Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol.

In the presence of phosphatidylserine, [20-3H]-phorbol 12,13-dibutyrate [( 3H]PBt2) bound specifically to a single class of binding sites in mouse brain cytosol (supernatant at 100,000 X g). The dissociation constant for binding was 3.1 X 10(-9) M, and at saturation 23.2 pmol of [3H]PBt2 was bound per mg of cytosolic protein. Less than 1 pmol of [3H]PBt2 per mg bound in the absence of phospholipids. Phosphatidic acid, sphingomyelin, and phosphatidylinositol also were able to reconstitute binding activity, whereas phosphatidylcholine and phosphatidylethanolamine were relatively ineffective. [3H]PBt2 binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 4.4 X 10(-11) M), phorbol 12,13-didecanoate (Ki = 7.7 X 10(-9) M), phorbol 12,13-diacetate (Ki = 4.4 X 10(-7) M) and 4-O-methylphorbol 12-myristate 13-acetate (Ki = 5.1 X 10(-7) M). The apparent Ki values of the phorbol-related diterpenes for inhibiting binding agreed reasonably closely with the values previously determined for mouse brain membrane binding. The biologically inactive derivatives phorbol (30 microM) and 4 alpha-phorbol 12,13-didecanoate (30 microM) did not inhibit binding. The aporeceptor was eluted in one peak during Ultrogel 44 column chromatography, corresponding to a molecular weight of approximately equal to 77,000. Calcium phospholipid-dependent protein kinase C activity was eluted with a profile similar to that of the cytosolic aporeceptor-binding activity.