Van de Vijver M J, Kumar R, Mendelsohn J
Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1991 Apr 25;266(12):7503-8.
A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.
A431细胞表达大量表皮生长因子（EGF）受体，并产生这些受体的一种配体，即转化生长因子-α（TGF-α）。我们已获得证据表明，这些细胞上的EGF受体可能通过配体经“自分泌”途径被激活，并且研究了内源性产生的TGF-α对受体磷酸化的激活是发生在细胞内还是细胞表面。当A431细胞在无血清条件下培养，且不存在外源性配体时，发现EGF受体具有基础水平的磷酸化。当细胞在持续存在阻断TGF-α与EGF受体结合的单克隆抗体的情况下用32Pi培养进行标记时，磷酸化水平降至基础水平的30±10%。这种降低不能用由于抗受体单克隆抗体结合导致的EGF受体下调和分解代谢引起的受体含量减少来解释。抗体介导的受体磷酸化降低伴随着培养基中分泌的TGF-α种类水平的增加。我们还用[35S]半胱氨酸对A431细胞进行了15分钟的脉冲标记，并在不同的追踪期后用抗磷酸酪氨酸抗体对细胞裂解物进行免疫沉淀。追踪40分钟后可检测到酪氨酸磷酸化的EGF受体，4 - 6小时后达到最大值；这些时间与EGF受体合成后到达细胞表面然后达到最大表达所需的时间间隔一致。此外，用抗磷酸酪氨酸抗体免疫沉淀的只有170 kDa的成熟EGF受体种类，而不是160 kDa的细胞内前体。这些脉冲追踪实验的结果以及抗受体单克隆抗体可阻断受体磷酸化的发现表明，EGF受体的激活可能是由于内源性配体（可能是TGF-α）的结合，这种结合发生在细胞表面，而不是在受体生物合成和细胞内加工过程中。
