Fig. 6.

ERK1/2-dependent βarr2 phosphorylation on S14 and T276 induces CXCR4 intracellular sequestration. (A–C) CXCR4 cell-surface expression was assessed by dual-flow cytometry in WT MEFs or βarr1/2-KO MEFs transiently transfected with HA-CXCR4-vYFP in the absence (Control) or presence of the indicated Ras mutants (A) and with or without βarr1 and/or βarr2 complementation (B and C). (D) Phosphorylation of βarrs was performed with recombinant bovine βarr1 or βarr2 as a substrate and recombinant active ERK1 in the presence of [γ³²P]-ATP for 15 min. The gel was stained with Coomassie, and ³²P incorporation was quantified using a PhosphorImager (Left) or during kinetics using scintillation counting (Right). The data shown are representative of three independent experiments. (E and F) CXCR4 cell-surface expression was assessed by dual-flow cytometry in βarr1/2-KO MEFs transiently transfected with HA-CXCR4-vYFP, βarr2 WT, or the indicated βarr2 mutants, with or without Ras CA. Data shown represent the mean ± SEM of at least three independent experiments and were normalized to the control condition (100%). Expression of βarr2 WT and mutants were assayed by immunoblotting. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.