Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO # 1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 x 10⁶ sites/cell) and a low-affinity class (affinity constant = 4.880 nM; 7 x 10⁷ sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [³H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [³H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.