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)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 two classes of binding sites, one with Kd = 8 nM and about 1--2 x 10(5) sites per cell and the other with Kd = 710 nM and about 3 x 10(6) sites 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 135-fold purification of an inhibitory factor with a molecular weight in the range 40,000 to 80,000.
D000818:Animals; D001667:Binding, Competitive; D001769:Blood; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002478:Cells, Cultured; D003043:Cocarcinogenesis; D007700:Kinetics; D010703:Phorbol Esters / Q000378:metabolism*; D010704:Phorbols / Q000378:metabolism*; D011493:Protein Kinase C; D051381:Rats; D011955:Receptors, Drug / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010703:Phorbol Esters; D010704:Phorbols; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C