Reference : A fluorescence cell biology approach to map the second integrin-binding site of talin...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/10993/4924
A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain
English
Tremuth, L. A [Lab. de Biol. et Physiol. Integree, CNRS/GDRE-ITI, Université du Luxembourg, 162A, Avenue de la Faïencerie, Luxembourg]
Kreis, Stephanie mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Melchior, C. A [Pharmacol./Physchim. Intrac. C./M., UMR CNRS 7034, Univ. Louis Pasteur de Strasbourg, Illkirch 67401, France]
Hoebeke, J. B [Institut Gilbert Laustriat (IFR 85), Illkirch 67401, France]
Rondé, P. C D [> >]
Plançon, Sébastien mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Takeda, K. C [> >]
Kieffer, N. A [> >]
2004
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology
279
21
22258-22266
Yes (verified by ORBilu)
International
0021-9258
1083-351X
Baltimore
MD
[en] Adhesion; Amino acids; Fluorescence; Mutagenesis; Proteins ; Cell biology; Cytoplasms ; Cells ; actin; alpha integrin; alpha2beta3 integrin; beta integrin; complementary DNA; fibrinogen; glutathione transferase; green fluorescent protein; hybrid protein; integrin; talin; unclassified drug; vinculin ; actin filament; amino acid sequence; animal cell; article; binding site; cell granule; cell spreading; CHO cell; cytology; cytoskeleton; fluorescence; fluorescence resonance energy transfer; focal adhesion; gene expression; genetic transfection; human; human cell; nonhuman; nucleotide sequence; priority journal; protein domain; protein localization; staining; surface plasmon resonance ; Actins; Animals; Binding Sites; Cell Line; CHO Cells; Coloring Agents; Cricetinae; Cytoskeleton; DNA ; Complementary; Escherichia coli; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique ; Indirect; Glutathione Transferase; Green Fluorescent Proteins; Humans; Integrin beta Chains; Kinetics; Luminescent Proteins; Microscopy ; Fluorescence; Models ; Biological; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Protein Structure ; Tertiary; Recombinant Fusion Proteins; Surface Plasmon Resonance; Talin; Time Factors; Transfection; Vinculin ; Animalia; Cricetinae; Cricetulus griseus
[en] The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing α IIbβ 3, linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with β 3-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin α IIbβ 3 or with the recombinant wild type, but not the Y747A mutant β 3 cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between β 3-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin- binding site within the talin rod domain is important for β 3-cytoskeletal interactions but does not participate in α IIbβ 3 activation.
http://hdl.handle.net/10993/4924
10.1074/jbc.M400947200
http://www.scopus.com/inward/record.url?eid=2-s2.0-2542419770&partnerID=40&md5=74131dcf70f1e4d5f8d60987b51d9a8d
cited By (since 1996)50
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