Identification of Receptors for Phorbol Ester Tumor Promoters in intact Mammalian Cells and of an Inhibitor of Receptor Binding in Biologic Fluids

Utilizing [3H]phorbol dibutyrate [P(Bu)2], we have developed an assay for high-affinity phorbol ester receptors in intact rat embryo fibroblasts. At 37 degrees C, binding of [3H]P(Bu)2reached a maximum within 10 min and was rapidly reversible. The tumor promoters 12-O-tetradecanoyl-phorbol 13-acetate, teleocidin B, and mezerein were potent inhibitors of [3H]P(Bu)2binding. Phorbol and 4-α -phorbol didecanoate, which lack tumor-promoting activity, did not inhibit [3H]P(Bu)2binding. Epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, arginine and lysine vasopressin, luteinizing-hormone releasing hormone, and diazepam did not inhibit [3H]P(Bu)2binding. A Scatchard analysis was compatible with two classes of binding sites, one with Kd=8 nM and about 1-2 × 105sites per cell and the other with Kd=710 nM and about 3× 106sites per cell. Sera from various species, human amniotic fluid, and certain tissue extracts inhibited specific binding of [3H]P(Bu)2. Fractionation of human serum led to a 135-fold purification of an inhibitory factor with a molecular weight in the range 40,000 to 80,000.