| Our laboratory's overall goal is to define the parameters which comprise the receptor-mediated, phosphoinositide-signaling system. To this end, we use the WRK-1 hormone-responsive rat mammary tumor cell line as a model system. These cells have a very active phosphoinositide cycle which we and others have extensively characterized. Working with this system, we determined that a close relationship exists between the extent of receptor occupation and the specific phosphoinoisitide which is recruited for cycling. In addition, we determined that the resynthesis of phosphatidylinositol, which follows agonist-induced hydrolysis of phosphatidylinositol bisphosphate 1) does not require the presence of agonist but 2) does require the activity of a diacylglycerol kinase. To further characterize the mechanisms which regulate the resynthesis of phosphatidylinositol, we recently purified phosphatidylinositol synthase and are currently designing a purification scheme for phosphatidic acid cytidylyltransferase. Additionally, to determine whether prolonged cycling requires transport of phosphatidylinositol to the plasma membrane to replenish phosphatidylinositol bisphosphate, we designed a system to measure phosphatidylinositol transport to the plasma membrane and selected WRK-1 variants which have differing levels of the putative phosphatidylinositol transport protein following transfection with cDNA for the transfer protein in the sense and antisense direction.
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