HDL3-signalling in HepG2 cells involves glycosyl-phosphatidylinositol-anchored proteins
| Autor: | Gaelle Pinchon, Stéphane Nion, Françoise Nazih-Sanderson, Jean-Charles Fruchart, Christiane Delbart |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Glycosylphosphatidylinositols
Biophysics Biochemistry Filipin Antibodies Diglycerides chemistry.chemical_compound Endocrinology Phosphatidylcholine Tumor Cells Cultured Humans Protease Inhibitors Phospholipase C Phospholipase D Leupeptin Membrane Proteins Phosphatidic acid Molecular biology Culture Media Digitonin chemistry Type C Phospholipases Phosphatidylcholines lipids (amino acids peptides and proteins) PMSF Lipoproteins HDL Signal Transduction |
| Zdroj: | Biochimica et biophysica acta. 1346(1) |
| ISSN: | 0006-3002 |
| Popis: | In [3H]phosphatidylcholine (PC) prelabelled HepG2 cells, HDL3 stimulates a biphasic increase in 1.2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 microM D 609, RHC-80267 or U-73122 and less by 100 microM propranolol. A phospholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 microM propranolol. Cellular preincubation with 200 microg/ml antibodies against the inositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab(IPG)), or depletion 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 membrane cholesterol content by filipin (5 microg/ml), digitonin (5 microg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL3-signal, suggesting the involvement of a lipolytic cleavage of GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response time to HDL3, with a DAG peak at 2-3 min. In the presence of protease-inhibitors, HDL3 releases in the culture medium several proteins with a residual IPG that binds Ab(IPG) after SDS-PAGE analysis and immunoblotting. HDL3-signalling pathways comprise tyrosine kinases, as preincubation with 100 microg/ml genistein or tyrphostin inhibits the HDL3-signal. HDL3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins. |
| Databáze: | OpenAIRE |
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