Fig. 4

The interplay between phosphate and activation sensors in receptor-independent and receptor-dependent signaling. a Size exclusion chromatography (SEC) of the ΔN_IP6 and ΔC_IP6 mutants measured on a Superdex S200 Increase 10/300 GL column (24 mL). Arrestin-3 (1–393) runs anomalously on size exclusion chromatography, but exhibits a characteristic shift in molecular weight upon the addition of IP₆. In the absence of IP₆, both the ΔN_IP6 and the ΔC_IP6 mutants are monodisperse and have a similar elution volume to wild-type, but in the presence of IP₆, no mobility shift is observed. b Temperature-jump binding curve for the Cys-less-T222C arrestin-3. c Overlay of finger loop of arrestin structures. Basal (gray): PDB entries 1CF1⁷, 1JSY³⁵, 1ZSH²⁴, 3P2D⁶, 1G4M⁴; active (green): PDB entries 4ZRG¹², 4JQI¹¹, 4J2Q¹⁰. Bound to receptor (magenta): 4ZWJ¹³. Bound to IP₆ (blue) d The conserved motif EDL/(I)D folds into an α-helix. e,f Evaluation of mean binding ± SEM of wild-type and mutant Venus-arrestin-3 binding to the luciferase-tagged e M2 muscarinic or f D2 dopamine receptor by BRET. In arrestin-3-KNC (K11A, K12A, L49A, D51A, R52A, L69A, Y239A, D241A, C252A, P253A, D260A and Q262A), two key phosphate-binding lysines and 10 residues that bind other parts of the receptor were mutated to alanines. This precludes GPCR binding as described^{22, 34}, making this an appropriate negative control. Data from three experiments were compared to wild-type by one-way ANOVA. ***p ≤ 0.001