title: R-Ras interacts with filamin a to maintain endothelial barrier function.
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.
authors.affiliation: Department of Cell and Molecular Biology, Cardiovascular Research Center and the John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Griffiths
authors.forename: M
authors.initials: M
authors.lastname: Grundl
authors.forename: J S
authors.initials: JS
authors.lastname: Allen
authors.forename: M L
authors.initials: ML
authors.lastname: Matter
mesh_terms: D015820:Cadherins / Q000378:metabolism; D002199:Capillary Permeability; D003285:Contractile Proteins / Q000378:metabolism*; D003331:Coronary Vessels / Q000166:cytology; D003599:Cytoskeleton / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D064448:Filamins; D055785:Gene Knockdown Techniques; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D015820:Cadherins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D010768:Phosphoserine; D016392:Proto-Oncogene Proteins pp60(c-src); C085929:RRAS protein, human; D018631:ras Proteins