Figure 5.

TrkA, ARMS and CrkL association is enhanced by NGF. (A) Deletion of the Shc-binding site abolished NGF-induced FRS-2 interaction, but did not affect complex formation between TrkA and CrkL/c-Crk II. PC12^nnr5 cells stably expressing wild-type TrkA or a mutant Trk (ΔShc) carrying a deletion (ENPQY⁴⁹⁰F) that does not bind Shc or FRS2 were treated with NGF for 10 min. Lysates were immunoprecipitated either with an anti-Crk antibody, followed by blotting with an anti-Trk antiserum (RTA), or with anti-FRS2 antiserum and blotting with antiphosphotyrosine (p-Tyr). Reprobing the blots with anti-CrkL and anti-FRS2 verified sample loading. (B) TrkA, ARMS and CrkL association in PC12 cells is enhanced by NGF treatment. PC12-615 cells were treated for the indicated times with 100 ng/ml NGF. Detergent lysates were immunoprecipitated with anti-ARMS polyclonal antibody. Western blotting was performed with Trk (C-14) and CrkL polyclonal antibodies. (C) CrkL increases the binding of C3G and Trk upon NGF treatment. PC12-615 cells were stimulated for the indicated time with NGF and extracts were immunoprecipitated with pan-Crk antibodies. Western blotting analysis was performed with C3G, Abl, CrkL and TrkA to detect these proteins. Note that the increasing levels of C3G and activated TrkA were pulled down together with CrkL in response to NGF treatment. (D) Association of Trk, ARMS and CrkL. HEK293T cells were transfected with the indicated plasmids. Anti-FLAG immunoprecipitates were blotted with the indicated antibodies. The association of TrkA with CrkL was enhanced by ARMS expression (lane 1 versus lane 4).