Figure 4. Divergent actions on β-arrestin2 recruitment by OBP versus EBP mutations.

Examination of β-arrestin2 recruitment activity at OBP T140A^3.37 and A225G^5.46 mutations versus EBP mutation L362F^7.35. a) Methylergonovine Gq-mediated calcium flux (left panel) comparing T140A^3.37 (red) to 5-HT_2BR WT (black, EC₅₀ = 21 nM). β-arrestin2 recruitment (right panel) comparing T140A^3.37 (red) to 5-HT_2BR WT (black, EC₅₀ = 1.2 nM). b) Methysergide Gq-mediated calcium flux (left panel) comparing A225G^5.46 (green, EC₅₀ = 33 nM) to 5-HT_2BR WT (black). β-arrestin2 recruitment (right panel) comparing A225G^5.46 (green, EC₅₀ = 1.7 nM) to 5-HT_2BR WT (black). c) Lisuride Gq-mediated calcium flux (left panel) comparing L362F^7.35 (purple, EC₅₀ = 65 nM) to 5-HT_2BR WT (black). β-arrestin2 recruitment (right panel) comparing L362F^7.35 (purple) to 5-HT_2BR WT (black). d) LSD Gq-mediated calcium flux (left panel) comparing L362F^7.35 (purple, EC₅₀ = 40 nM) to 5-HT_2BR WT (black, EC₅₀ = 42 nM). β-arrestin2 recruitment (right panel) comparing L362F^7.35 (purple) to 5-HT_2BR WT (black, EC₅₀ = 0.97 nM). Data in panels a-d are expressed as fold-over-basal and represent mean and S.E.M from three independent experiments (N=3) performed in triplicate. e) LSD dissociation comparing 5-HT_2BR wild-type (black, k_off = 0.015 min⁻¹) to the L362F^7.35 mutant (purple, k_off = 0.240 min⁻¹). Data represent percent specific binding indicating mean and S.E.M from three independent experiments (N=3) performed in duplicate. f) Schematic comparing the location of the EBP residues L209^EL2 and L362^7.35 (purple), which can result in either Gq or β-arrestin2 recruitment preference, to the location of OBP residues T140^3.37 and A225^5.46 (green), which result in equal contributions to Gq activity and β-arrestin2 recruitment.