Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits.

Abstract

Abstract. Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and iaminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed a and 13, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed a and 13, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the ct subunits. Pulsechase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the a and 13 subunits of the class I and II antigens by peptide mapping indicated that the 13 subunits were identical while the ct subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody.The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I13 and 1113 subunits were structurally related to the 13 subunit of the fibronectin receptor described by others. However, none of these receptors shared the same ct subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.These results suggest that the class I and II receptors are two new members of a family of cell surface receptors for the extracellular matrix involved in mediating cell adhesion and shall be referred to as ECMRI and II. Each member of this family possesses a common 13 subunit and a unique a subunit. The class II receptor appears to be a primary mediator of specific cell adhesion to collagen. Th...

The α2β1 integrin mediates the malignant phenotype on type I collagen in pancreatic cancer cell lines

“…Figure 5A and B demonstrate clearly that pancreatic cancer cell adhesion and migration on type I collagen is mediated exclusively by the a 2 b 1 integrin, and that neither the a 1 , a 3 , a 5 , or a 6 integrin subunits, nor the a v b 3 or a v b 5 integrins appear to be involved in the adhesive interactions between AsPC-1, BxPC-3, CFPAC, or FG pancreatic cancer cells and type I collagen. Immunoprecipitation studies shown in Figure 4 and previous studies by us and other laboratories (Cheresh and Spiro, 1987;Wayner and Carter, 1987;Sonnenberg et al, 1988;Wayner et al, 1988Wayner et al, , 1993Grzesiak et al, 1992Grzesiak et al, , 2005aFabbri et al, 1996) demonstrate that the monoclonal antibodies used for the type I collagen inhibition studies are functional.…”

“…The function-blocking monoclonal antibodies directed against particular integrin subunits and integrins used in these studies have been described (Cheresh and Spiro, 1987;Wayner and Carter, 1987;Sonnenberg et al, 1988;Wayner et al, 1988Wayner et al, , 1993Fabbri et al, 1996), and include FB12 (a 1 ), P1E6 (a 2 ), P1B5 (a 3 ), P1D6 (a 5 ), GoH3 (a 6 ), P5D2 (b 1 ), LM609 (a v b 3 ), P1F6 (a v b 5 ), and ASC-3 (b 4 ) (Chemicon International, Temecula, CA, USA). For immunoprecipitation (IP) experiments, we used the monoclonal antibodies described above along with rabbit polyclonal antisera against the a 5 integrin subunit, which has also been described previously (Ruoslahti and Pierschbacher, 1987).…”

Fibronectin receptor overexpression and loss of transformed phenotype in a stable variant of the K562 cell line.

“…No increases in surface expression of the collagen-binding heparan sulfate proteoglycan, CD44/ECMR-111, were detected by immunoprecipitation or immunofluorescence ( Figure 3D and data not shown) (Wayner and Carter, 1987). Finally, because FA-K562 adhesion to FN is trypsin insensitive and RGD inhibitable, syndecan is unlikely to be the receptor mediating FA-K562 adhesion to FN (Saunders and Bernfield, 1988).…”

“…Additional FN-specific sequences or conformation were also required, as FA-K562 did not adhere to plastic coated with thrombin; GRGDS peptide; or the RGD-containing proteins laminin, collagen, or fibrinogen (Figure 1 F and data not shown). Although transforming growth factor-beta (TGF-beta) has been detected in some FN preparations (Fava and McClure, 1987) and could be responsible for morphologic changes, no bioactive TGF-beta was found in the FN preparation used in this study (personal communication, Dr. A. Purchio (Wayner and Carter, 1987;Wayner et al, 1989). Cells with similar phenotypes and FNR expression levels have been independently generated three times.…”

“…Soluble ligand or antibody to alpha5beta1 may displace this inhibitory substance, allowing normal signal transduction to occur. A central role of alpha5beta1 in growth control may explain why diverse transforming agents have been found to affect the structure, expression, or function of this particular integrin (Hirst eta/., 1986;Plantefaber and Hynes, 1989;Symington et al, 1989 (Hemler et a/, 1987;Wayner and Carter, 1987;Wayner et al, 1988). Monoclonal antibodies to the platelet glycoproteins (anti-llbill.…”