title: R-Ras regulates beta1-integrin trafficking via effects on membrane ruffling and endocytosis.
abstract: BACKGROUND
  Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins.
  
  
  RESULTS
  GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, beta1-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of beta1-integrin, and decreased beta1-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras.
  
  
  CONCLUSIONS
  Our results suggest that integrin function involves integrin trafficking via a cycle of membrane protrusion, ruffling, and endocytosis regulated by R-Ras, providing a novel mechanism by which integrins are linked to R-Ras through control of membrane dynamics.
authors.affiliation: Dept of Pharmacology, Laboratory for Molecular Biology and University of Wisconsin Carbone Cancer Center, University of Wisconsin, 1525 Linden Dr, Madison, WI 53706, USA.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Conklin
authors.forename: Aude
authors.initials: A
authors.lastname: Ada-Nguema
authors.forename: Maddy
authors.initials: M
authors.lastname: Parsons
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Riching
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Keely
mesh_terms: D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000502:physiology; D002522:Chlorocebus aethiops; D004705:Endocytosis; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism; D018631:ras Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D049452:Green Fluorescent Proteins; D019012:Integrin beta1; D034741:RNA, Small Interfering; D020691:rab GTP-Binding Proteins; D018631:ras Proteins; D000099193:rab11 GTP-Binding Proteins