Synthetic peptide GD-2 is a sequence of amino acids derived from the carboxy-terminal long arm of the A chain of laminin. Previous studies have shown that peptide GD-2 promotes the adhesion of human squamous cell carcinoma (SCC) cells as well as a variety of other cell lines. In this study, we attempted to identify the receptor that SCC cells use to adhere to peptide GD-2. Monoclonal antibodies (mAbs) against a human SCC cell line were generated. One of these mAbs, ASC-1, bound to the surface of SCC cells as determined by flow cytometry. This mAb inhibited SCC cell adhesion to peptide GD-2 and laminin, but not fibronectin or type IV collagen, suggesting that mAb ASC-1 binds to the SCC receptor for the peptide GD-2 sequence of laminin. MAb ASC-1 immunoprecipitated a complex composed of two components of 135 and 116 kDa. Immunoadsorption of ASC-1-binding material from the SCC cell extract by incubation with mAb ASC-1 resulted in the removal of the α3β1 integrin from the extract. Immunohistochemical staining of tissue from a normal human tongue and from a patient with SCC of the tongue revealed that mAb ASC-1 stained the surface of epithelial cells that were in contact with the basement membrane, as well as those cells located two to three layers above the basement membrane. This mAb also stained blood vessels in the squamous tissue. This staining pattern was identical to that observed when the same tissues were stained by a mAb against the α3 integrin subunit. In summary, by use of a new mAb, ASC-1, that recognizes the α3β1 integrin, we have determined that the α3β1 integrin mediates SCC cell adhesion to the peptide GD-2 sequence within laminin.
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We gratefully acknowledge the contribution of the human laminin by Dr. Kurt Gehlsen, human plasma ®bronectin by Dr. James McCarthy, and type IV collagen by Dr. Sally Palm. We also thank Drs. Arnoud Sonnenberg, Leo Furcht, and Elizabeth Wayner for providing us with mAbs against the various integrin subunits; Dr. Carlos Mani-vel for providing us with the patient's SCC specimen; Paula Gross-man and Michael Grossman for their excellent technical assistance; and Rahn Kollander for help with the immunohistochemistry tissue sample preparations. We also thank Drs. Mark Wilke, Robert Milius, and Gregg Fields for their helpful discussions. This study was supported by NIH/NCI Grant CA60658 (A.P.N.S.), a grant from the University of Minnesota Graduate School Grants-in-Aid Program (A.P.N.S.), a grant from the American Heart Association (K.M.S.), and a scholarship from Chulalongkorn University, Bangkok, Thailand (S.P.).