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

Abstract

A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a calcium/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 micrograms of phosphatidylserine per ml or 200 micrograms of phosphatidylinositol per ml) and is greatly enhanced by addition of calcium (0.5 mM) and magnesium (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 +/- 2.5 nM with 1.8 +/- 0.6 x 10(4) sites per cell compared to a Kd of 11.9 +/- 4.4 nM and 4.1 +/- 1.0 x 10(4) sites per cell for the membrane receptor. Consistent with the existence of at least two 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 has been observed for intact cells. These results further support the similarity between phorbol ester receptors and the calcium/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.