Figure 1.

Analysis of receptor/G protein interaction by FRET. (A) FRET was measured between YFP-tagged receptors and CFP-tagged Gβγ subunits. When these constructs are excited at 436 nm, emission is shifted from 480 to 535 nm when both fluorophores are close enough to each other to permit FRET. (B) In HEK293T cells transiently expressing α_2A-YFP and CFP-γ₂ together with Gαi₁β₂ (μg transfected DNA: α_2A-YFP 0.4, Gαi₁ 2, Gβ₁ 0.5, Gγ₂ 0.25), α_2A-YFP (left) and CFP-γ₂ (right) colocalize at the cell membrane (scale bar: 5 μM). (C) Upon stimulation with 100 μM NE (bar), a decrease in CFP fluorescence (F_CFP) and an increase in corrected YFP fluorescence (F_YFP) were observed. This resulted in an increase in FRET, assessed as the ratio of F_YFP over F_CFP. The FRET increase is readily reversible upon agonist washout. (D) The average increase in FRET ratio was ∼0.022 (n=8). (E) Fluorescence and FRET changes in a cell (as in panel C) in response to different NE concentrations. The FRET signal is stable over more than 400 s, and the amplitude of the FRET change depends on agonist concentration (concentrations indicated in μM; representative experiment out of eight shown). The agonist-independent increase in the YFP and CFP traces at ∼270 s is due to removal of solution from the coverslip holder; note that no increase is seen in the ratiometric FRET trace.