References of "Kieffer, N."
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See detailGreen fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.
Plançon, Sébastien UL; Morel-Kopp, M. C.; Schaffner-Reckinger, Elisabeth UL et al

in The Biochemical journal (2001), 357(Pt 2), 529-36

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either ... [more ▼]

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions. [less ▲]

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See detailEvidence from site-directed mutagenesis that the cytoplasmic domain of the beta3 subunit influences the conformational state of the alphaVbeta3 integrin ectodomain.
Schaffner-Reckinger, Elisabeth UL; Brons, N. H.; Kieffer, N.

in Thrombosis and haemostasis (2001), 85(4), 716-23

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes ... [more ▼]

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire beta3 cytoplasmic domain induced constitutive LIBS exposure on alphavbeta3 and alphaIIbeta3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on alphavbeta3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the beta3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the alphavbeta3 ectodomain, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes. [less ▲]

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See detailDistinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.
Schaffner-Reckinger, Elisabeth UL; Gouon, V.; Melchior, Chantal UL et al

in The Journal of biological chemistry (1998), 273(20), 12623-32

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3 ... [more ▼]

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events. [less ▲]

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