Figure 5. Retromer and arrestin regulate PTH-mediated cAMP production in bone cells.

a, Averaged cAMP response measured by FRET changes in ROS17/2.8 cells expressing either epac^CFP/YFP alone or co-expressing epac^CFP/YFP with either β-arrestin1[IV-AA]^tom or Vps26 and Vps29. Cells were treated with control buffer or a brief challenge of 100 nM PTH(1–34) (arrow). Data represent the mean ± s.e.m. of N = 4 independent experiments and n = 20 cells.

b, Expression level of wild-type β-arrestin1 influences cAMP signaling. ROS cells co-transfected with epac^CFP/YFP and β-arrestin1^tom were challenged with PTH (100 nM) as in (a) and time-courses of cAMP response were measured as described in Figure 1. Integrated signaling was estimated by summing the area under the curve from 0–15 min after ligand challenge. Arrestin expression levels were estimated by epifluorescence imaging of tdTomato fluorescence. Integrated cAMP values were then binned according to arrestin expression level and plotted as averages (mean ± s.e.m. N = 4 independent experiments). The dotted line represents the 95% confidence interval.

c, The total PTH-induced cAMP responses for experiments represented in the Supplementary Figure 6 were determined by measuring the area under the curve from 0–10 min. Bars represent the mean ± s.e.m. of N = 4 independent experiments and n = 12 (control), n = 14 (U0126), n = 15 (rolipram), and n = 20 (βarr1[IV-AA]) cells.

d, Representative Western blots of time courses of ERK1/2 activation in ROS 17/2.8 cells control (Ctrl) or transiently transfected to express βarr1[IV-AA] in response to 100 nM PTH(1–34). The data are the mean ± s.e.m. of N = 3 separate experiments; Statistical comparison of the curves was performed by a two-way ANOVA (***, P < 0.001).