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. 2021 Mar 5;296:100503. doi: 10.1016/j.jbc.2021.100503

Figure 2.

Figure 2

MeNArC assay with unmodified class A receptors.A, schematic overview of the arrestin membrane-recruitment assay. The N-terminal NanoLuc fragment is fused to a doubly palmitoylated fragment of GAP43, thereby tethering it to the plasma membrane (MeN), and the C-terminal NanoLuc fragment is fused to the N terminus of β-arrestin (ArC). B, β-arrestin2 recruitment shown in fold change of luminescence for D2R with time course for 1 μM quinpirole-induced β-arrestin2 recruitment in the absence (blue) or presence (purple) of 10 μM sulpiride injected 18 min after agonist treatment. Steady state is reached after ∼40 min. Dose–response curves of (C) β-arrestin2 and (D) β-arrestin1 recruitment by D2R with dopamine alone (blue line, top plots) and with 1 μM dopamine post treatment with increasing concentration of the antagonist sulpiride (purple line, bottom plots). Experiments were carried out with D2R cotransfected with GRK2. The pEC50 for β-arrestin2 with quinpirole was: −6.8 ± 0.16 and IC50 sulpiride: −7.50 ± 0.35. For β-arrestin1 the pEC50 for quinpirole: −7.35 ± 0.17 and IC50 sulpiride: −8.37 ± 0.95. E, β-arrestin2 recruitment tested with the D2R partial agonists quinpirole, bromocriptine, terguride, and PPP yielding pEC50 ± SD compared with dopamine of −6.68 ± 0.09, −7.18 ± 0.32, −8.02 ± 0.25, and −5.61 ± 1.01, respectively. F, dose–response curves of β-arrestin2 recruitment by wild-type (WT) MOR (blue, square) and MOR 11S/T-A (black, circle) with increasing concentration of agonist DAMGO; pEC50 ± SD: WT MOR, −7.46 ± 0.11, and MOR 11S/T-A, −6.82 ± 0.19. Global fits from three to eight independent experiments each performed in triplicate. Error bars represent SD.