Fig. 3.

Sortase ligation of a phosphopeptide onto the β₂AR restores its allosteric interaction with β-arrestin1. (A) Cartoon schematic of sortase ligation method. A synthetic phosphopeptide (pp) derived from the vasopressin-2-receptor (V₂R) with three N-terminal glycine residues (GGG-V₂Rpp) is ligated onto receptors containing a C-terminal LPETGGH recognition motif. In the sequence of GGG-V₂Rpp below the schematic, phosphorylated residues are highlighted in red. (B) Coomassie-stained gel showing the coimmunoprecipitation of heterotrimeric Gs and β-arrestin1 (βarr1) with isoproterenol (ISO)-bound, phosphopeptide-ligated FLAG-β₂AR (β₂ARpp). Fab30 binds specifically to V₂Rpp-bound βarr1 (10). Loading controls represent 10% of input. (C and D) Competition binding experiments using radiolabeled [¹²⁵I]-cyanopindolol (CYP) with HDLs containing (C) β₂ARpp or (D) β₂AR ligated to a nonphosphorylated version of the V₂R peptide (β₂ARnp). Gs increases the affinity of ISO for both β₂ARpp and β₂ARnp HDLs (log IC₅₀: −9.15 ± 0.03, −9.02 ± 0.04, respectively) compared with no transducer (log IC₅₀: −6.24 ± 0.04, −6.42 ± 0.09, respectively), but βarr1 only increases ISO affinity for β₂ARpp HDLs (log IC₅₀: −7.14 ± 0.07) and not β₂ARnp HDLs (log IC₅₀: −6.49 ± 0.04). Data shown in C and D are the mean of at least three independent experiments, with error bars representing SE, and asterisks (*) indicate a log IC₅₀ value significantly different from the control curve (P < 0.05, one-way ANOVA). (E) The effects of ISO on the HDL-β₂ARpp-bimane fluorescence emission spectrum are enhanced by Gs and βarr1. Data shown are representative of three independent experiments.