Isolation and characterization of a specific receptor for biologically active phorbol and ingenol esters

A high-affinity, specific receptor for biologically active phorbol and ingenol esters has been purified to electrophoretic homogeneity from murine brains using ammonium sulfate fractionation, and DEAE-cellulose, Sephadex G-200, Affi-Gel Blue, and phenyl-Sepharose chromatographies. The receptor is a single-chain hydrophobic protein with a molecular weight (Mr) of 81,500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor has a sedimentation coefficient of 5.2 S and Stokes radius of 30.3 A. It has an isoelectric point (pI) of 5.5. The receptor is heat and acid labile. The receptor absolutely depends upon phosphatidylserine or phosphatidylinositol (optimum concentration approximately 4-8 micrograms/ml) for its activity. A variety of divalent cations stimulates the binding activity of the receptor. A molecule of receptor binds 1-2 molecules of phorbol-12, 13-dibutyrate (PDBu) with a Kd value of 4.2 nM. Those phorbol and ingenol esters which stimulate cell growth in culture and have tumor-promoting activity in vivo inhibit the binding of labeled PDBu to its homogeneous receptor, while the biologically inactive derivatives fail to do so. The homogeneous receptor protein contains a protein kinase activity.