Fig. 1.

ICL1–9 is a potent β-arrestin–biased pepducin. (A) β-Arrestin recruitment was assessed by BRET2 in HEK293 cells transiently transfected with β₂AR–RLucII and GFP10–β-arrestin2. β-Arrestin2 recruitment is reported at 10 min postagonist stimulation with 1 μM isoproterenol (Iso), 5 μM salbutamol (Sal), or 10 μM pepducin. The sequences of these pepducins and initial BRET analysis for isoproterenol, salbutamol, ICL1–4, ICL1–11, ICL1–15, and ICL1–20 have been previously reported, albeit as time courses (5). Although ICL1–9 exhibited a modest ability to promote β-arrestin recruitment in our previous primary screen (5), subsequent analysis shows that it has comparable efficacy to ICL1–4, ICL1–11, and ICL1–20. The data are represented by the mean ± SD from three independent experiments. (B) ICL1–9 is a high-potency β-arrestin–biased pepducin with an EC₅₀ of 96 ± 14 nM with a sequence of LVITAIAKFERLQTVTNY containing an N-terminal palmitate and C-terminal amide (5). ICL1–4 (1.9 ± 0.5 μM), ICL1–11 (1.7 ± 0.5 μM), and ICL1–20 (1.1 ± 0.3 μM) demonstrated comparable efficacy to ICL1–9 but operated with lower potency. The data are represented by the mean ± SD from three independent experiments. (C) cAMP production in HEK293 cells using Iso, Sal, and the pepducins that promoted β-arrestin recruitment in A. The data represent the mean ± SD from three independent experiments and are primarily derived from our previous report (5).