalization trajectories of βarr2–PLCδ-PH, V2R–βarr2 and V2R–PLCδ-PH. Colocalization of two molecules (i) and four diffusion state of each molecule (ii, iii). Scale bars, 1 µm. f–h, The difference in diffusion state step count of colocalized molecules among the three particle pairs between basal conditions and after 15 min of ligand stimulation: βarr2–PLCδ-PH (f), V2R–βarr2 (g) and V2R–PLCδ-PH (h). WT AVP (−), n = 19; WT AVP (+), n = 36; C-3Q AVP (−), n = 19; C-3Q AVP (+), n = 32; NC-4Q AVP (−), n = 18; NC-4Q AVP (+), n = 23; n represents individual cells analyzed across two independent biological replicates. AVP (−), vehicle stimulation; AVP (+), AVP stimulation. In a–c,f–h, bars and error bars represent the mean and s.e.m., respectively. Statistical significance was calculated using a two-way ANOVA followed by Tukey’s test for multiple-comparison analysis. *P < 0.05, **P < 0.01 and ***P < 0.001.
