title: Human carcinoma cells bind thrombospondin through a Mr 80,000/105,000 receptor.
abstract: The metastatic 11B squamous carcinoma cell line synthesizes and secretes high levels of the extracellular matrix glycoprotein thrombospondin (TSP) and displays aggressive invasiveness in a nude mouse model forming highly undifferentiated tumors. The importance of adhesion events involving extracellular matrix proteins and the tumorigenic cell surface in metastasis led us to investigate the nature of the 11B cell surface receptor for TSP. Using TSP affinity chromatography, a cell surface complex of molecular weight 80,000 and 105,000 was isolated that appears to function as a receptor for TSP. Binding was specific for the COOH-terminal Mr 140,000 fragment of TSP. TSP and the Mr 140,000 fragment competed for the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP-coated microtiter wells in a dose-dependent manner with half-maximal inhibition observed at 16 and 40 micrograms/ml, respectively. In contrast, the NH2-terminal heparin-binding domain did not inhibit binding in a dose-dependent manner. Other extracellular matrix proteins, such as laminin, vitronectin, or type I collagen, were also unable to inhibit the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP. The specificity of the Mr 80,000/105,000 receptor for the Mr 140,000 fragment of TSP was further confirmed through the use of monoclonal antibodies. Monoclonal antibody C6.7 specific for the distal COOH terminus of TSP, but not monoclonal antibody A2.5 specific for the heparin-binding domain, inhibited binding. Binding was observed to be strongly Ca2+ dependent, slightly Mg2+ dependent, and independent of Mn2+. Immunoprecipitation analyses demonstrated no apparent cross-reactivity between the Mr 80,000/105,000 TSP receptor and members of the beta 1 or beta 3 integrin receptor families. Additionally, V-8 protease mapping demonstrated that the Mr 80,000 and 105,000 polypeptide bands are not related to each other through proteolytic processing. This initial identification and characterization of a carcinoma cell TSP receptor should allow a more detailed examination of the role of TSP in metastatic adhesion and motility.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.
authors.forename: R
authors.initials: R
authors.lastname: Yabkowitz
authors.forename: V M
authors.initials: VM
authors.lastname: Dixit
mesh_terms: D018955:CD36 Antigens; D002118:Calcium / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D002448:Cell Adhesion; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D016024:Receptors, Cytoadhesin / Q000032:analysis* / Q000276:immunology; D011971:Receptors, Immunologic / Q000032:analysis / Q000276:immunology; D019030:Receptors, Vitronectin; D019699:Thrombospondins; D014407:Tumor Cells, Cultured
chemical_list: D018955:CD36 Antigens; D010980:Platelet Membrane Glycoproteins; D016024:Receptors, Cytoadhesin; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D019699:Thrombospondins; D008345:Manganese; D006493:Heparin; D002118:Calcium