Cellular distribution and biological activity of epidermal growth factor receptors in A431 cells are influenced by cell-cell contact

The potential significance of cell-cell interactions on EGF receptor (EGFR) activity was investigated in cultured adherent A431 cells seeded as single-cell suspensions with different initial cell densities. In dense cultures, EGFRs were mainly localised at cell boundaries and in microvilli as shown by immunofluorescence analysis with an EGFR-specific antibody while in sparse cultures the distribution of EGFRs was more diffuse. Scatchard analysis showed that as cell density decreased the number of high-affinity receptors increased considerably. Upon treatment of adherent intact cells with EGF all cells in sparse cultures contained activated EGFRs as demonstrated by immunofluorescence analysis with a phosphotyrosine-specific antibody, while in dense cultures mainly cells at the periphery of a cluster and especially at their expanding borders exhibited activated EGFRs. EGF-induced phosphorylation in intact cells was greatly enhanced in sparse compared with dense cultures as demonstrated by immunoprecipitation with a phosphotyrosine-specific antibody. In contrast to intact cells, in cytoskeleton preparations, obtained after mild detergent treatment of adherent cells, EGFRs were able to undergo EGF-independent phosphorylation. Pretreatment of cells with EGF led to enhanced tyrosine phosphorylation of cytoskeletal-associated proteins. Our observations suggest that cell density has a considerable effect on the subcellular localisation as well as biological activity of the EGFR. Thus, in intact A431 cells growing with extensive cell-cell interactions some negative control mechanisms preventing EGFR activation may be exerted by adjacent cells.