Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids.

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Abstract
A specific high-affinity phorbol ester binding component was identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a Ca/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 .mu.g of phosphatidylserine/ml or 200 .mu.g of phosphatidylinositol/ml) and is gratly enhanced by addition of Ca (0.5 mM) and Mg (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 .+-. 2.5 nM with 1.8 .+-. 0.6 .times. 104 sites/cell compared to a Kd of 11.9 .+-. 4.4 nM and 4.1 .+-. 1.0 .times. 104 sites/cell for the membrane receptor. Consistent with the existence of at least 2 phorbol ester binding sites in EL4 cells was the observation of curvilinear Scatchard plots for binding to whole homogenates. The cytosolic binding showed the same order of potency for binding phorbol ester analogs as was observed for intact cells. These results further support the similarity between phorbol ester receptors and the Ca/phospholipid-dependent protein kinase and suggest that the cytosolic receptor may be involved in subsequent phorbol ester effects in EL4 cells including loss of the kinase activity from the cytosol and production of T-cell growth factor.

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