INHIBITION BY TOXIN-B OF INOSITOL PHOSPHATE FORMATION INDUCED BY G-PROTEIN-COUPLED AND TYROSINE KINASE RECEPTORS IN N1E-115 NEUROBLASTOMA-CELLS - INVOLVEMENT OF RHO-PROTEIN

G protein-coupled receptors activate phospholipase G (PLC)-beta isofor ms by the alpha or beta gamma subunits of G proteins, whereas growth-f actor receptors activate PLC-gamma isoforms by phosphorylating tyrosin e residues of the enzyme. As a common substrate for PLC enzymes, phosp hatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P-2] may play a pivotal r ole in the regulation of cellular PLC activity. Because small-molecula r-weight G proteins have been implicated in the synthesis of Ptdlns(4, 5)P-2, we studied the effect of Clostridium difficile toxin B, which g lucosylates and thereby inactivates small G proteins of the Rho family , on receptor-stimulated PLC activity. We report here that in N1E-115 neuroblastoma cells, stimulation of inositol phosphate formation by th e G protein-coupled receptor agonists bradykinin and lysophosphatidic acid and by the tyrosine kinase receptor agonist platelet-derived grow th factor is largely attenuated by toxin B treatment. Furthermore, ino sitol phosphate production stimulated by the stable GTP analog guanosi ne 5'-O-(3-thio)-triphosphate in permeabilized N1E-115 cells was inhib ited by C3 exoenzyme, which specifically inactivates Rho proteins. The inhibition by toxin B was apparently not caused by its effect on the cytoskeleton. In addition, the level of platelet-derived growth factor receptors, which was studied with immunoblotting, was unaffected by t oxin B. Using exogenous Ptdlns(4,5)P-2 as PLC substrate, it was found that the intrinsic enzymatic activity of PLC activated either by Ca2or by guanosine 5'-O(3-thio)triphosphate was not altered by toxin B. H owever, toxin B decreased strongly, by up to 80%, the cellular level o f Ptdlns(4,5)P-2 in a concentration-dependent manner, without changing 'chose of phosphatidylinositol and phosphatidylinositol 4-phosphate. T hese results, together with the recent finding that Rho family protein s can regulate phosphatidylinositol 4-phosphate 5-kinase activity, dem onstrate that Rho proteins are presumably important regulators of Ptdl ns(4,5)P-2 synthesis and, thereby, play an integral role in the regula tion of cellular signaling by PLC enzymes.