Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle.
The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated G(q/11), G(13), and G(12), and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G(13) and inhibited by Galpha(13) 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 Galpha(13) 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)-alpha inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G(12) was not coupled to RhoA or ARF, and Galpha(12) antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G(13)-dependent RhoA and by ARF and PKC-alpha and is modulated by an inhibitory G(12)-dependent pathway.
Murthy
K S
KS
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Zhou
H
H
Grider
J R
JR
Makhlouf
G M
GM
D036002:ADP Ribose Transferases / Q000494:pharmacology; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001905:Botulinum Toxins; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007422:Intestines; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D012844:Sincalide / Q000378:metabolism / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000906:Antibodies; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011493:Protein Kinase C; D010739:Phospholipase D; D001905:Botulinum Toxins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020559:Monomeric GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D012844:Sincalide