Fig. 6.

Timecourse, reversibility and concentration dependence of Y1 receptor β-arrestin association measured by sfGFP BiFC. Quantitative analysis was performed on automated plate reader images (see Materials and methods), to determine BiFC responses resulting from interaction of the Y1 receptor with β-arrestin1 (Y1 A1 cells; panels A, C, E) or β-arrestin2 (Y1 A2 cells; panels B, D, F). In A and B, 100 nM NPY timecourses (n = 4–5) were performed over 2 h at 37 °C (open circles), compared to vehicle controls (closed circles). Estimated half times from curve fitting (one phase association) are given in the text. In C and D, 100 nM NPY (solid symbols) timecourses were performed as before, together with vehicle controls (open symbols). Subsequently cells were washed with medium (2 × rinse, 1 × 60 min at 37 °C) to remove agonist, before fixation. NPY BiFC responses in the pooled data from treated and washed cells (n = 4–5) are compared with the original curve fits to the timecourse data without agonist removal (dotted line, from A or B). E and F show NPY concentration–response curves (n = 4) based on 60 min agonist treatment at 37 °C. pEC₅₀ values are quoted in the text, and were very similar to those previously observed for YFP BiFC responses (Supplementary Fig. 1).