A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain.
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 beta 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 alpha(IIb)beta(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 beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(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 beta(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 beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.
Tremuth
Laurent
L
Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), Université du Luxembourg, 162A, Avenue de la Faïencerie, L-1511, Luxembourg, France.
Kreis
Stephanie
S
Melchior
Chantal
C
Hoebeke
Johan
J
Rondé
Philippe
P
Plançon
Sébastien
S
Takeda
Kenneth
K
Kieffer
Nelly
N
D000199:Actins / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D004396:Coloring Agents / Q000494:pharmacology; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D039641:Integrin beta Chains / Q000737:chemistry*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D016608:Talin / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D016596:Vinculin / Q000737:chemistry
D000199:Actins; D004396:Coloring Agents; D018076:DNA, Complementary; D039641:Integrin beta Chains; D008164:Luminescent Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D016608:Talin; C413663:fluorescent protein 583; D016596:Vinculin; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase