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R-Ras interacts with filamin a to maintain endothelial barrier function.
- Source :
-
Journal of cellular physiology [J Cell Physiol] 2011 Sep; Vol. 226 (9), pp. 2287-96. - Publication Year :
- 2011
-
Abstract
- The molecular mechanisms regulating vascular barrier integrity remain incompletely elucidated. We have previously reported an association between the GTPase R-Ras and repeat 3 of Filamin A (FLNa). Loss of FLNa has been linked to increased vascular permeability. We sought to determine whether FLNa's association with R-Ras affects endothelial barrier function. We report that in endothelial cells endogenous R-Ras interacts with endogenous FLNa as determined by co-immunoprecipitations and pulldowns with the FLNa-GST fusion protein repeats 1-10. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In these cells FLNa and R-Ras co-localize at the plasma membrane. Knockdown of R-Ras and/or FLNa by siRNA promotes vascular permeability, as determined by TransEndothelial Electrical Resistance and FITC-dextran transwell assays. Re-expression of FLNa restored endothelial barrier function in cells lacking FLNa whereas re-expression of FLNaΔ3 did not. Immunostaining for VE-Cadherin in cells with knocked down R-Ras and FLNa demonstrated a disorganization of VE-Cadherin at adherens junctions. Loss of R-Ras and FLNa or blocking R-Ras function via GGTI-2133, a selective R-Ras inhibitor, induced vascular permeability and increased phosphorylation of VE-Cadherin (Y731) and Src (Y416). Expression of dominant negative R-Ras promoted vascular permeability that was blocked by the Src inhibitor PP2. These findings demonstrate that maintaining endothelial barrier function is dependent upon active R-Ras and association between R-Ras and FLNa and that loss of this interaction promotes VE-Cadherin phosphorylation and changes in downstream effectors that lead to endothelial leakiness.<br /> (Copyright © 2010 Wiley-Liss, Inc.)
- Subjects :
- Cadherins metabolism
Capillary Permeability
Coronary Vessels cytology
Cytoskeleton metabolism
Endothelial Cells cytology
Filamins
Gene Knockdown Techniques
Humans
Phosphorylation
Phosphoserine metabolism
Protein Binding
Protein Transport
Proto-Oncogene Proteins pp60(c-src) metabolism
RNA, Small Interfering metabolism
Contractile Proteins metabolism
Endothelial Cells metabolism
Microfilament Proteins metabolism
ras Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1097-4652
- Volume :
- 226
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Journal of cellular physiology
- Publication Type :
- Academic Journal
- Accession number :
- 21660952
- Full Text :
- https://doi.org/10.1002/jcp.22565