Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation.
Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.
Garcia-Pardo
A
A
Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.
Ferreira
O C
OC
Valinsky
J
J
Bianco
C
C
D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D009000:Monocytes / Q000378:metabolism*; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*
D005353:Fibronectins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic