We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclindependent kinase substrate p13 suc1 , and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase C␥-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosinedependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-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 activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkAShc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.

To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14‐amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC(Ki14) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild‐type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Ki14). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)‐mediated SHC tyrosine phosphorylation and a loss of high‐affinity TrkA‐SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF‐dependent tyrosine phosphorylation of the signaling molecules phospholipase Cγ‐1, MAP kinase/ERK‐1, and SNT. TrkAKi receptors internalize NGF comparable to wild‐type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3‐kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF‐dependent cell survival under serum‐free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate‐early genes (NGF1A, NGF1B, and c‐fos), suggesting that these gene products are not required for NGF‐dependent cell survival responses.

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