Identification of a novel cell attachment domain in the HIV- I Tat37-72 protein and its 90-kDa cell surface binding protein

Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/1993; 268(7):5279-84.
Source: PubMed


The HIV-1 transactivator protein Tat is essential for viral gene expression and replication. Tat is taken up by cells and transactivates the HIV-LTR promoter in the cell nucleus. The present studies show that cells adhere to both synthetic and recombinant Tat, and, using synthetic peptides, we localize the binding site to a region spanning amino acid residues 49-57 (peptide Tat49-57). Tat49-57 also inhibited cell attachment to solid phase full-length Tat peptide and to recombinant Tat protein. Using Tat peptide affinity chromatography, we identified a 90-kDa cell surface protein that binds to Tat. The 90-kDa protein could be eluted from the Tat column using the Tat49-57 peptide. A 90-kDa cell surface Tat binding protein was also identified by coprecipitation with Tat after incubation with radiolabeled cell membrane preparations. Co-precipitation of the 90-kDa protein was inhibited by competition with a Tat49-65 peptide, but not with Tat55-86. Our findings suggest that cellular attachment to Tat is mediated through a 90-kDa cell surface protein that binds to a Tat domain between amino acids 49 and 57.

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Available from: Hynda K Kleinman
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    • "All these observations point to a cooperation between integrin recognition sequences and basic amino acids in mediating the binding of adhesive proteins to integrin receptors. This kind of cooperation has been well demonstrated for the HIV-1 Tat protein in which one RGD sequence and the basic domain mediate integrindependent cell adhesion (Voegel et al., 1993; Weeks et al., 1993). "
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    ABSTRACT: Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.
    Full-text · Article · Jan 1998 · Molecular Biology of the Cell
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    • "While integrin and non-integrin receptors in cell-Tat interaction have been implicated (Brake et al., 1990; Barillari et al., 1993; Vogel et al., 1993; Weeks et al., 1993), Mann and Frankel (1991) "
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    ABSTRACT: In order to understand the importance of cell attachment to HIV-1 Tat, we quantified the strength of cell attachment to immobilized Tat in microtiter plate wells by the application of buoyant force. By replacing the attachment medium with dense medium, and subjecting the attached cells in the microtiter plates to centrifugal force in the conventional upright position, weakly binding and strongly binding cells could be discriminated (and separated) by varying the centrifugal speed. The strength of attachment of HT1080 cells to Tat was compared with that of the well-known extracellular matrix (ECM) proteins fibronectin and vitronectin. We observed that all three proteins mediated significant attachment of HT1080 cells both at 4 degrees C and 37 degrees C. However, unlike the ECM proteins, Tat was unable to engage in higher strength binding when the temperature was raised to 37 degrees C. The relatively weak binding of HT1080 cells to Tat (in the order of 3.0 mudynes/picomole of coated Tat) and lack of strengthening of binding to Tat at physiologic temperature suggests that this protein does not mimic adhesion molecule function. We anticipate that the methodology developed and described here will be useful in a wide variety of cell-matrix and cell-cell interaction studies.
    Full-text · Article · Feb 1997 · Journal of Cell Science
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    • "Although Tat is best known for transcription, it has other functions (Huang et al., 1994) and has been reported to be a secretable factor that promotes the growth of Kaposi-like cells (Ensoli et al., 1990). There is evidence that Tat can be taken up actively into cells (Frankel and Pabo, 1988) through binding to a cell-surface protein (Weeks et al., 1993) implicated to be avb5 integrin (Vogel et al., 1993). Once inside cells, Tat interacts with multiple partners in activating transcription. "

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