Specific binding of phorbol esters to Friend erythroleukemia cells -- general properties, down regulation and relationship to cell differentiation

Specific and saturable binding sites for 20-³H]phorbol 12,13-dibutyrate(³H]PDBu) were demonstrated in intact Friend erythroleukemiacells (FELC), in which inducible erythroid differentiation isreversibly inhibited by phorbol esters. The binding of ³H]PDButo intact cells was maximal within only 15 min of incubationat 37°C, after which there was a gradual decrease; bindingat 4°C however, was a slow process, requiring > 180 minfor maximal binding. A Scatchard analysis showed that the dissociationconstant for binding of ³H]PDBu is 8.3 nM; at saturation, 1.75x 10⁵ molecules of ³H]PDBu are bound per cell. The bindingof ³H]PDBu is blocked by 12-O-tetradecanoyl phorbol-13-acetate,phorbol 12,13-didecanoate, mezerein, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and resiniferatoxin, but not by phorbol or4-phorbol 12,13-didecanoate. There was, in general, a good correlationbetween the potency of these agents in inhibiting ³H]PDBu bindingand their activity in promoting tumors on mouse skin. Inducersof differentiation, such as hexamethylene bisacetamide, dimethylsulfoxide and butyric acid, as well as inhibitors of cell differentiation,dexamethasone and local anesthetics, did not significantly blockthe binding of ³H]PDBu to intact FELC. When FELC were inducedto differentiate with 4 mM hexamethylene bisacetamide (80% ofcells were benzidine-positive), a slight decrease (10–20%)in the number of binding sites at saturation was seen, but thedissociation constant was not changed. When the cells were preculturedwith non-radioactive phorbol esters, a significant decreasein ³H]PDBu binding was observed, suggesting a homologous downregulation of phorbol ester receptors. Scatchard analysis indicatedthat the decrease in ³H]PDBu binding was due to a decreasein the number of binding sites and not to a change in affinity.Such specific phorbol ester binding sites might mediate a numberof biochemical and biological effects of phorbol esters on FELC.