title: Regulation of phospholipase C-gamma activity by glycosphingolipids.
abstract: Glycosphingolipid-enriched domains are hot spots for cell signaling within plasma membranes and are characterized by the enrichment of glycosphingolipids. A role for glucosylceramide-based glycosphingolipids in phospholipase C-mediated inositol 1,4,5-trisphosphate formation has been previously documented. These earlier studies utilized a first generation glucosylceramide synthase inhibitor to deplete cells of their glycosphingolipids. Recently, more active and specific glucosylceramide synthase inhibitors, including d-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol (d-t-EtDO-P4), have been designed. d-t-EtDO-P4 has the advantage of blocking glucosylceramide synthase at low nanomolar concentrations but does not cause secondary elevations in cell ceramide levels. In the present study, d-t-EtDO-P4 depleted cellular glucosylceramide and lactosylceramide in cultured ECV304 cells at nanomolar concentrations without obvious cellular toxicity. The expression of several signaling proteins was evaluated in glycosphingolipid-depleted ECV304 cells to study the role of glycosphingolipids in phospholipase C-mediated signaling. No difference was observed in the cellular expression of phospholipase C-gamma between controls and glycolipid-depleted cells. Western blot analysis, however, revealed that depletion of endogenous glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 induced tyrosine phosphorylation of phospholipase C-gamma in a concentration-dependent manner with maximum induction at 100 nm. The phosphorylation of phospholipase C-gamma induced by d-t-EtDO-P4 was abolished by exogenously added glucosylceramide, consistent with a specific glycosphingolipid-phospholipase C-gamma interaction. The phospholipase C-gamma phosphorylation was maximally enhanced by bradykinin when cells were exposed to 100 nm d-t-EtDO-P4. The measurement of cellular activity of phospholipase C-gamma, by myo-inositol 1,4,5-trisphosphate radioreceptor assay, demonstrated that depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 resulted in significantly increased formation of inositol 1,4,5-trisphosphate above base line, and an increased sensitivity of phospholipase C-gamma to bradykinin stimulation. Thus, the activation of phospholipase C-gamma is negatively regulated by membrane glycosphingolipids in ECV304 cells.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Liming
authors.initials: L
authors.lastname: Shu
authors.forename: Lihsueh
authors.initials: L
authors.lastname: Lee
authors.forename: James A
authors.initials: JA
authors.lastname: Shayman
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006028:Glycosphingolipids / Q000502:physiology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011412:Propanolamines / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: C404730:3',4'-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; D004791:Enzyme Inhibitors; D006028:Glycosphingolipids; D007527:Isoenzymes; D011412:Propanolamines; D011759:Pyrrolidines; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D051966:Phospholipase C gamma