The integrin a6134 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express a6f34 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human a6 and 134, it was, instead, colocalized with a6,64 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of a6f34 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of 134 that forms a complex containing HD1 in vitro, since the expression of a6 with a mutant 134 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of a6134 with HD1, the cytoplasmic domain of 134 is essential. We suggest that this association may be crucial for hemidesmosome assembly.
Abstract. Lymphocyte attachment to fibronectin is mainly mediated by the interaction of ix5/31 and ot4/31 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-/31 mAb TS2/16 can convert the partly active a4B1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects ot4B1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack ix5/31) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence, mAbs anti-oM and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying ol4/31 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of ot4/31, we tested whether TS2/16-aetivated o~4/31 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated ceils to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated ot4/31. These results emphasize the role of ot4/31 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.YMPHOCYTE interactions with fibronectin (Fn) l are important for their differentiation, migration, activation, and biological function (reviewed in 20, 46). The specific sequences in Fn that mediate cell attachment are located within two main regions of the molecule: the central cell-binding domain contains the RGD site which acts in synergy with at least two other regions within this domain (56).
The region of fibronectin encompassing type III repeats 4 -6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact Fibronectin (Fn) 1 is an extracellular matrix and plasma protein composed of structural domains that contain binding sites for other macromolecules (fibrin, heparin/proteoglycans, collagen), as well as for cells (reviewed in Hynes (1)). The NH 2 -terminal heparin binding domain or Hep I (see Fig. 1) also interacts with cell surfaces by binding to uncharacterized molecules and is mainly involved in formation of Fn matrices (1, 2). The Hep II domain displays the highest avidity for heparin and contains specific sequences which bind proteoglycans and/or the ␣41 integrin at the cell surface and induce cell adhesion (3-5). One of these sites is H1, which is a ligand for ␣41 in melanoma (6) and lymphoid cells (7). Two other sequences, CS-1 and CS-5, located within the IIICS segment (outside the Hep II domain) are also ligands for ␣41 (8 -11) and bind with different affinities (12). These previous studies have established that ␣41 may bind several sequences in the COOHterminal region of Fn and that these interactions are particularly important in lymphoid cells, where ␣41 is highly expressed.The central Hep III domain is less well studied because it binds heparin only at low salt concentration, and the physiological significance of this interaction is unclear. Using DNA affinity chromatography, we and others previously isolated proteolytic fragments from this region of Fn, which displayed low affinity binding to heparin. These include a 14-kDa fragment corresponding to FN-III1 repeat (13) and two fragments of 18 -20 kDa (FN-III4-1/2 5 repeats) (14) and 30 kDa (FN-III4 -6 repeats) (15) from human and bovine plasma Fn, respectively. We also previously reported that an 80-kDa tryptic fragment (FN-III4-1/2 FN-III11) binds heparin with low avidity (16). It is not known whether this heparin binding domain also interacts with cells.The present study was undertaken to further characterize the properties of the Hep III domain of Fn. To this effect, we have prepared recombinant fragments encompassing type III homology repeats from this region and have studied their cell binding activities. We show that a fragment containing the FN-III5 repeat mediates adhesion of T and B lymphoid cells efficiently. Furthermore, we have identified a novel 6-amino acid-sequence as the active site in FN-III5, and we show that activated ␣41 and ␣47 integrins are receptors for this sequence and the FN-III5 fragment. These results therefore establish a novel function for the Hep III Fn domain.
As reported previously, we have extensively studied FoxJ2, a member of the Fork Head transcription factors family. While the biochemical and functional structures of this transcription factor are well understood, its biological function remains unknown. Here, we present data that address this point using transgenic mouse technology. We found that the birth rate and the number of transgenic animals obtained when transferring embryos over-expressing the FoxJ2 protein were lower than those obtained with embryos over-expressing a control protein, suggesting FoxJ2 overexpression has a negative effect on embryonic development. Transient FoxJ2 transgenesis experiments have confirmed that FoxJ2 over-expression has a lethal effect on embryonic development from E10.5. Moreover, in vitro culture of FoxJ2-microinjected embryos demonstrated a significant developmental blockage, indicating that FoxJ2 could also have an effect on preimplantation stages. Most probably, these negative effects of FoxJ2 over-expression during development also explain the low percentage of adult transgenic mice obtained. Furthermore, most of the transgenic mice that lived to adulthood did not show transgene expression. In fact, the only two adult transgenic animals (one male and one female) in which FoxJ2 transgene expression was detected showed a mosaic expression and died prematurely as a result of cardio-respiratory failure. Postmortem analysis of these animals revealed a hypertrophic heart and abnormal testes in the male. In order to identify genes regulated by FoxJ2 consistent with the phenotypes observed for FoxJ2 transgenic mice, EMSA assays and co-transfection experiments were carried out. Our data indicate that the genes coding for the gap junction protein Connexin-43 and the cellcell contact protein E-Cadherin, may be good candidates for FoxJ2-regulated genes. Interestingly, Connexin-43 and E-Cadherin show expression patterns similar to FoxJ2, and the phenotypes of Connexin-43 and E-Cadherin mutants resemble those of our FoxJ2 transgenic animals. These data suggest that the lethal effect on embryonic development of FoxJ2 overexpression, as well as the alterations observed in the heart and testes of adult transgenic mice, could be determined by changes in the transcription of genes such as Connexin-43 and/or E-Cadherin.
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