title: The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of beta1 integrins.
abstract: The small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including beta1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility. The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs). Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro, but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of beta1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of beta1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of beta1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization.
authors.affiliation: Department of Cell Biology, University of Virginia Health Sciences Center, Box 800732, Charlottesville, Virginia 22908, USA.
authors.forename: Jillian L
authors.initials: JL
authors.lastname: Dunphy
authors.forename: Radim
authors.initials: R
authors.lastname: Moravec
authors.forename: Kim
authors.initials: K
authors.lastname: Ly
authors.forename: Troy K
authors.initials: TK
authors.lastname: Lasell
authors.forename: Paul
authors.initials: P
authors.lastname: Melancon
authors.forename: James E
authors.initials: JE
authors.lastname: Casanova
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D017931:DNA Primers; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D000089982:ADP-Ribosylation Factor 6; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; C504483:IQSEC1 protein, human; D019012:Integrin beta1; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human