title: The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions.
abstract: Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.
authors.affiliation: LBPI: Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), Université du Luxembourg, 162A, avenue de la Faiencerie, L-1511 Luxembourg.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kreis
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Schönfeld
authors.forename: Chantal
authors.initials: C
authors.lastname: Melchior
authors.forename: Beat
authors.initials: B
authors.lastname: Steiner
authors.forename: Nelly
authors.initials: N
authors.lastname: Kieffer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen; D003593:Cytoplasm / Q000502:physiology; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014471:Umbilical Veins; D014746:Vimentin / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D038982:Integrin alpha2beta1; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D003094:Collagen