Nerve growth factor induces a multimeric TrkA receptor complex in neuronal cells that includes Crk, SHC and PLC-gamma...

Binding of nerve growth factor (NGF) to the TrkA receptor results in homodimerization, and activation of the intrinsic tyrosine kinase activity of the receptor, leading to multiple phosphorylations. We investigated the in vivo formation and composition of the receptor complex induced by NGF in a central nervous system-derived cell line (B104-neo), transfected with the human cDNA for TrkA. Using receptor-activated immunoprecipitation followed by analysis of the immune complexes by SDS-PAGE and immunoblotting, we show that NGF induces the association of TrkA with c-Crk-II in a multimeric complex that also includes SHC and PLC-gamma 1. While the tyrosine phosphorylation of TrkA, SHC and PLC-gamma 1 increased with time in the presence of NGF and was inhibited by the tyrosine kinase inhibitor K252a, the state of tyrosine phosphorylation of c-Crk-II did not appear to change with NGF treatment. Immunodepletion studies demonstrated that the interaction of c-Crk-II with TrkA not only occurs indirectly through the SHC proteins, but may also involve another mode of binding. Furthermore, we show that c-Crk-II is associated with tyrosine phosphorylated p130CAS in unstimulated cells and that NGF treatment results in the de-association of p130CAS/c-Crk-II complex in the absence of an apparent change in the tyrosine phosphorylation of p130CAS. These results clearly implicate c-Crk-II in the NGF signaling pathway and support the concept that more than one signaling molecule known to participate in the activation of Ras associates with TrkA upon NGF treatment.