Abstract. Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing ,x,20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wildtype cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity.Surface labeling with 125I and immunoaflinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2 % FnR. Nevertheless, /3 subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR/3 chain as well as enhanced amounts of a pre-/3 moiety in the variants, ct chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of ct chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR ct chain eDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of c~ chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.T HE cell surface receptor for fibronectin (FnR) ~ plays a major role in mediating adhesive interactions between cells and the extracellular matrix (11,31,45). This intrinsic membrane glycoprotein of ~140 kD is a member of the integrin superfamily of cell adhesion receptors. Integrins share a number of common features; for example, they are composed of noncovalenfly linked beterodimers with large extracellular domains. In mammalian cells, the integrin receptor superfamily is divided into three ...

Abstract. Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four-to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not.Neither the number of cell surface FnRs nor the receptor affinity, as measured by ~25I-fibronectin and '25I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectindependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.T HE adhesion of cells to the extracellular matrix is a complex process which involves metabolic and cytoskeletal activities as well as cell surface receptors for matrix macromolecules (11,29). Within the last few years two major families of matrix adhesion receptors have been described. The integrin or "cytoadhesin" family (3, 27, 49) is comprised of a number of heterodimeric receptors of ,~ 140 kD in molecular mass. These molecules are involved in a wide variety of cell-matrix and cell-cell adhesive processes in neuronal (18), immune (56, 57), connective tissue (7, 14), and hematopoietic (40, 41) cellular systems. The integrin superfamily includes mammalian receptors for fibronectin (7, 43), vitronectin (44), collagen, and laminin (58, 59). The interactions of many, but not all, integrins with their specific ligands can be inhibited by short peptides containing the RGD adhesive sequence (48,56,60). High affinity mammalian cell receptors for laminin and elastin seem to be members of another family of matrix adhesion receptors (22,36,45). These molecules are in the 65-70-kD range and, in the case of laminin rece...

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Human red blood cells contain protein kinase C (PKC) which acts exclusively on the membrane skeletal proteins band 4.1, band 4.9 and adducin. PKC activity can be stimulated by the addition of the phorbol ester 12-O-tetradecanoyl phorbol IEacetate to intact c&s. Phosphorylation of band 4.1 by PKC in vitro results in a dramatic reduction in band 4.1 binding to spectrin and actin, as well as to the cytoplasmic domain of band 3. Here we show that the lectin wheat germ agglutinin (WGA), which binds to the extracellular domain of glycophorin results in the inhibition of PKC catalyzed phosphorylation of band 4.1, band 4.9 and likely adducin as well. The lectin concanavalin A, which binds to band 3 was without effect. Our results suggest that the binding of WGA to glycophorin results in a major rearrangement of the membrane skeletal network which correlates with reduced phosphorylation of membrane skeletal proteins by PKC.Protein kinase C; Phosphorylation; Erythrocyte; Band 4.1

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