Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neur otrophin receptor tyrosine kinase, TrkA Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase C gamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipita tion assays. Grb2 binding to TrkA is mediated by the central SH2 domain, re quires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carbox yl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the act ivation loop tyrosines on TrkA, we can stimulate constitutive kinase activi ty and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. T hus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct eviden ce that the activation loop tyrosines of a receptor tyrosine kinase, in add ition to their essential role in kinase activation, also serve a direct rol e in the recruitment of intracellular signaling molecules.