HDL3-SIGNALING IN HEPG(2) CELLS INVOLVES GLYCOSYL-PHOSPATIDYLINOSITOL-ANCHORED PROTEINS

In [H-3]phosphatidylcholine (PC) prelabelled HepG(2) cells, HDL3 stimu lates a biphasic increase in 1,2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 mu M D 609, RHC-80267 or U-73122 and less by 100 mu M propranolol. A phos pholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 mu M propran olol. Cellular preincubation with 200 mu g/ml antibodies against the i nositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab(IPG)), or depl etion in GPI-anchored proteins by cellular pretreatment with 0.5 U/ml PI-PLC: 1 mM insulin and 2 HU/ml streptolysin-O, or depletion in membr ane cholesterol content by filipin (5 mu g/ml), digitonin (5 mu g/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL3-signal, suggesti ng the involvement of a lipolytic cleavage off GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response t ime to HDL3, with a DAG peak at 2-3 min. In the presence of protease-i nhibitors, HDL3 releases in the culture medium several proteins with a residual IPG that binds Ab(IPG) after SDS-PAGE analysis and immunoblo tting. HDL3-signalling pathways comprise tyrosine kinases, as preincub ation with 100 mu g/ml genistein or tyrphostin inhibits the HDL3-signa l. HDL3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.