Fig. 5.

Pre-assembly and dissociation of Y₁ receptor to inhibitory G proteins studied by saturation and kinetic BRET experiments. a Saturation BRET experiments of Y₁-RLuc8 with different concentrations of chimeric Gα_∆6qi4myr(Gα_iq)-Venus, Gα_i1-Venus or Gα_o-Venus in transiently transfected HEK293 cells. BRET signal in the basal state in particular for Gα_i1 and Gα_io is saturable and indicates pre-assembly of the complex. Stimulation with 1 µM NPY for 10 min hardly changes BRET ratios for Gα_iq but decreases BRET for Gα_i1 and Gα_io. Right panel: Comparison of BRET signal at a F/L ratio of 0.2 prior to stimulation (−) and after agonist stimulation for 10 min ( +). Columns are compared by two-tailed t-test. Shown is mean ± SEM of n ≥ 3 experiments conducted in technical quadruplicate. b Kinetic BRET experiments of the same constructs at saturating F/L ratio (F/L > 0.2) to resolve ligand effects on Y₁R- Gα interactions. The BRET signal prior to stimulation was recorded for 2 min and the baseline was set to 0. After ligand stimulation (technical lag time 30 s), there was a short increase of the BRET signal, interpreted as recruitment, followed by a decrease of the BRET signal to (Gα_iq) and below the baseline (Gα_i1 and Gα_io), interpreted as complex dissociation. Shown are buffer-corrected representative examples of n = 3 independent experiments conducted in technical quadruplicate