Fig. 6

Fig. 6 Figure 6. Src Requirement for TVA Release from CMG2 and RhoA Recruitment (A) Control MEFs and SYF MEFs were either treated with or without FBS overnight. Cells were then treated or not (control) with 500 ng/mL of PA for 1 h and lysed, and CMG2 was immunoprecipitated (IP). IPs were analyzed by SDS-PAGE and western blotting against actin, PA, and mouse CMG2. (B) Control MEFs and SYF MEFs were transfected for 24 h with cDNA expressing an empty control (Ctrl), GFP-fyn, GFP-src, or GFP-yes, then grown with or without FBS overnight, and CMG2 was immunoprecipitated. IPs and cell lysates were analyzed by SDS-PAGE and western blotting against actin, mouse CMG2, and GFP for fyn, src, and yes expression. (C–F) RPE1 cells were transfected with control siRNA (−) or with siRNA against (D) src and fyn or (F) RhoA for 72 h, then transfected for 24 h with cDNA expressing CMG2-V5 WT, W396L, or K417S and serum starved overnight. The cells were then treated or not with 500 ng/mL of PA (1 h 4°C plus 20 min at 37°C) or (E) the src-like kinase inhibitor PP1 (50 μM in serum-free medium) for 1 h at 37°C and lysed, and CMG2 was immunoprecipitated. IPs were analyzed by SDS-PAGE and western blotting against actin, RhoA, total src, active src, phospho-tyrosine, PA, and V5. For all experiments, n = 3 replicates were performed, and a representative experiment is shown. (G) Schematic representation of the herein identified steps that CMG2 undergoes when evolving from a ligand-free to a ligand-bound state. (1) Ligand binding to CMG2 leads to signal transduction across the plasma membrane and causes (2) release of src and phosphorylation of an unknown component. This leads to (3) talin, vinculin, and actin release from CMG2. (4) RhoA is then able to bind CMG2 lysine 417 and (5) to recruit active src. (6) src will then phosphorylate CMG2 for receptor endocytosis. (6′) Additional effector proteins are also recruited by RhoA and contribute to CMG2 function.