A ROLE FOR RHO IN RECEPTOR-STIMULATED AND G-PROTEIN-STIMULATED PHOSPHOLIPASE-C - REDUCTION IN PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE BY CLOSTRIDIUM-DIFFICILE TOXIN-B

Receptors coupled to heterotrimeric guanine nucleotide-binding protein s (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns( 4,5)P-2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha or free beta gamma subunits of the relevant G proteins. To study wheth er low molecular weight G proteins of the Rho family are involved in r eceptor signalling to PLC, we examined the effect of Clostridium diffi cile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (I-ILK) c ells stably expressing the m3 muscarinic acetylcholine receptor (mAChR ) subtype. Toxin B treatment of HEK cells did not affect basal PLC act ivity, but potently and efficiently inhibited mAChR-stimulated inosito l phosphate formation. PLC activation by the endogenously expressed th rombin receptor and by the direct G protein activators, AIF(4)(-) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLC activi ty. Finally, both toxin B and C3 exoenzyme significantly reduced, by 4 0 to 50%, the total level of PtdIns(4,5)P-2 in HEK cells, without affe cting the levels of phosphatidylinositol and phosphatidylinositol 4-ph osphate. Accordingly, when PLC activity was measured with exogenous Pt dIns(4,5)P-2 as enzyme substrate, Ca2+- as well as GTP gamma S- or AlF 4--stimulated PLC activities were not altered by prior toxin B treatme nt. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled re ceptor signalling to PLC, most likely by reducing the cellular substra te supply.