Identification of receptors for phorbol ester tumor promoters in intact mammalian cells and of an inhibitor of receptor binding in biologic fluids.

Abstract

Using [3H]phorbol dibutyrate [P(Bu)2], an assay was developed for high-affinity phorbol ester receptors in intact rat embryo fibroblasts. At 37.degree. C, binding of [3H]P(Bu)2 reached 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)2 binding. Phorbol and 4-.alpha.-phorbol didecanoate, which lack tumor-promoting activity, did not inhibit [3H]P(Bu)2 binding. 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)2 binding. A Scatchard analysis was compatible with 2 classes of binding sites, one with Kd = 8 nM and about 1-2 .times. 105 sites/cell and the other with Kd = 710 nM and about 3 .times. 106 sites/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 MW in the range 40,000-80,000.