A role for Rho in receptor- and G protein-stimulated phospholipase C. Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B.
Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha of free beta gamma subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signaling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, A1F-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 activity. Finally both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, When PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca(2+)- as well as GTP gamma S- or A1F-4-stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.
Schmidt
M
M
Institut für Pharmakologie, Universitätsklinikum Essen, Germany.
Bienek
C
C
Rümenapp
U
U
Zhang
C
C
Lümmen
G
G
Jakobs
K H
KH
Just
I
I
Aktories
K
K
Moos
M
M
von Eichel-Streiber
C
C
D036002:ADP Ribose Transferases / Q000494:pharmacology; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D001905:Botulinum Toxins; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000502:physiology*; D010738:Type C Phospholipases / Q000502:physiology*; D020742:rhoA GTP-Binding Protein
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011976:Receptors, Muscarinic; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D010738:Type C Phospholipases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein