Specific lipid components in membrane receptors of 12-O-tetradecanoylphorbol-13-acetate as revealed by direct photoaffinity labelling.

The use of [20-3H]-12-O-tetradecanoylphorbol-13-acetate (3H-TPA) as a direct photoaffinity probe of receptors was investigated. When membrane receptors in a particulate fraction from mouse brain were loaded with the high-affinity agonist 3H-TPA, irradiation of such preparations with ultraviolet (UV) light resulted in specific irreversible binding of the label to the membrane lipids phosphatidylethanolamine (PE) and phosphatidylserine (PS); evidence for labelling of proteins was lacking. The labelled lipids were tentatively identified by co-chromatography; they corresponded to selected reference lipids obtained by photoaffinity labelling. When a variety of natural and synthetic reference lipids was irradiated in the presence of 3H-TPA, photoadducts were obtained primarily from natural lipids containing unsaturated acyl chains in position 2. The synthetic 1,2-dipalmitoyl derivatives of phosphatidylcholine (PC), PE and phosphatidyl glycerol were refractory. When the photoadducts from PC and PE were treated with phospholipases A2 and C, the chromatographic pattern of cleavage products obtained was in accordance with that of phospholipids carrying the 3H-TPA label on the acyl moiety in the 2-position. Since previous evidence had suggested that a lipid-protein complex was the phorbol ester receptor, it may, therefore, be concluded that the receptor(s) contain PE and PS in specific interaction with protein(s). Accordingly, PE- and PS-dependent enzymes (or other proteins) are considered prime candidates for phorbol ester receptors.