Characterization of specific binding of [3H]phorbol 12,13-dibutyrate and [3H]phorbol 12-myristate 13-acetate to mouse brain.

[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBU) and [20-3H]-phorbol 12-myristate 13-acetate ([3H]PMA) bound specifically and with high affinity to one class of saturable binding sites in particulate preparations from mouse brain. The dissociation constants for binding of [3H]PDBU and [3H]PMA were 7 nM and 66 pm, respectively. At half-maximal specific binding, binding of [3H]PDBU was 96% specific and binding of [3H]PMA was 91% specific. At saturation, 28 pmol of [3H]PDBU were bound per mg protein. Nonradioactive phorbol 12-myristate 13-acetate blocked > 95% of the specific [3H]PDBU binding. Conversely, nonradioactive phorbol 12,13-dibutyrate blocked > 95% of the specific [3H]PMA binding. The binding sites in mouse brain for [3H]PDBU and [3H]PMA thus appear to be equivalent. In addition to phorbol 12-myritate 13-acetate, seven other biologically active phorbol-related diterpenes inhibited [3H]PDBU binding with potencies which correlated with their biological activities. In the case of phorbol 12,13-diacetate, inhibition of [3H]PDBU binding was shown to be competitive. Phorbol (20 microgram/ml) and 4 alpha-phorbol 12,13-didecanoate (20 microgram/ml), two compounds devoid of biological activity, had no effect on binding. Binding was sensitive to heat, papain, and phospholipase A2 but was insensitive to neuraminidase. Based on the high level of specific phorbol ester binding in brain, we postulate that the phorbol ester target plays a functional rather than information-transducing role in the cell.