Specific binding of [20-3H]12-deoxyphorbol 13-isobutyrate to phorbol ester receptor subclasses in mouse skin particulate preparations.

Abstract

[20-3H]12-Deoxyphorbol 13-isobutyrate ([3H]DPB), an inflammatory but relatively nonpromoting analogue of the phorbol ester tumor promoters, bound to mouse skin particulate preparations in a specific, saturable, and reversible manner. Analysis of the binding yielded curvilinear Scatchard plots, consistent with two binding sites present at 0.14 (Site 1) and 1.6 (Site 2) pmol/mg protein and possessing binding affinities of 6.9 and 86 nM, respectively. Structure-activity analysis yielded good correlation (r = 0.94) for a series of 15 diterpene derivatives, including mezerein, between binding affinities at Site 2 and literature values for mouse ear inflammatory potencies. Comparison of binding by [3H]DPB with that by the typical phorbol ester [20-3H]phorbol 12,13-dibutyrate ([3H]PDBU) indicated that PDBU also bound to the sites recognized by [3H]DPB, with affinities of 0.7 and 10 nM, respectively. In addition, a third PDBU binding site was present in mouse skin at 1.9 pmol/mg protein (Site 3) and possessed an affinity of 53 nM. The affinity of DPB for Site 3, determined from competition of [3H]PDBU binding, was 5400 nM. Despite problems in quantitation, the structure-activity relations for Site 3 appeared to differ from those at Site 2 and resembled more closely those expected for complete promoters. Whether the different binding sites represent distinct protein receptors or the same receptor differentially modified remains to be determined.