Figure 5: Truncation of the third intracellular loop in β₂V₂R ablates core interaction with βarr1.

(a) Cross-linking experiments and electron microscopy based structural model of β₂V₂R–βarr1 complex has identified the third intracellular loop of the β₂V₂R as prominent interface for core interaction through docking of the finger loop of βarr1. Residues that are identified to cross-link with each other in β₂V₂R–βarr1 complex are labelled and their side chains are highlighted as space fill model. (b) Cross-linking studies and X-ray crystal structure of rhodopsin-visual arrestin also displays the vicinity of the third intracellular loop in rhodopsin with the finger loop of visual arrestin. (c) Sequence alignment of β₂V₂R and β₂V₂R^ΔICL3 (third intracellular loop truncated receptor) to highlight the deleted amino acids (Gly²³⁸-Lys²⁶⁷) (red box). (d) Confocal microscopy of HEK-293 cells expressing either β₂V₂R or β₂V₂R^ΔICL3 with β-arr1-YFP. Agonist stimulation leads to accumulation of endocytotic vesicles that indicates recruitment of βarr1 to activated receptor. Nuclear staining is shown using 4,6-diamidino-2-phenylindole. Compared with β₂V₂R, β₂V₂R^ΔICL3 exhibits somewhat weaker recruitment of βarr1 as reflected by less punctate appearance. Scale bar, 10 μm. (e) Coimmunoprecipitation of β₂V₂R^ΔICL3 with βarr1 expressed in HEK-293 cells further confirms the recruitment of βarr1 to the truncated receptor upon agonist stimulation. Cells were stimulated with agonist (Isoproterenol, 10 μM for 30 min at 37 °C) followed by cross-linking using dithiobis(succinimidyl-propionate) (1 mM for 30 min at room-temperature) and subsequently, receptor–βarr1 complex was coimmunoprecipitation using anti-FLAG antibody beads. (f) Assembly of β₂V₂R^ΔICL3+β-arr1+Fab30 complex as measured using ELISA approach and (g) coimmunoprecipitation experiment. Similar to β₂V₂R, β₂V₂R^ΔICL3 also forms a stable complex with βarr1 in the presence of Fab30. (h) Quantification of β₂V₂R^ΔICL3–βarr1 complex formation as assessed by coimmunoprecipitation. (i) Bimane fluorescence spectroscopy on β₂V₂R^ΔICL3 complex reveals the absence of fluorescence quenching even in the presence of agonist and thereby suggests the lack of core interaction. (j) Bimane fluorescence at emission λ_max as measured in i is presented as bar graph. Data in f represents mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (***P<0.001). Data in g and h represent two independent experiments.