Fig. 1: V2RppWT and V2RppT360 impart different conformations on βarr1.
From: Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody
a Schematic representation of the inability of V2RT360A mutant to promote endosomal trafficking of βarr1 as published previously. b Amino acid sequences of the V2RWT and Thr360 mutant phospho-peptides (V2RppT360-1 and V2RppT360-2) were used in this study. Phosphorylated residues (serine, S; threonine, T) are highlighted in red, and the position 360 is indicated by an arrow. V2RppT360-1 and V2RppT360-2 contain a non-phosphorylated threonine and an alanine at position 360, respectively. c Limited trypsin proteolysis of βarr1 (5 min) in the absence or presence of indicated phospho-peptides at two different trypsin: βarr1 ratio followed by visualization of the bands on SDS-PAGE. A representative gel from four independent experiments (left panel) and a schematic of the proteolysis pattern corresponding to 1: 25 ratio of trypsin:βarr1 (right panel) is shown here. d Densitometry-based quantification (mean ± SEM) of indicated bands from four independent experiments, normalized with respect to V2RppWT condition (treated as 100%) (One-way ANOVA, Dunnett’s multiple comparisons test). The exact p values are as follows: Gly−8 to Arg418 (48 kDa) band (1: 25)- Apo (p < 0.0001), V2RT360-1 (p = 0.0159), V2RT360-2 (p = 0.1539); Gly−8 to Arg418 (48 kDa) band (1: 50)- Apo (p = 0.0039), V2RT360-1 (p = 0.0566), V2RT360-2 (p = 0.385); Leu1 to Arg418 (47 kDa) band (1: 25)-Apo (p = 0.0132), V2RT360-1 (p < 0.0001), V2RT360-2 (p = 0.0844); Leu1 to Arg418 (47 kDa) band (1: 50)-Apo (p < 0.0024), V2RT360-1 (p = 0.0001), V2RT360-2 (p = 0.059); Leul1 to Arg285 (32 kDa) band (1: 25)-Apo(p < 0.0001), V2RT360-1 (p = 0.0006), V2RT360-2 (p < 0.0001); Leul1 to Arg285 (32 kDa) band (1: 50)-Apo(p = 0.0072), V2RT360-1 (p = 0.0005), V2RT360-2 (p = 0.0036); Leul1 to Arg188 (21 kDa) band (1: 25)-Apo (p < 0.0001), V2RT360-1 (p = 0.0028), V2RT360-2 (p = 0.0057); Leul1 to Arg188 (21 kDa) band (1: 50)-Apo(p < 0.0001), V2RT360-1 (p = 0.0051), V2RT360-2 (p = 0.0044). Source data are provided as a Source Data file (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = non-significant).
