Abstract
Differential inhibitors of Gbetagamma-effector regions are required to dissect the biological contribution of specific Gbetagamma-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gbetagamma-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gbetagamma, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3beta, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase gamma (PI3Kgamma) to the plasma membrane and the signaling to AKT in response to Gbetagamma overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kgamma and Gbetagamma. Other PhLP-derived constructs interacted with Gbetagamma but were not effective inhibitors of Gbetagamma signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gbetagamma-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gbetagamma-PI3Kgamma interaction.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Aorta / cytology
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Line
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Cell Movement / physiology
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Dimerization
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Endothelial Cells / cytology*
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Endothelial Cells / metabolism*
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Extracellular Signal-Regulated MAP Kinases / metabolism*
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GTP-Binding Protein beta Subunits / chemistry
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GTP-Binding Protein beta Subunits / genetics
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GTP-Binding Protein beta Subunits / metabolism*
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GTP-Binding Protein gamma Subunits / chemistry
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GTP-Binding Protein gamma Subunits / genetics
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GTP-Binding Protein gamma Subunits / metabolism*
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Green Fluorescent Proteins / genetics
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Humans
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In Vitro Techniques
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Kidney / cytology
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Lysophospholipids / metabolism
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Lysophospholipids / pharmacology
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / physiology
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Mutagenesis, Site-Directed
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Neovascularization, Physiologic / physiology
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Nerve Tissue Proteins / chemistry
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Pertussis Toxin / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphorylation / physiology
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-akt / metabolism*
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Sphingosine / analogs & derivatives
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Sphingosine / metabolism
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Sphingosine / pharmacology
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Swine
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Transfection
Substances
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Carrier Proteins
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G-protein Beta gamma
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GTP-Binding Protein beta Subunits
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GTP-Binding Protein gamma Subunits
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Lysophospholipids
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Nerve Tissue Proteins
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PDCL protein, human
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Green Fluorescent Proteins
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sphingosine 1-phosphate
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Pertussis Toxin
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Phosphatidylinositol 3-Kinases
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Proto-Oncogene Proteins c-akt
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Extracellular Signal-Regulated MAP Kinases
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Sphingosine