Specific binding of phorbol ester tumor promoters to mouse tissues and cultured cells

Phorbol esters bind to mouse tissues and chick embryo fibroblasts in a specific, saturable, and reversible fashion. The binding site, located in the membrane fraction, is heat and protease sensitive. Binding can be measured most readily with [3H]PDBu. Binding of [3H]PDBu is of high affinity and is inhibited competitively by nonradioactive phorbol esters; the dissociation constants of the phorbol esters correspond quantitatively to their respective biological and tumor-promoting activities. Of particular significance, highly inflammatory but weakly promoting or nonpromoting diterpene esters are much less potent than PMA. Binding of [3H]PMA has been measured directly. The results confirm that PMA and PDBu interact at the same major high-affinity binding site. [3H]PDBu binding is entropy driven. The equilibrium dissociation constant is independent of temperature, whereas the off-rate is highly temperature dependent. In vivo, specific binding activity increases during embryonic development. It also shows considerable variation among tissues. In the mouse, highest binding activity, 28 pmole/mg, is in the brain (skin, for example, binds 3.9 pmole/mg). Between regions of he brain, 10-fold differences in binding activity are found. The high level of phorbol ester binding in brain suggests that the phorbol ester receptor plays a functional rather than an exclusively information-transducing role in the cell. Growth of cells in the presence of PDBu causes marked down-modulation of phorbol ester receptors. In the GH4C1 rat pituitary cell line, receptor number is decreased by 80% in 24 hr. In membrane preparations, the phorbol ester binding affinity is calcium sensitive. It has been speculated that endogenous ligands interacting at the phorbol ester receptor may exist. The finding that the supernatant fraction from boiled or acidified brain inhibits [3H]PDBu binding is therefore exciting.