We have analysed the interactions of three proteoglycans of the decorin family, decorin, biglycan and fibromodulin, with transforming growth factor beta (TGF-beta). The proteoglycan core proteins, expressed from human cDNAs as fusion proteins with Escherichia coli maltose-binding protein, each bound TGF-beta 1. They showed only negligible binding to several other growth factors. Intact decorin, biglycan and fibromodulin isolated from bovine tissues competed with the fusion proteins for the TGF-beta binding. Affinity measurements suggest a two-site binding model with Kd values ranging from 1 to 20 nM for a high-affinity binding site and 50 to 200 nM for the lower-affinity binding site. The stoichiometry indicated that the high-affinity binding site was present in one of ten proteoglycan core molecules and that each molecule contained a low-affinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-beta binding than fibromodulin or the non-glycosylated fusion proteins; removal of the chondroitin/dermatan sulphate chains of decorin and biglycan (fibromodulin is a keratan sulphate proteoglycan) increased the activities of decorin and biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-beta. The fusion proteins competed for the binding of radiolabelled TGF-beta to Mv 1 Lu cells and endothelial cells. Affinity labelling showed that the binding of TGF-beta to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type-II receptors was unaffected. TGF-beta 2 and 3 also bound to all three fusion proteins. Latent recombinant TGF-beta 1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan. The results show that the three decorin-type proteoglycans each bind TGF-beta isoforms and that slight differences exist in their binding properties. They may regulate TGF-beta activities by sequestering TGF-beta into extracellular matrix.

Abstract. Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, cxv/35, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an aviS5 ligand, vitronectin, also bound c~,/35 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The a~/35 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; tXv/35 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a/3t integrin did bind to these peptides. The interaction of c~v/35 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences. TH~ tripeptide arg-gly-asp (RGD) 1 is required for cell adhesion to a number of proteins, including fibronectin, vitronectin, and fibrinogen (Pierschbacher and Ruoslahti, 1984;Ruoslahti and Pierschbacher, 1987). This adhesion is mediated by integrins, a family oftransmembrane receptors composed of two subunits, ot and/3 (Hemler, 1990;Ruoslahti, 1991;Hynes, 1992).The Tat protein of human immunodeficiency virus (HIV-1) contains an RGD sequence and can mediate cell attachment in an RGD-dependent manner (Brake et al., 1990). Extracellular Tat is internalized by cells and transported to the nucleus, where it retains the ability to transactivate the HIV promoter (Frankel and Pabo, 1988). Furthermore, extracellular Tat has been shown to modulate cell proliferation, both in the suppression of proliferation of antigen-activated T-cells (Viscidi et al., 1989) and in the stimulation of proliferation of Kaposi's sarcoma (KS)-derived cells (Ensoli et al., 1990 We felt that the possibility of an RGD-binding integrin mediating some of the interactions of Tat with cell surfaces was of a considerable interest and set out to identify such an integrin. We found that the otvl3~ integrin bound to the Tat protein, but that this interaction was not significant in the uptake of Tat by cells. Surprisingly, our results indicate that the binding of this integrin to Tat requires the basic region, whereas the RGD sequence is silent. We also provide evidence that a basic sequence in vitronectin can serve as a binding site...