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Human Carcinoma Cells Bind Thrombosphondin through a M_r 80,000/105,000 Receptor¹

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

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 M_r 140,000 fragment of TSP. TSP and the M_r 140,000 fragment competed for the binding of the ¹²⁵I-labeled M_r80,000/105,000 cell surface complex to TSP-coated microtiter wells in a dose-dependent manner with half-maximal inhibition observed at 16 and 40 µg/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 M_r 80,000/105,000 cell surface complex to TSP. The specificity of the M_r 80,000/105,000 receptor for the M_r 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 M_r 80,000/105,000 TSP receptor and members of the ß₁ or ß₃ integrin receptor families. Additionally, V-8 protease mapping demonstrated that the M_r 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.

¹ This work was supported by NIH Grant CA 51888.

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 2/18/91. Accepted 5/ 1/91.

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