Biosynthesis and acquisition of biological activity of the fibronectin receptor

The biosynthesis of the 140-kilodalton fibronectin receptor complex by cultured 3T3 mouse cells was characterized and compared with that of chick embryo fibroblasts. Three murine glycoprotein components of 140-150 kilodaltons (band 1), 125 kilodaltons (band 2), and 105 kilodaltons (band 3) could be immunoprecipitated from metabolically labeled 3T3 cells using polyclonal antibodies. In pulse-chase experiments, bands 1 and 3 of the mouse receptor were labeled to maximal levels immediately after completion of the labeling pulse. However, band 2 was not detected at short chase times, and it reached maximal labeling only after approximately 12 h of chase. The appearance of band 2 occurred at the same rate as the disappearance of band 3. Only bands 1 and 2 could be affinity purified by binding to immobilized fibronectin cell-binding fragment, indicating that they represent mature functional receptor components. When 3T3 cells were incubated with radioactive sugars, band 1 of the receptor labeled well with both [3H]mannose and [3H]glucosamine. However, band 2 labeled well with [3H]glucosamine but contained low amounts of mannose, and band 3 labeled well with [3H]mannose but contained low amounts of glucosamine. Digestion of both bands 2 and 3 with endoglycosidase F yielded similar-sized products of approximately 88,000 daltons, suggesting that post-translational asparagine-linked oligosaccharide processing can account for most of the size difference between these bands. These data suggest that in the mouse fibronectin receptor, band 3 is a biologically inactive precursor of band 2 that does not appear on the cell surface. In contrast, pulse-chase experiments using chicken embryo fibroblasts indicated that the three components of the chicken 140k complex were distinct moieties. Our results demonstrate distinct types of processing for fibronectin receptor complexes from different species. In mammalian cells, this receptor undergoes a surprisingly long (20 h) maturation process involving asparagine-linked oligosaccharides before reaching its final, biologically active form.     

The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.     

Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin

The 75,000-dalton cell-binding domain of fibronectin (f75k) and synthetic peptides derived from its sequence have been used to examine the cell-surface fibronectin receptor. The spreading of baby hamster kidney fibrolasts on f75k-coated substrates and the direct binding of [3H]f75k to these cells were both competitively inhibited by a synthetic peptide of fibronectin with the sequence Gly-Arg-Gly-Asp-Ser, indicating that the peptide and f75k interact with the same cell-surface sites. Related peptides, including the inverted sequence Ser-Asp-Gly-Arg, also competed for f75k binding. Our results provide the first evidence that fibroblastic baby hamster kidney cells recognize the same fibronectin amino acid sequence in direct binding of soluble ligand and in indirect inhibitory biological assays, thus correlating our direct fibronectin-receptor binding assay with biological functional assays for the fibronectin receptor in fibroblasts.     

The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex

A monoclonal antibody, P1H5, to the human fibroblast class II extracellular matrix receptor (ECMR II) specifically inhibits human fibroblast adhesion to collagen and immunoprecipitates a cell surface receptor containing an alpha and beta subunit of approximately 140 kilodaltons each (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). We report here that P1H5 also specifically inhibits adhesion of unactivated human platelets to type I and III collagens, but not to fibronectin. Immunoprecipitation of the class II ECMR from Triton X-100 detergent lysates of platelets, after cell surface iodination, identified the platelet collagen receptor. Peptide mapping confirmed that the II alpha and II beta subunits immunoprecipitated from platelets are structurally homologous with those derived from fibroblasts. The platelet ECMR II alpha and -beta subunits comigrate with platelet membrane glycoproteins Ia and IIa, respectively, on two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. These results indicate that platelet and fibroblast adhesion to collagen are both mediated by a similar receptor and that the alpha and beta subunits of this receptor are identical to platelet membrane glycoproteins Ia and IIa, respectively. Although glycoprotein Ia has been previously implicated as a collagen binding protein, our results are the first direct evidence that platelet glycoprotein Ia is associated with glycoprotein IIa in a heterodimer complex and that this complex, by mediating platelet attachment, is an actual receptor for platelet adhesion to collagen.     

Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth

Mouse-hatched blastocysts cultured in vitro will attach and form outgrowths of trophoblast cells on appropriate substrates, providing a model for implantation. Immediately after hatching, the surfaces of blastocysts are quiescent and are not adhesive. Over the period 24-36 h post-hatching, blastocysts cultured in serum-free medium become adhesive and attach and spread on the extracellular matrix components fibronectin, laminin, and collagen type IV in a ligand specific manner. Attachment and trophoblast outgrowth on these substrates can be inhibited by addition to the culture medium of an antibody, anti-ECMr (anti-extracellular matrix receptor), that recognizes a group of 140-kD glycoproteins similar to those of the 140-kD extracellular matrix receptor complex (integrin) recognized in avian cells by CSAT and JG22 monoclonal antibodies. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibits trophoblast outgrowth on both laminin and fibronectin. However, the presence of the peptide does not affect attachment of the blastocysts to either ligand. Immunoprecipitation of 125I surface-labeled embryos using anti-ECMr reveals that antigens recognized by this antibody are exposed on the surfaces of embryos at a time when they are spreading on the substrate, but are not detectable immediately after hatching. Immunofluorescence experiments show that both the ECMr antigens and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophectoderm cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblast cells to the substratum.     

Tenascin interferes with fibronectin action

Primary chick embryo fibroblasts attach to a tenascin substrate, but remain rounded and do not spread out. The proportion between tenascin and fibronectin in mixtures used to coat the substrate determines the shape of the cells. Tenascin inhibits integrin-mediated chick fibroblast attachment to fibronectin, laminin, and the GRGDS peptide. Rat fibroblast attachment to fibronectin, but not to laminin, is inhibited by tenascin. A monoclonal antibody against tenascin, as well as its Fab fragments, is able to neutralize the inhibitory activity on cell attachment and is therefore assumed to mask the cell-binding site of tenascin. On electron micrographs showing this monoclonal antibody bound to tenascin, its epitope can be localized to the terminal knob at the distal ends of the tenascin arms.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Integrin heterodimer and receptor complexity in avian and mammalian cells

We report data showing that the integrin receptor complex in chickens contains several discrete heterodimers all sharing the beta 1-integrin subunit combined separately with different alpha-subunits. Using antisera to synthetic peptides based on cDNA sequences of chicken and human alpha-integrin subunits to analyze the integrin complement of avian and mammalian cells, we show that band 2 of the chicken integrin complex contains alpha-subunits related to both alpha 3- and alpha 5-subunits of human integrins. alpha 3 beta 1 and alpha 5 beta 1 have both previously been shown in human cells to be fibronectin receptors and alpha 3 beta 1 can also act as a receptor for laminin and collagen. We also provide evidence for the presence, in band 1 of the chicken integrin complex, of a third integrin alpha-subunit which is also alpha 5 related. This integrin subunit exists in a separate heterodimer complex with beta 1 and binds to fibronectin-affinity columns. These results provide explanations for published data showing that the avian integrin complex contains receptor activity for a variety of extracellular matrix proteins. We conclude that the chicken integrin complex comprises a set of beta 1-integrin heterodimers equivalent to the human VLA antigens and includes at least two fibronectin receptors. Finally, we show that chicken embryo fibroblasts also contain a beta 3-class integrin related to the RGD receptors defined in various human cells.     

Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin"

Platelet membrane glycoprotein (GP) IIIa forms a Ca2+-dependent heterodimer complex with GP IIb. The GP IIb-IIIa complex constitutes the fibrinogen and fibronectin receptor on stimulated platelets. A biochemically and immunologically similar membrane glycoprotein complex is present on endothelial cells. A human umbilical vein endothelial cell cDNA library was screened using oligonucleotide probes designed from peptide sequences obtained from platelet GP IIIa. A cDNA clone was sequenced and found to encode a protein of 84.5 kDa. The translated endothelial cDNA contained five sequences that corresponded to peptide sequences in platelet GP IIIa, including the amino-terminal 19 residues. Thus, the endothelial and platelet forms of GP IIIa are apparently identical. Glycoprotein IIIa consists of a long amino-terminal extracellular domain with several potential N-linked glycosylation sites and four cysteine-rich tandem repeats, a 29-residue hydrophobic transmembrane segment, and a short carboxyl-terminal cytoplasmic domain. Glycoprotein IIIa has a 47% amino acid sequence homology to "integrin," a fibronectin receptor from chicken embryo fibroblasts. This homology suggests that GP IIIa is a member of a family of cell-surface adhesion receptors.     

Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.     

Evidence for a dual mechanism of chick embryo fibroblast adhesion on fibronectin and laminin substrata

Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule

Nr-CAM is a membrane glycoprotein that is expressed on neurons. It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region. We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule. To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence. After transfection, L cells expressed Nr-CAM on their surface and aggregated. Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments. To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Escherichia coli. Addition of FGTNr to the transfected cells blocked their aggregation. Further analysis using a combination of cell aggregation assays, binding of cells to FGTNr-coated substrates, aggregation of FGTNr-coated Covaspheres and binding of FGTNr-coated Covaspheres to FGTNr-coated substrates revealed that Nr-CAM mediates two types of cell interactions: a homophilic, divalent cation-independent binding, and a heterophilic, divalent cation-dependent binding. Homophilic binding was demonstrated between transfected L cells, between chick embryo brain cells and FGTNr, and between Covaspheres to which FGTNr was covalently attached. Heterophilic binding was shown to occur between transfected and untransfected L cells, and between FGTNr and primary chick embryo fibroblasts; in all cases, it was dependent on the presence of either calcium or magnesium. Primary chick embryo glia or a human glial cell line did not bind to FGTNr-coated substrates. The results indicate that Nr-CAM is a cell adhesion molecule of the nervous system that can bind by two distinct mechanisms, a homophilic mechanism that can mediate interactions between neurons and a heterophilic mechanism that can mediate binding between neurons and other cells such as fibroblasts.     

Tenascin mediates cell attachment through an RGD-dependent receptor

Tenascin is an extracellular matrix glycoprotein expressed in association with mesenchymal-epithelial interactions during development and in the neovasculature and stroma of undifferentiated tumors. This selective expression of tenascin indicates a specific role in cell matrix interactions. We now show that tenascin can support the adhesion of a variety of cell types, including various human tumor cells, normal fibroblasts, and endothelial cells, all of which can attach to a substrate coated with tenascin. Detailed studies on the mechanism of the tenascin-promoted cell attachment were carried out with the human glioma cell line U251MG. The attachment of these cells and others to tenascin were inhibited specifically by peptides containing the RGD cell attachment signal. Affinity chromatography procedures similar to those that have been used to isolate other adhesion receptors yielded a heterodimeric cell surface protein which bound to a tenascin affinity matrix in an RGD-dependent fashion. One of the subunits of this putative tenascin receptor comigrates with the beta subunit of the fibronectin receptor in SDS-PAGE and cross reacts with antibodies prepared against the fibronectin receptor in immunoblotting. These results identify the tenascin receptor as a member of the fibronectin receptor family within the integrin superfamily of receptors. The cell attachment response on tenascin is distinctly different from that seen on fibronectin, suggesting that cell adhesion and motility may be modulated at those sites where tenascin is expressed in the extracellular matrix.     

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.     

Recruitment of a heparan sulfate subunit to the interleukin-1 receptor complex. Regulation by fibronectin attachment.

In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.     

Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets

Soluble fibronectin binds specifically to glycoprotein (GP) IIb-IIIa on thrombin-activated platelets, and this binding is not observed with platelets of patients with Glanzmann's thrombasthenia (GT) which lack GPIIb-IIIa. Here we report that GT platelets retain the ability to interact with fibronectin-coated surfaces. Adhesion to fibronectin does not require platelet activation and is inhibited by soluble fibronectin, antibodies specific for fibronectin, peptides containing the sequence Arg-Gly-Asp and polyclonal antibodies specific for band 3 of the chicken embryo fibroblast fibronectin receptor (anti-band 3). Using anti-band 3, we have purified a second fibronectin receptor from human platelets, a heterodimer composed of glycoproteins previously designated GPIc and GPIIa. The GPIc-IIa complex is found on both GT and normal platelets and appears to be identical to the GP138 kD-GP160 kD complex recently immunopurified by Giancotti et al. (1986. Exp. Cell Res. 163:47-62) and by Sonnenberg et al. (1987. J. Biol. Chem. 268:10376-10383). In this report, we provide the first evidence that GPIc-IIa actually mediates adhesion of platelets to fibronectin-coated surfaces. GPIc-IIa thus represents a second functional fibronectin receptor, distinct from GPIIb-IIIa, that is largely responsible for the adhesion of nonactivated platelets to fibronectin-coated surfaces.     

Collagens support embryo attachment and outgrowth in vitro: effects of the Arg-Gly-Asp sequence

Collagen types I through VI support attachment and outgrowth of mouse blastocysts in vitro. We found that embryos acquire the ability to attach to collagens type II and VI relatively early in their developmental program. The time at which half of the embryos displayed outgrowth formation and the morphology of outgrowths formed on these two collagen types are similar to those observed for laminin, fibronectin, and hyaluronate. Embryos acquire the ability to outgrow on the other collagen types at a later time in culture. Both "native" and denatured collagens support embryo attachment and outgrowth, indicating that this activity is intrinsic to the primary collagens' structure. A synthetic peptide containing the sequence Arg-Gly-Asp inhibits embryo outgrowth on collagen type II and denatured collagen type IV, whereas a peptide containing the related sequence, Arg-Gly-Glu, has relatively little effect on embryo outgrowth. In contrast, embryo attachment to collagen types I, V, and VI was not inhibited specifically by the Arg-Gly-Asp peptide sequence. Consequently, it appears that embryos use multiple adhesion systems to attach to collagens. Among these are adhesion systems that have a peptide recognition specificity similar to that of fibronectin receptors. These studies indicate that embryo interactions with collagens may be one aspect of the tissue invasion processes that take place during implantation.     

The role of extracellular matrix in the migration and differentiation of parietal endoderm from teratocarcinoma embryoid bodies

Embryoid bodies formed from teratocarcinoma stem cells differentiate an outer layer consisting of parietal and visceral endoderm or of visceral endoderm exclusively. We have previously shown that when these embryoid bodies are plated on collagen-coated substrates a parietal endoderm-like cell migrates onto the substrate, whereas all of the visceral endoderm remains associated with the stem cell mass, suggesting a role for substrate contact in parietal endoderm differentiation. We now identify fibronectin as the migration-promoting component in these cultures, and note that laminin and collagen type IV are 10-fold less effective at promoting both attachment and endoderm outgrowth. The RGDS tetrapeptide (arg-gly-asp-ser) from the cell attachment domain of fibronectin can specifically block attachment and outgrowth on both fibronectin- and laminin-coated substrates. In addition, the involvement of the 140-kD fibronectin receptor is demonstrated using an antibody directed against this molecule.