Thrombospondin-1 (TSP1) is a matricellular protein that displays both pro-and anti-adhesive activities. Binding to sulfated glycoconjugates mediates most high affinity binding of soluble TSP1 to MDA-MB-435 cells, but attachment and spreading of these cells on immobilized TSP1 is primarily  1 integrin-dependent. The integrin ␣ 3  1 is the major mediator of breast carcinoma cell adhesion and chemotaxis to TSP1. This integrin is partially active in MDA-MB-435 cells but is mostly inactive in MDA-MB-231 and MCF-7 cells, which require  1 integrin activation to induce spreading on TSP1. Integrinmediated cell spreading on TSP1 is accompanied by extension of filopodia containing  1 integrins. TSP1 binding activity of the ␣ 3  1 integrin is not stimulated by CD47-binding peptides from TSP1 or by protein kinase C activation, which activate ␣ v  3 integrin function in the same cells. In MDA-MB-231 but not MDA-MB-435 cells, this integrin is activated by pertussis toxin, whereas serum, insulin, insulin-like growth factor-1, and ligation of CD98 increase activity of this integrin in both cell lines. Serum stimulation is accompanied by increased surface expression of CD98, whereas insulinlike growth factor-1 does not increase CD98 expression. Thus, the pro-adhesive activity of TSP1 for breast carcinoma cells is controlled by several signals that regulate activity of the ␣ 3  1 integrin. Thrombospondin-1 (TSP1)1 is an extracellular matrix glycoprotein that has diverse effects on cell behavior (reviewed in Refs. 1 and 2). The five known thrombospondin genes display distinct patterns of expression during development and in several disease states. Disruption of the thbs1 gene in mice results in lordosis of the spine and abnormal proliferation and inflammatory responses in the lung (3). Suppression of THBS1 expression by loss of wild type p53, by activated Ras, Myc, nickel, and in metastatic clones of several tumor cell lines suggested that loss of TSP1 expression may contribute to tumor progression (reviewed in Ref. 4). Consistent with this hypothesis, overexpression of THBS1 in breast carcinoma cells (5), a transformed endothelial cell line (6), fibroblasts from Li Fraumini patients (7), and glioblastoma cells (8) decreases tumor growth in animal models. This suppressive activity is due at least in part to the anti-angiogenic activity of TSP1 (reviewed in Refs. 4, 9, and 10). TSP1 antagonizes growth factor-stimulated proliferation and migration of endothelial cells. Its anti-angiogenic activity is thought to be the major mechanism for suppression of tumor growth in THBS1-transfected MDA-MB-435 breast carcinoma cells, because thrombospondin overexpression strongly inhibited tumor growth in vivo but did not significantly alter in vitro proliferation, motility, or the ability of the tumor cells to form colonies in soft agar (5). However, higher doses of exogenous TSP1 and some TSP1 peptides can directly inhibit proliferation of these cells in vitro (11).Defining the receptors that recognize TSP1 on endothelial and tumor cells ...
The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by ␣ 4  1 and ␣ 5  1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by ␣ 3  1 integrin and required that the ␣ 3  1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified ␣ 3  1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by ␣ 3  1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.
Conformational changes induced in thrombospondin-1 by removal of calcium regulate interactions with some ligands of its N-modules. Because calcium binds primarily to elements of the C-terminal signature domain of thrombospondin-1, which are distant from the N-modules, such regulation was unexpected. To clarify the mechanism for this regulation, we compared ligand binding to the Nmodules of thrombospondin-1 in the full-length protein and recombinant trimeric thrombospondin-1 truncated prior to the signature domain. Three monoclonal antibodies were identified that recognize the N-modules, two of which exhibit calcium-dependent binding to native thrombospondin-1 but not to the truncated trimeric protein. These antibodies or calcium selectively modulate interactions of fibronectin, heparin, sulfatide, α3β1 integrin, tumor necrosis factor-α-stimulated gene-6 protein, and, to a lesser extent, α4β1 integrin with native thrombospondin-1 but not with the truncated protein.These results indicate connectivity between calcium binding sites in the C-terminal signature domain and the N-modules of thrombospondin-1 that regulates ligand binding and functional activities of the N-modules.
Hemoglobin specifically induces fibronectin (FN) binding to the pathogenic yeast Candida albicans. When grown in the complex medium Sabouraud broth, C. albicans expresses receptors that bind to several domains of FN. Growth in defined medium supplemented with 0.1% hemoglobin, however, enhanced the binding of FN to a single class of receptors, with a Kd = 4.6 × 10−8 M. Competitive binding assays using recombinant and proteolytic fragments of FN revealed that the cell-binding domain mediated this interaction. A recombinant 40-kDa fragment of FN consisting of type III repeats 9 to 13 had an inhibitory activity similar to that of the entire 120-kDa cell-binding domain, indicating that the C-terminal portion of the cell-binding domain contains the binding site. A recombinant 33-kDa fragment of the cell-binding domain and a 33-kDa fragment with the RGD sequence deleted had the same inhibitory activities, demonstrating that the RGD sequence recognized by some mammalian integrins is not required. The addition of hemoglobin to the culture medium also enhanced Candida cell adhesion to immobilized FN and to 120- and 40-kDa fragments of FN but not to the collagen-binding or fibrin I domains. Using ligand protection, we identified a surface protein from C. albicans with an apparent molecular mass of 55 kDa that was protected by both FN and the 40-kDa fragment derived from the cell-binding domain. Therefore, hemoglobin both induces FN binding and changes the relative affinities of C. albicans for the cell- and collagen-binding domains of FN.
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