The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated Gq/11, G13, and G12, and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G13 and inhibited by Gα13 antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 ± 2% to 53 ± 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Gα13 antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-α inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G12 was not coupled to RhoA or ARF, and Gα12 antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G13-dependent RhoA and by ARF and PKC-α and is modulated by an inhibitory G12-dependent pathway.
Analysis of the cellular functions of the small GTP-binding protein rho p21 with Clostridium botulinum C3 exoenzyme.
