Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.
Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10(-6) M, 48 h; ; ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)gammaS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPgammaS-induced translocation of cytosolic RhoA () to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPgammaS, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.
Fujihara
H
H
Departments of Molecular Physiology and Biological Physics, Pathology and Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Walker
L A
LA
Gong
M C
MC
Lemichez
E
E
Boquet
P
P
Somlyo
A V
AV
Somlyo
A P
AP
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001905:Botulinum Toxins; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004167:Diphtheria Toxin / Q000494:pharmacology*; D016232:Endothelins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology; D010656:Phenylephrine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011169:Portal Vein / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004167:Diphtheria Toxin; D016232:Endothelins; D020730:Guanine Nucleotide Dissociation Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C039171:diphtheria toxin fragment B; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D010656:Phenylephrine; D012967:Sodium Dodecyl Sulfate; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; D002118:Calcium