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.