Figure 5.

Arrestin 3 and delta opioid receptors are pre-engaged on the cell membrane and ARM390 increases this association. A, Basal BRET signal between δOR-Rluc and Arr2-YFP or Arr3-YFP. HEK293 cells were cotransfected with a constant concentration of δOR-Rluc and increasing concentrations of Arr2-YFP or Arr3-YFP plasmids. The data shown are representatives of five independent experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site. BRET is expressed as a function of δOR expression for a constant amount of arrestin and shows increased basal interaction at the cell membrane between δOR-Rluc8 and Arr3-YFP, which is not observed between δOR-Rlub8 and Arr2-YFP. B–E, To determine the effect of ARM390 on delta opioid receptor-arrestin interactions, HEK293 cells were transfected with δOR-Rluc8 and Arr2-YFP (B), δOR-Rluc8 and Arr3-mOrange (C), or δOR-Rluc8, Arr2-YFP, and Arr3-mOrange (D, E). For the three partners-BRET experiment, D shows representative images from the Arr2-YFP channel and E those from the Arr3-mOrange channel. Cells were incubated with ARM390 (100 nm) and mean BRET intensity was quantified for the cell membrane (Mb) and cytosol (Cyto) at 0, 10, and 15 min after ARM390 administration. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA with Holm–Sidak post hoc analysis (n = 3–6 cells and 7 square regions per subcellular compartment). Delta opioid receptors are pre-engaged with Arr3 at the cell membrane and this interaction is strengthened after ARM390 binding.