title: Redistribution of epidermal growth factor receptor as a function of cell density, cell-cell adhesion and calcium in human (A-431) cells.
abstract: 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.
authors: Georgetown Medical Center, Department of Anatomy and Cell Biology, Washington, D.C. 20007.
authors: C A
authors: CA
authors: Suárez-Quian
authors: S W
authors: SW
authors: Byers
mesh_terms: D000595:Amino Acid Sequence; D015820:Cadherins / Q000276:immunology; D002118:Calcium / Q000032:analysis / Q000502:physiology*; D002294:Carcinoma, Squamous Cell; D002448:Cell Adhesion; D002452:Cell Count; D002460:Cell Line; D066246:ErbB Receptors / Q000032:analysis* / Q000276:immunology / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
chemical_list: D015820:Cadherins; D007106:Immune Sera; D066246:ErbB Receptors; D002118:Calcium