The small GTPase RhoA regulates the contraction of smooth muscle tissues by catalyzing the assembly of cytoskeletal signaling complexes at membrane adhesion sites.
The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/βPIX. Paxillin tyrosine phosphorylation and its association with βPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Wenwu
W
Zhang
Youliang
Y
Huang
Susan J
SJ
Gunst
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D020125:Mutation, Missense; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000235:genetics / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; C496061:PXN protein, human; D051419:Paxillin; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C066737:RHOA protein, human; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000109:Acetylcholine