Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.
One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.
Rebecchi
M J
MJ
Program in Molecular Biology, Developmental and Membrane Biology, Memorial Sloan-Kettering Cancer Research Center, New York, NY 10021.
Rosen
O M
OM
D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D010716:Phosphatidylinositols / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism
D006150:Guanine Nucleotides; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D013917:Thrombin; D002118:Calcium