Cunningham M E, Stephens R M, Kaplan D R, Greene L A
Department of Pathology and Center of Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Biol Chem. 1997 Apr 18;272(16):10957-67. doi: 10.1074/jbc.272.16.10957.
Many receptor tyrosine kinases possess an "activation loop" containing three similarly placed tyrosine autophosphorylation sites. To examine their roles in the TRK 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 >/= Y674F >> Y675F >/= YY670/674FF = YY670/675FF >> YY674/675FF > YYY670/674/675FFF. NGF-induced tyrosine phosphorylation of Shc, ERKs, and SNT and immediate early gene inductions generally paralleled neurogenic potential. However, activation of phosphatidylinositol 3'-kinase and tyrosine phosphorylation of phospholipase Cgamma-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 Cgamma-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.
许多受体酪氨酸激酶都拥有一个“激活环”，其中包含三个位置相近的酪氨酸自磷酸化位点。为了研究它们在TRK神经营养因子（NGF）受体中的作用，将这些残基（酪氨酸-670、酪氨酸-674和酪氨酸-675）分别以及全部组合突变为苯丙氨酸，并在缺乏Trk的PC12nnr5细胞中稳定表达。所有突变受体均显示神经生长因子（NGF）刺激的自磷酸化显著减少，表明催化活性受损。当通过相对受体表达比较转染子时，NGF诱导的神经突生长呈现剂量反应行为，并表现出功能等级：野生型＞Y670F≥Y674F＞＞Y675F≥YY670/674FF = YY670/675FF＞＞YY674/675FF＞YYY670/674/675FFF。NGF诱导的Shc、细胞外信号调节激酶（ERK）和SNT的酪氨酸磷酸化以及立即早期基因诱导通常与神经发生潜能平行。然而，磷脂酰肌醇3'-激酶的激活和磷脂酶Cγ-1的酪氨酸磷酸化基本被消除。后一种效应似乎是由于突变的TRK选择性地无法将结合磷脂酶Cγ-1所需的酪氨酸残基（酪氨酸-785）自磷酸化，这表明“激活环”酪氨酸参与了NGF依赖性的受体构象变化。我们的研究结果强调了表达水平在评估受体突变后果中的重要性，以及所有三个激活环酪氨酸在调节通过TRK的整体和特定NGF介导的信号传导中都发挥作用。
