Autophosphorylation of Activation Loop Tyrosines Regulates Signaling By the Trk Nerve Growth Factor Receptor

Many receptor tyrosine kinases possess an ''activation loop'' containing three similarly placed tyrosine autophosphorylation sites. To examine their roles in the TRI( NGF receptor, these residues (Tyr-670, Tyr-674, and Tyr-675) were mutated singly and in all combinations to phenylalanine and stably expressed in Trk-deficient PC12nnr5 cells. All mutant receptors showed significantly diminished nerve growth factor (NGF)-stimulated autophosphorylation, indicating impaired catalytic activity. NGF-induced neurite outgrowth exhibited dose-responsive behavior when transfectants were compared by relative receptor expression and exhibited a functional hierarchy: wild type > Y670F greater than or equal to Y674F much greater than Y675F greater than or equal to YY670/674FF = W670/675FF much greater than W674/675FF > YYY670/674/675FFF. NGF-induced tyrosine phosphorylation of She, ERKs, and SNT and immediate early gene inductions generally paralleled neurogenic potential. However, activation of phosphatidylinositol 3'-kinase and tyrosine phosphorylation of phospholipase C gamma-1 was essentially abolished. The latter effect appears due to selective inability of the mutated TRKs to autophosphorylate the tyrosine residue (Tyr-785) required for binding phospholipase C gamma-1 and indicates that the ''activation loop'' tyrosines participate in NGF-dependent changes in receptor conformation. Our findings stress the importance that expression levels play in assessing the consequences of receptor mutations and that all three activation loop tyrosines have roles regulating both overall and specific NGF-mediated signaling through TRK. [References: 91]

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