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PMID: 9398666 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.

Molecular biology of the cell ·Vol. 8 ·No. 12 ·1997-12-00 ·Pages 2437-47

Fujihara H, Walker LA, Gong MC, Lemichez E, Boquet P, Somlyo AV, Somlyo AP

Abstract

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.

MeSH Terms
ADP Ribose Transferases/pharmacology Adenosine Diphosphate/metabolism Animals Biological Transport/drug effects Botulinum Toxins Calcium/antagonists & inhibitors,metabolism,pharmacology Cell Membrane/drug effects,metabolism Cytosol/drug effects,metabolism Diphtheria Toxin/pharmacology Endothelins/antagonists & inhibitors,pharmacology Guanine Nucleotide Dissociation Inhibitors/metabolism Guanosine 5'-O-(3-Thiotriphosphate)/antagonists & inhibitors,metabolism,pharmacology In Vitro Techniques Isometric Contraction/drug effects Muscle, Smooth, Vascular/drug effects,metabolism,physiology Peptide Fragments/pharmacology Phenylephrine/antagonists & inhibitors,pharmacology Phorbol 12,13-Dibutyrate/pharmacology Portal Vein/drug effects,metabolism,physiology Precipitin Tests Protein Binding/drug effects Rabbits Recombinant Fusion Proteins/pharmacology Sodium Dodecyl Sulfate/pharmacology rho-Specific Guanine Nucleotide Dissociation Inhibitors rhoA GTP-Binding Protein/antagonists & inhibitors,metabolism
Chemicals
Diphtheria Toxin Endothelins Guanine Nucleotide Dissociation Inhibitors Peptide Fragments Recombinant Fusion Proteins diphtheria toxin fragment B rho-Specific Guanine Nucleotide Dissociation Inhibitors Phenylephrine Sodium Dodecyl Sulfate Phorbol 12,13-Dibutyrate Guanosine 5'-O-(3-Thiotriphosphate) Adenosine Diphosphate ADP Ribose Transferases exoenzyme C3, Clostridium botulinum Botulinum Toxins rhoA GTP-Binding Protein Calcium
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Fujihara 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
Gong M C
Lemichez E
Boquet P
Somlyo A V
Somlyo A P
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.8.12.2437
Published
1997-12-00
Pages
2437-47
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC25718
Subset
IM
Grants
NHLBI NIH HHS · P01-HL-48807 · United States
External Links
PubMed source DOI: 10.1091/mbc.8.12.2437 PMC Full Text View journal details
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