Thrombin and insulin receptor signalling via phosphoinositide (PI)-spe
cific phospholipase C (PLC) and PI 3-kinase was studied in [H-3]inosit
ol-labelle 1321N1 cells. Thrombin stimulated a dramatic, transient act
ivation of PLC which is probably mediated via receptors of the 'tether
ed-ligand' type, since it was both reproduced by, and abolished follow
ing, pretreatment of cells with a synthetic peptide (SFLLRN) correspon
ding to the ligand domain of the human thrombin receptor. However, nei
ther thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin
did not influence [H-3]InsP(3) concentrations but stimulated accumulat
ion of [H-3]PtdIns(3,4,5)P-3 and [H-3]PtdIns(3,4)P-2, the relative ste
ady-state concentrations of which may indicate degradation of [H-3]Ptd
Ins(3,4,5)P-3 by 5- and 3-phosphatases. The independent coupling of th
rombin and insulin receptors to PLC and PI 3-kinase respectively in 13
21N1 cells allowed interactions between these systems to be examined.
Thus insulin-stimulated [H-3]PtdIns(3,4,5)P-3 accumulation was attenua
ted on co-stimulation of the thrombin receptor, whereas concentrations
of [H-3]PtdIns(3,4)P-2 were transiently enhanced but then reduced. Th
ese results indicate that thrombin receptors in 1321N1 cells do not ac
tivate PI 3-kinase, but can modulate signalling by this enzyme.