Figure 2 - Transconformational switching of the _2A-AR by the MOR as a mechanism underlying direct inhibition of receptor activation.

(a) Receptor activation is monitored by recording changes in FRET between CFP (blue circle, the donor) and FlAsH (yellow circle, the acceptor) introduced respectively into the C-terminal tail and the third intracellular loop of the _2A-AR. (b) Time-resolved changes of the FRET ratio F_FlAsH/F_CFP in a single HEK 293 cell expressing the _2A-AR^FlAsH/CFP (left panels) or coexpressing _2A-AR^FlAsH/CFP and MOR (right panels). Yellow and blue traces represent emission intensities of FlAsH and CFP, respectively. The red trace represents the calculated FRET ratio corrected according to equation (1) with the initial value at t = 0 set to 1. Horizontal bars indicate the application of NE (50 M) or morphine (50 M) to the cell. Traces are representative of n 10 experiments. (c) Effect of morphine (50 M) on NE-activated _2A-AR^FlAsH/CFP in cells coexpressing _2A-AR^FlAsH/CFP and MOR and in the presence of pertussis toxin (PTX). The change in the corrected FRET ratio (FRET) was set to 100% (response to 50 M NE) (n = 3). (d) Bars represent the effects of NE and morphine added alone or together on the change in FRET (N_FRET values s.e.m. calculated according to equation (2)) occurring in membranes prepared from HEK cells expressing _2A-AR^FlAsH/CFP and MOR (n = 7). ^**P < 0.05 when comparing the values obtained after addition of NE versus NE + MOR.