Journal of Cellular Biochemistry 28:307-318 (1985) zy Characterization of a Membrane-Associated Glycoprotein Complex Implicated in Cell Adhesion to Fibronectin Takayuki Hasegawa, Etsuko Hasegawa, Wen-Tien Chen, and Kenneth M. Yamada zyxwvu Membrane Biochemistry Section, Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20205 (TH., E.H., K.M. V.) and Department of Anatomy, Georgetown University Medical Center, Washington, DC 20007 (W.-TC.) zyxw We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band l), 135,000 (band zyxwvutsrq 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band l), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a compo- sition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells. Key words: glycoprotein complex, cell adhesion, fibronectin Cell adhesion is a complex process involving a variety of possible molecular mechanisms zyxwvuts [ 1,2]. One approach to analyzing adhesion has been to characterize isolated extracellular proteins such as fibronectin that mediate cell-to-substrate adhe- sion [3-91. A complementary approach is to identify membrane proteins essential for adhesion by the use of inhibitory monoclonal antibodies [ 10-141. The JG22E antibody inhibits fibroblast attachment and spreading on fibronectin zyx [35]. In immunofluores- cence assays, the antigen co-localizes with fibronectin in attachment sites of fibro- blasts. In analogy to fibronectin itself, this otherwise inhibitory monoclonal antibody can also serve as an adhesive molecule if attached to substrates [35]. The JG22E Received December 29, 1984; accepted April 2, 1985. zyxwv 0 1985 Alan R. Liss, Inc.