Suárez-Quian C A, Byers S W
Georgetown Medical Center, Department of Anatomy and Cell Biology, Washington, D.C. 20007.
Tissue Cell. 1993;25(1):1-17. doi: 10.1016/0040-8166(93)90061-o.
Cell surface expression of the epidermal growth factor (EGF) receptor in several cell lines declines as a function of increased cell density and is associated with diminished responsiveness to EGF. However, the mechanism whereby this density-induced down regulation of receptors occurs has not been discerned. In the present study the distribution of the EGF receptor in A-431 cells as a function of cell density using (1) two polyclonal antibodies raised against peptide specific sequences of the EGF receptor that recognize either the cytoplasmic or extracellular domains of the receptor, respectively, and (2) biotinylated EGF, a specific probe for the cell surface receptor is now investigated. Immunolocalization of the receptor using the polyclonal antibodies or the biotin-EGF revealed that the receptor was homogeneously distributed on the cell surface of individual cells, or in cells plated at low density. In contrast, as cell density increased, prominent EGF immunoreactivity and biotin-EGF staining became limited to the periphery of the cells, at sites of cell-cell apposition, and was characterized by a honeycomb pattern, typical of a basolateral distribution. The effects of low Ca++ treatment, known to cause cells to round up and detach from one another, on EGF receptor distribution in cells at high cell density were then examined. Confocal microscopy of immunostained preparations revealed that incubation of high density cultures in Ca(++)-free media for as little as 10 min restored the homogeneous distribution of the EGF receptor and resulted in strong intracellular staining. Three-dimensional reconstruction of serial optical sections revealed that redistribution of the EGF receptor following low Ca++ treatment involved a heretofore undetected 'ruffling', an immunostaining pattern characterized by stripes of intense fluorescence signal interspersed with complete absence of fluorescence. Next, cell-cell adhesion was disrupted with antisera to the cell adhesion molecule E-cadherin. Although the antisera caused cells to detach from one another, eventually leading to cell rounding and redistribution of the EGF receptor, the receptor 'ruffling' immunostaining pattern rendered by the low Ca++ treatment was not detected. These results suggest that an association may exist between the plasma membrane EGF receptor distribution, density-induced EGF receptor down regulation, and the growth effects of low Ca++ observed in previous studies.
在几种细胞系中，表皮生长因子（EGF）受体的细胞表面表达随细胞密度增加而下降，并与对EGF的反应性降低相关。然而，受体这种密度诱导下调发生的机制尚未明了。在本研究中，使用（1）两种针对EGF受体肽特异性序列产生的多克隆抗体，分别识别受体的胞质或胞外结构域，以及（2）生物素化EGF（一种细胞表面受体的特异性探针），研究了A-431细胞中EGF受体随细胞密度的分布情况。使用多克隆抗体或生物素化EGF对受体进行免疫定位显示，受体在单个细胞或低密度接种的细胞的细胞表面均匀分布。相反，随着细胞密度增加，显著的EGF免疫反应性和生物素化EGF染色局限于细胞周边、细胞-细胞接触部位，并呈现蜂窝状模式，这是典型的基底外侧分布特征。然后研究了低钙处理（已知会导致细胞变圆并彼此分离）对高细胞密度细胞中EGF受体分布的影响。免疫染色制剂的共聚焦显微镜检查显示，将高密度培养物在无钙培养基中孵育仅10分钟，就能恢复EGF受体的均匀分布，并导致强烈的细胞内染色。连续光学切片的三维重建显示，低钙处理后EGF受体的重新分布涉及一种迄今未被发现的“褶皱”现象，这种免疫染色模式的特征是强烈荧光信号条带与完全无荧光区域相间分布。接下来，用抗细胞粘附分子E-钙粘蛋白的抗血清破坏细胞-细胞粘附。尽管抗血清导致细胞彼此分离，最终导致细胞变圆和EGF受体重新分布，但未检测到低钙处理产生的受体“褶皱”免疫染色模式。这些结果表明，质膜EGF受体分布、密度诱导的EGF受体下调与先前研究中观察到的低钙生长效应之间可能存在关联。
