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Specific Binding of [20-³H]12-Deoxyphorbol 13-Isobutyrate to Phorbol Ester Receptor Subclasses in Mouse Skin Particulate Preparations¹

Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20205^,3

[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.

¹ This research was supported in part by Grant BC 345 from the American Cancer Society and by Grant CA 22895 from the NIH.

² To whom requests for reprints should be addressed.

³ Current address.

Received 4/25/83. Accepted 7/12/83.