HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways

Previous studies have indicated that in HepG(2) cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG(2) cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (Ab(HBP)). HDL3, when binding to HepG(2) cells, promoted the release in the culture medium of a 110 kDa protein that binds Ab(HBP), while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (Ab(IPG)), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [H-3]-PC prelabeled HepG(2) cells, Ab(HBP) were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with Ab(IPG) was found to inhibit only the HDL3-signal and not the Ab(HBP)-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and Ab(HBP)-signal. Moreover cellular preincubation with Ab(HBP) for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites. (C) 1997 Elsevier Science B.V.