Figure 4

(A) Schematic depiction of the (rhod-)opsin activation and phosphorylation states contained within ROS membranes. Light-activated states of the receptor are indicated via a box containing the structure of the chromophore, retinal. (B) WT, single, double, triple and two quadruple mutant arrestin-mCherry fusion proteins were expressed in HEK293 cells and purified. Each protein was used in a direct binding assay with membranes containing the depicted activation and phosphorylation states of the (rhod-)opsin receptor. Fluorescence units indicate the level of bound arrestin-1–mCherry fusion protein to the specific receptor state after membrane pull down and two washing steps. Membranes containing P-ROS, ROS and P-opsin were kept in the dark throughout the experiment, whereas P-ROS* and ROS* were illuminated for 2.5 min using light, filtered with a 495 nm long-pass filter before pull down. The experiments were carried out in triplicates and normalized to the amount of purified arrestin-mCherry protein (1.5 µg). The statistical significance of measurements is indicated in comparison with P-ROS* binding and determined by one-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.