Abstract
Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.
Keywords:
Calcium; Guanine Nucleotide Exchange Factor (GEF); Rho; Signal Transduction; Smooth Muscle.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Calcium / metabolism*
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Cell Line
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Gene Silencing / drug effects
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Guanine Nucleotide Exchange Factors / agonists*
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Guanine Nucleotide Exchange Factors / genetics
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Guanine Nucleotide Exchange Factors / metabolism
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Guanosine 5'-O-(3-Thiotriphosphate) / analogs & derivatives*
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Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
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Humans
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Mice
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Mice, Knockout
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Organ Culture Techniques
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Phenylephrine / analogs & derivatives*
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Phenylephrine / pharmacology
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Protein Multimerization / drug effects
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Protein Structure, Tertiary
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Rabbits
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Rats
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Receptor, Endothelin A / genetics
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Receptor, Endothelin A / metabolism
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Receptors, Thromboxane A2, Prostaglandin H2 / genetics
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Receptors, Thromboxane A2, Prostaglandin H2 / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Rho Guanine Nucleotide Exchange Factors / agonists*
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Rho Guanine Nucleotide Exchange Factors / genetics
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Rho Guanine Nucleotide Exchange Factors / metabolism
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rho GTP-Binding Proteins / genetics
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rho GTP-Binding Proteins / metabolism
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rho-Specific Guanine Nucleotide Dissociation Inhibitors / genetics
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rho-Specific Guanine Nucleotide Dissociation Inhibitors / metabolism
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rhoA GTP-Binding Protein / genetics
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rhoA GTP-Binding Protein / metabolism
Substances
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1-(2-nitro)phenylethyl-P(3)-guanosine 5'-O-(3-thio)triphosphate
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ARHGEF11 protein, human
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ARHGEF11 protein, mouse
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ARHGEF12 protein, human
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Arhgef12 protein, mouse
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Arhgef12 protein, rat
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Guanine Nucleotide Exchange Factors
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PDZ-RhoGEF, rat
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Receptor, Endothelin A
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Receptors, Thromboxane A2, Prostaglandin H2
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Recombinant Proteins
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Rho Guanine Nucleotide Exchange Factors
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rho-Specific Guanine Nucleotide Dissociation Inhibitors
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RHOA protein, human
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N-2-nitrobenzylphenylephrine
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Phenylephrine
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Guanosine 5'-O-(3-Thiotriphosphate)
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RhoA protein, mouse
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rho GTP-Binding Proteins
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rhoA GTP-Binding Protein
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Calcium