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Human Carcinoma Cells Express Receptors for Distinct Domains of Thrombospondin¹

Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan 48109

Thrombospondin (TSP), an adhesive glycoprotein, is incorporated into the extracellular matrix, mediates cell attachment and spreading, chemotaxis, haptotaxis, and may participate in the directed movement of cells in metastasis. Evidence from several model systems suggests that these functions may be mediated by different domains within the TSP molecule. Radioligand binding assays on 11B squamous carcinoma cells with ¹²⁵I-TSP demonstrated the presence of 1.2 x 10⁶ sites/cell with an apparent K_d of 74 nM. Binding studies using TSP fragments demonstrated that both the NH₂ terminal heparin-binding domain (HBD) and the COOH terminal fragment with a molecular weight of 140,000 (140K) retained the ability to bind 11B cells in a time-dependent, dose-dependent, saturable, and specific manner. The HBD bound to 11B cells with an apparent K_d of 1.2 µM at 1.4 x 10⁶ sites/cell. Binding of 140K to cells demonstrated half-maximal binding at 36 nM and a B_max of 1.9 x 10⁵ sites/cell. The binding of 140K also showed a high degree of positive cooperativity with a Hill slope of +3.5, suggesting that binding one 140K molecule to cells leads to increased binding of additional 140K molecules. In addition, the HBD and 140K showed no cross-competition in binding assays. Therefore, it appears likely that these distinct TSP domains bind to separate sites on the cell surface. Neither vitronectin or the peptide RGDS were able to inhibit the binding of TSP or 140K to 11B cells. Based on these data, there appears to be more than one distinct receptor on 11B cells for TSP: one receptor class which mediates the binding of the HBD and a second receptor class which mediates the binding of the M_r 140,000 fragment.

¹ This work was supported in part by Grant PDT-324 from the American Cancer Society (V. M. D.) and National Institutes of Health Postdoctoral Traineeship HL-07517 (R. Y.). V. M. D. is an Established Investigator of the American Heart Association.

² To whom requests for reprints should be addressed, at Department of Pathology, University of Michigan Medical School, 1301 Catherine Street, Box 0602, Ann Arbor, MI 48109-0602.

Received 8/ 3/90. Accepted 1/ 9/91.

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