Haugh J M, Schooler K, Wells A, Wiley H S, Lauffenburger D A
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1999 Mar 26;274(13):8958-65. doi: 10.1074/jbc.274.13.8958.
The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFalpha-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-gamma1 (PLC-gamma1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFalpha stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFalpha. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-gamma1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-gamma1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-gamma1 to its substrate in endocytic trafficking organelles.
表皮生长因子受体（EGFR）配体，即表皮生长因子（EGF）和转化生长因子-α（TGFα），引发配体和受体分子不同的内吞后加工过程，这会影响长期的细胞信号转导结果。这些差异源于在分选内体的酸性条件下，EGF-EGFR相互作用的亲和力高于TGFα-EGFR。为了确定内体中的EGFR占据情况是否也会影响短期信号转导事件，我们研究了磷脂酶C-γ1（PLC-γ1）途径的激活，这一事件已被证明对生长因子诱导的细胞运动至关重要。我们发现，EGF在内化后继续刺激EGFR的最大酪氨酸磷酸化，而正如预期的那样，TGFα的刺激明显较少。然而，EGF导致的受体激活水平较高，并未产生比TGFα刺激更高水平的磷脂酰肌醇（4,5）-二磷酸（PIP2）水解。通过改变细胞表面和内化区室之间活化受体的比例，我们发现只有细胞表面受体有效地参与PLC功能。与PIP2水解相反，PLC-γ1酪氨酸磷酸化与任一配体刺激的Tyr(P)-EGFR总水平呈线性相关，表明内化的EGFR功能缺陷不能归因于无法与信号蛋白相互作用并使其磷酸化。我们得出结论，通过PLC途径的EGFR信号转导在PLC-γ1磷酸化和PIP2水解之间的某个点受到空间限制，这可能是因为内吞运输细胞器中与EGFR结合的PLC-γ1接近其底物的机会有限。
