Foehr E D, Raffioni S, Murray-Rust J, Bradshaw R A
Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California 92697, USA.
J Biol Chem. 2001 Oct 5;276(40):37529-36. doi: 10.1074/jbc.M103234200. Epub 2001 Jul 17.
To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.
为了评估成纤维细胞生长因子受体1（FGFR1）细胞内结构域酪氨酸残基对信号传导能力的贡献，通过酪氨酸到苯丙氨酸阻断和单个转换系统地改变了稳定转染的嵌合体，该嵌合体带有血小板衍生生长因子受体（PDGFR）的胞外结构域和FGFR1的胞内结构域。该构建体（PFR1）胞内结构域的15个酪氨酸残基被分为四个线性片段（分别标记为A、B、C和D），它们分别包含4、4、2和5个酪氨酸残基。当受到血小板衍生生长因子刺激时，在转染的PC12细胞的神经突生长试验中，酪氨酸A、B或A + B片段发生突变的衍生物在48小时时的活性约为未修饰嵌合体的三分之二，但在96小时时达到完全活性。仅消除两个激活环酪氨酸（C片段）也会使受体失活。D片段中4个（或5个）酪氨酸发生突变的所有衍生物在诱导分化方面均无活性，但在体外试验中显示出低水平的激酶活性。对D片段中1、2或3个酪氨酸以不同组合进行系统改变的衍生物表明，2个残基（使用人FGFR1编号为Tyr(677)和Tyr(701)）对生物活性至关重要，但其余3个残基，包括先前确定的磷脂酶Cγ（PLCγ）激活位点Tyr(766)，并非如此。FRS2、SOS和ERK1/2的激活（磷酸化）与分化活性平行。PLCγ活性依赖于Tyr(766)的存在，但也需要Tyr(677)和/或Tyr(701)。尽管完全活性的嵌合体不需要PLCγ，但该活性显著增强了显示FRS2激活降低的嵌合体的反应。这些结果表明，PFR1不利用任何酪氨酸残基（无论是否磷酸化）来激活FRS2。然而，它确实需要Tyr(677)和/或Tyr(701)，它们可能直接或间接地起到稳定活性构象的作用。
