Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells

Thrombin and insulin receptor signalling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [H-3]inositol-labelle 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the 'tethered-ligand' type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [H-3]InsP(3) concentrations but stimulated accumulation of [H-3]PtdIns(3,4,5)P-3 and [H-3]PtdIns(3,4)P-2, the relative steady-state concentrations of which may indicate degradation of [H-3]PtdIns(3,4,5)P-3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [H-3]PtdIns(3,4,5)P-3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [H-3]PtdIns(3,4)P-2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.