Jis. Macdonald et al., Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA, J BIOL CHEM, 275(24), 2000, pp. 18225-18233
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.