[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Tremuth, Laurent A; Lab. de Biol. et Physiol. Integree, CNRS/GDRE-ITI, Université du Luxembourg, 162A, Avenue de la Faïencerie, Luxembourg
Kreis, Stephanie ; University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Melchior, Chantal; Pharmacol./Physchim. Intrac. C./M., UMR CNRS 7034, Univ. Louis Pasteur de Strasbourg, Illkirch 67401, France
Hoebeke, Johan; Institut Gilbert Laustriat (IFR 85), Illkirch 67401, France
Rondé, Philippe; Université Louis Pasteur (Strasbourg) - ULP ; Institut Gilbert Laustriat > Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires
Plançon, Sébastien ; University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Takeda, Kenneth; Université Louis Pasteur (Strasbourg) - ULP > Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires
Kieffer, Nelly ; University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
External co-authors :
yes
Language :
English
Title :
A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain
Publication date :
2004
Journal title :
Journal of Biological Chemistry
ISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States