An MBoC Favorite: Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein

This paper marks the early stages of understanding the molecular mechanisms of intracellular signaling activated by the extracellular matrix (ECM). I like it also as an exploration of a new area through closely linked questions. By 1991, many integrins had been cloned and their ECM ligand specificities studied. A trio of papers from 1989 to 1990 reported that integrin occupancy could affect complex cell behaviors, such as differentiation; however, the mechanisms of these behaviors remained unknown. In separate research, phosphotyrosinecontaining proteins had been observed to be concentrated in focal adhesions. In this paper, Guan et al. (1991) linked these areas by demonstrating that fibroblast adhesion to fibronectin correlated with dynamic, environment-dependent, tyrosine phosphorylation of a 120-kDa protein. The results included important clues for future researchers: multiple domains of fibronectin were needed for the strongest effect, and the cytoplasmic domain of β1 integrin was also necessary. The 120-kDa protein was identified as focal adhesion kinase in 1992 publications from other laboratories. By identifying an intracellular biochemical event correlated with ECM–integrin interactions and adhesive functions, this paper presaged multiple top-cited MBoC papers of the 1990s.


Introduction
The interactions of cells with extracellular matrix (ECM) proteins play important roles in a variety of biological processes, including embryonic development, wound healing, and malignant transformation. Both in vivo and in vitro, ECM proteins markedly influence the growth, migration, and differentiation of higher eukaryotic cells (Hay, 1985;Mosher, 1989;Hynes, 1990). For example, the proliferation of nontransformed fibroblasts in culture is dependent on their adhesion and spreading on a solid surface (Folkman and Moscona, 1978). This property, termed anchorage dependence, is reduced or lost as cells become increasingly transformed (Tucker et al., 1981;Wittelsberger et al., 1981).
Many of the effects of ECM proteins on cellular properties are mediated by their binding to integrins. Integrins are cell surface receptor proteins for a variety of ligands, including ECM proteins such as fibronectins (FNs), laminin, and collagens (Buck and Horwitz, 1987;Hynes, 1987;Ruoslahti and Pierschbacher, 1987;Ruoslahti, 1988). They are heterodimers of noncovalently associated a and , subunits. Both subunits of integrins are transmembrane glycoproteins containing a large extracellular domain, a single transmembrane segment, and generally a short cytoplasmic domain. There are at least 11 different integrin a subunits and 7 a subunits so far identified in mammalian cells (Hemler, 1990). In general, the combination of particular a and : subunits determines the Iigand specificity of the integrin complex. For example, a5j31 integrin serves specifically as a FN receptor on many cell types, whereas avf33 acts as a vitronectin receptor (Dejana et al., 1988;Singer et al., 1988). Furthermore, different integrins may recognize distinct sites within the same ECM protein. Thus, a5j31 interacts with FNs at the central RGDS (arg-gly-asp-ser) sequence, whereas a4,f1 integrin binds to the sequence EILDV in the alternatively spliced V (or III.CS) region (Wayner et al., 1989;Guan and Hynes, 1990;Mould et al., 1990).
In contrast with the great progress made in recent years identifying various integrin complexes and their ligand specificity, little is known about the molecular mechanisms by which integrins transmit information across the plasma membrane. Although they are presumably responsible for triggering the subsequent events leading to alterations in cell growth, migration, and differentiation, the immediate intracellular biochemical events induced by integrin/ECM Iigand interactions are poorly understood.
Reversible phosphorylation of proteins has long been recognized to regulate various cellular metabolic pathways (Edelman et aL, 1987). More recently, protein tyrosine phosphorylation has been implicated in transmitting signals that regulate cell growth and differentiation (Hunter and Cooper, 1985;Hunter, 1987;Ullrich and Schlessinger, 1990). In nontransformed fibroblasts, proteins phosphorylated at tyrosines are concentrated in cell-cell or cell-matrix contacts (Maher et al., 1985). This raises the possibility that protein tyrosine phosphorylation is involved in transducing signals across membranes when integrins bind to ECM proteins and aggregate into focal contacts. Ferrell and Martin (1989) and Golden et al. (1990) have reported tyrosine phosphorylation of several proteins in activated platelets whose phosphorylation relies at least in part on integrin-mediated events. In the present study, we have examined protein tyrosine phosphorylation in fibroblastic cells during attachment and spreading mediated by FNs and/ or integrins.

Major sites of tyrosine phosphorylation in attached cells
To study the effects on tyrosine phosphorylation of FN/integrin interactions, we first examined the distribution of tyrosine phosphorylated proteins in NIH3T3 cells on their adhesion to plasma FN (pFN). Cells were plated on coverslips coated with pFN and allowed to attach and spread for 2 h in the absence of serum. They were then fixed, permeabilized, and stained by double-label immunofluorescence using a rabbit serum, 363, against the integrin f3, subunit and a monoclonal antibody, py2O, specific for phosphorylated tyrosines (Glenney et al., 1988). The results are shown in Figure 1. Figure Figure 1A illustrates the staining pattern of tyrosine phosphorylated proteins detected by py2O. Their distribution colocalizes extensively, although not completely, with the integrin #, subunit, especially in focal contacts at cell edges (arrows).
These results show that tyrosine phosphorylation occurs on proteins in focal contacts in agreement with earlier results (Maher et al., 1985). Focal contacts are likely sites of signal transduction by f3, integrins on their interaction with the multivalent ligand FN (Burridge et al., 1988).
To identify the tyrosine phosphorylated proteins detected by py2O using immunofluorescence, we next examined the profile of proteins phosphorylated on tyrosines after cell attachment to pFN by western blotting using the same antibody. Tissue culture dishes were coated with either pFN or poly-L-lysine (PLL), and excess sites on the substrate were then blocked by incubation with bovine serum albumin (BSA). The incubation with BSA effectively blocked attachment of NIH3T3 cells to dishes in the absence of adhesive substrates. NIH3T3 cells were then plated in Dulbecco's Modified Eagle's Medium (DMEM) without serum and incubated for 25 min. After washing, cells were solubilized by RIPA buffer, and nuclei were removed by centrifugation. Protein concentrations of the detergent lysates were then determined, and equal amounts of protein were electrophoresed on polyacrylamide gels and transferred to nitrocellulose membranes ( Figure 2B). Western blots were carried out with py2O followed by 1251-labeled sheep anti-mouse IgG. The autoradiograph of the blotted membrane is shown in Figure 2A. A 1 20-kDa protein was the major protein detected by py2O and was much more prominent in cells attached to pFN than in those attached to PLL (Figure 2A). Therefore this protein, designated as ppl 20, is a prime candidate for being a protein detected by the same antibody in the immunofluorescence analysis of cells plated on pFN (Figure 1). Figure 2C shows the morphology of cells attached to PLL (top) or pFN (bottom) just before lysis in RIPA buffer.
At these early times there is little difference in morphology of cells on the two substrates. However, at later times pFN induces spreading of cells, whereas PLL does not (see below). Similar results were obtained by immunoprecipitation with py2O (data not shown). This precipitation was blocked by inclusion of phosphotyrosine but not phosphoserine or phosphothreonine, confirming the specificity of the antibody. Furthermore, several other antiphosphotyrosine antibodies also detected ppl 20 as a major band in the lysates of cells plated on FN although the spectrum of other bands varied with different antibodies (data not shown).
The integrin receptors for pFN on NIH3T3 cells include a3f1 and a5f1, and the common f1 subunit contains a site for tyrosine phosphorylation in the cytoplasmic domain (Hirst et al., 1986;Tamkun et aL, 1986;Tapley et al., 1989). To determine whether ppl 20 is the in- tegrin f3, subunit itself, we carried out the experiments shown in Figure 3. Detergent lysates from cells plated on pFN were incubated with py2O-coupled agarose beads at 4°C for 1 h. The bound fractions (B) were eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer and loaded on gels along with the total starting material (T) and the unbound fractions (U.B.). Duplicate gels were transferred to nitrocellulose membranes and probed with either py2O (left) or 363 (anti-:1 serum, right).
The asterisk on the left marks pp120, which was largely immunoprecipitated by py2O- tein was not recognized by the anti-f,1 antiserum. In contrast, the 3 , B subunit as well as its precursor (B and preB marked at the right) was found exclusively in the unbound fraction. Reciprocally, when integrins were precipitated by anti-:, antibody (Marcantonio and Hynes, 1988) and the precipitate blotted with py20, no band was detected (data not shown). These distinctions between ppl 20 and f,B as well as the apparent differences in mobility on SDSgels indicated that the ppl 20 protein is not the integrin flu subunit. Using similar immunological and electrophoretic criteria, we also ruled out the possibility of ppl20 being vinculin or a-actinin (data not shown but see below). The subcellular localization of ppl20 was examined in experiments shown in Figure 4. NIH3T3 cells plated on pFN were broken by homogenization in hypotonic buffers. After removing the nuclei by low-speed centrifugation, the supernatants were subjected to ultracentrifugation at 100 000 x g for 1 h at 4°C to separate the membranes (pellet) from the cytoplasmic (soluble) fractions. These two fractions were adjusted to equal volumes, and aliquots were electrophoresed, transferred, and blotted with various antibodies. Figure 4, left, shows that ppl 20 distributes approximately equally in both membrane (P) and cytosol (S) fractions. Vinculin was similarly distributed in both fractions with more in the cytosol but does not comigrate with pp120 (middle), whereas the integrin f31 subunit was fractionated exclusively into the membrane fraction as expected (right). Thus, ppl20 does not appear to be identical with any of the known major constituents of focal contacts and does not behave as an integral membrane protein.

Involvement of specific segments of fibronectins
To determine the particular segments of FN that are involved in inducing ppl 20 phosphorylation, NIH3T3 cells were plated on a variety of adhesive substrates for 1 h and ppl 20 phosphorylation was then quantitated by western blotting using py20 as described above. As shown in Figure 5A, pFN (column C) induced significant levels of ppl 20 phosphorylation above the background level observed on PLL (column B). Another nonspecific cell adhesive substrate, concanavalin A (column D), failed to induce ppl 20 phosphorylation. Furthermore, neither collagen type IV (column E) nor laminin (column F) was able to induce the change, although these cells contain an integrin receptor reported to recognize them (a331).
Columns G-J show results from cells plated on several different FN fragments, including an RGD-containing pep-tide called peptite-2000 (column G); the 120-kDa chymotryptic fragment, which contains the central cell-binding domain but lacks the heparin-binding domain (column H); and two recombinant FN fragments consisting of type Ill repeats 1117-11115 comprising both the cell-and heparin-binding domains of FN, with (7-15.BAV, column J) or without (7-15.0, column 1) the alternatively spliced regions (generous gift of Paul Johnson and John Peters, MIT, Cambridge, MA). These results showed that the recombinant FN fragments containing both the central cell-binding domain and the carboxyl heparin-binding domain were fully functional, whereas the 120-kDa fragment used at the same concentration was active but at a significantly reduced level. Interestingly, the RGD peptide peptite-2000 did not induce any significant level of ppl 20 phosphorylation at doses from 0.1 to 100 ,g/ml and for various lengths of time (see also below). We also noted that the ability of various substrates to induce ppl 20 phosphorylation seemed to be correlated with their ability to induce extensive cell spreading (see Figure 5, panels B-J). There was no obvious effect of the three alternatively spliced segments of FN either on spreading of NIH3T3 cells or on phosphorylation of pp12O (compare I and J).
To explore further the possible links between cell spreading and ppl 20 phosphorylation, time course experiments were carried out for cells plated on pFN and peptite-2000 as shown in Figure 6. Cells were photographed and lysed at various times after plating as indicated. Equal amounts of protein were then analyzed by western blotting with py2O as described above. The top panel shows that ppl 20 phosphorylation (marked *) is significant in attached growing cells (G), falls to low levels on trypsinization of  Figure 6 also shows that, by 10 min after plating, cells attached to both pFN and peptite-2000 without spreading or apparent distinctions in morphology on these two substrates. This indicated that ppl 20 phosphorylation was not a consequence of cell spreading. At 3 h after plating, however, cells were fully spread on pFN but remained round on peptite-2000 suggesting a possible link between ppl 20 phosphorylation and later cell spreading.

Involvement of integrin f1 subunit
The integrin f3, subunit is the common subunit of cell surface integrin receptors for pFN. To investigate its role in mediating ppl 20 phosphorylation, we used NIH3T3 cell lines stably expressing the heterologous chicken integrin fl, subunit or its mutated forms (Solowska et al., 1989;Marcantonio et al., 1990). Cells were plated on dishes coated with the monoclonal antibody CSAT that interacts with the ligandbinding site of the integrin ,B1 subunit and is chicken-specific. Cells plated on PLL or pFN were used as negative and positive controls. The location of ppl 20, vinculin, and integrin 3,B and its precursor are marked at the sides.
Cell lysates were analyzed by western blotting with py2O as described, and an autoradiograph is shown in Figure 7 with ppl20 marked by an asterisk at the left. All cells showed phosphorylation of pp12O when attached to pFN via endogenous integrins (lanes F). However, when the effects of attachment via the transfected chicken integrins were examined by attachment to CSAT antibody (lanes C), marked differences were observed among the different mutated forms. The wild type (WT) chicken integrin subunit was able to induce partial ppl20 phosphorylation on cell attachment to CSAT. However, the deletion mutants Al, A2, A4, and A5 lacking various lengths of the cytoplasmic domain failed to induce the same change. A mutation changing tyr788 to phe (YF) did not abolish the ability of the chicken f3, to induce ppl20 phosphorylation on cells plated on CSAT. These results demonstrated that the integrin subunit is at least partially responsible for mediating pFN-induced ppl 20 tyrosine phosphorylation and that its cytoplasmic interactions are critical for this activity. Furthermore, the hydroxyl group of tyr788 seems not to be important (YF), whereas the last four amino acids missing in mutant A5 might be involved in the cytoplasmic interactions of integrin 3 , B necessary for ppl20 phosphorylation. Figure  on CSAT monoclonal antibody. At 30 min after plating, all cells attached to the antibody substrates as well as to PLL or pFN. However, we noticed that Al, A2, A4, and A5 transfected cells appeared to be rounder than WT and YF transfected cells. Furthermore, these cells detached from the substrates by 3 h, whereas cells transfected with WT and YF remained attached. These results suggested a correlation between pp120 phosphorylation and stable attachment to substrates in integrin-mediated adhesion. ... o s1 _ . . pFN peptite200;..

Discussion
In multicellular organisms, the behavior of individual cells such as their growth, differentiation, and migration is tightly controlled by their extracellular environments. Two major classes of controlling components are diffusible peptide growth factors and insoluble extracellular matrix proteins. These two classes of molecules influence cellular behavior both independently and cooperatively (Ruoslahti and Yamaguchi, 1991;Yayon et al., 1991). Rapid progress has been made in recent years elucidating the molecular 3 h I4 mechanisms of the soluble growth factors. They bind to their cell surface receptors, which results in the activation of several second messenger pathways. These pathways often involve a series of phosphorylation events that are dependent on the activation of protein kinases. The activation of the second messenger pathways in turn triggers a series of downstream events that alter gene expression and cellular structures to affect cell properties (Sibley et al., 1987;Yarden and Ullrich, 1988;Williams, 1989). In  transduce signals across the plasma membrane. Recent identification of integrins as the cell surface receptors for many extracellular matrix proteins and molecular cloning of the genes for integrins has opened the way to investigate these questions at the molecular level.
In this report, we showed that attachment of fibroblasts to FNs induced tyrosine phosphorylation of a protein, ppl 20, in the absence of any added soluble growth factors. This induction was rapid and preceded cell spreading. The central regions of FN molecules, including both the cell binding domain and high-affinity heparin binding domain, appeared to be sufficient to induce the response, whereas attachment of cells to RGD-containing peptides or to several other adhesive ligands was insufficient. We also showed that phosphorylation of ppl 20 can be mediated by integrin receptors on the cell surface and that the cytoplasmic domain of the integrin f3, subunit is essential for this. These results implicate protein tyrosine kinases in signal transduction by integrin receptors for FN.
The possibility that the FN or CSAT preparations contain soluble growth factors that are responsible for the induction of phosphorylation of ppl 20 is rendered unlikely by several observations. The same results are obtained with plasma FN and with several recombinant FN fragments produced in Chinese hamster ovary (CHO) cells grown in serum-free medium ( Figure   5). Furthermore, phosphorylation of ppl 20 is also induced by CSAT antibody in a fashion dependent on the form of chicken integrin #I subunit expressed by the cells (Figure 7). If the effect of the CSAT preparation was due to contaminating growth factors, there is no obvious reason for the dependence on the exact form of exogenous integrin expressed by the cells.
We noted that the induction of the tyrosine phosphorylation of pp120 is rapid and occurs before significant changes in cell shape. It reaches maximum levels at 10 min after cell plating, which is about the time it takes for fibroblasts to attach to FN-coated surfaces. Thus, it is possible that the phosphorylation of ppl 20 on tyrosine plays a regulatory role in the process of stable cell attachment. Alternatively, ppl 20 phosphorylation might be involved in events required for the subsequent cell spreading on substrates resulting in significant changes in cell shape. Indeed, we observed a correlation between induction of ppl 20 phosphorylation and of cell spreading by various substrates ( Figure 5). Interestingly, the 1 20-kDa chymotryptic fragment of pFN induces only intermediate levels of ppl 20 phosphorylation while apparently supporting cell spreading. Previous experiments by LeBaron et al. (1988) and Woods et al. (1988) have shown that the cell-binding domain alone does not cause full cell spreading in the sense that focal contacts are not observed in fibroblasts plated on it. The heparin-binding domain in combination with the cell-binding domain is necessary to induce full cell spreading and focal contact formation. In agreement with this, two recombinant FN segments, 7-15.0 and 7-15.BAV, containing both the cell-binding domain and the heparin-binding domain induced high levels of ppl 20 phosphorylation ( Figure 5).
The extent of cell spreading has previously been linked to the growth control of nontransformed cells (Folkman and Moscona, 1978). Besides the obvious possibility that cell spreading controls cell proliferation directly through its influence on the cytoskeleton, it is possible that both parameters are dependent on a third factor related in some way to substrate, as the authors pointed out. The phosphorylation of ppl 20 might be a candidate for such a third factor that regulates both cell spreading and proliferation. Therefore, ppl 20 phosphorylation could influence cellular behavior either directly or through its effects on cell spreading. The resolution of these possibilities requires further studies using reagents specific for ppl 20.
Our results also point to a critical role of the cytoplasmic domain of integrin f,8 in mediating phosphorylation of ppl 20. This raises the question as to whether this domain interacts with a protein-tyrosine kinase. Although the v-src gene product can be localized with integrin A, in focal contacts, there is as yet no evidence that this kinase associates with integrin fl, directly (Burridge et al., 1988). Recently, the CD4 and CD8 receptors on T lymphocytes have been shown to associate and regulate the src-related protein-tyrosine kinase, Ick (Veillette et al., 1988). Similarly, IgM on B lymphocytes has been shown to associate with another src-related kinase, lyn (Yamanashi et al., 1991). Thus, the coupling of cell surface receptors having short cytoplasmic domains with submembranous protein tyrosine kinases could represent a common mechanism for mediating signal transduction events triggered by cell-cell and cell-matrix interactions. This mode of action would be analogous with that of soluble growth factor receptors that generally have large cytoplasmic domains with intrinsic protein tyrosine kinase activities regulated by extracellular ligand binding. Alternatively, the cytoplasmic domain of the integrin AI subunit may mediate intracellular phosphorylation events by associating with the putative substrates and bringing them to their physiological kinases on cell binding to FNs. Focal contacts in fibroblasts are likely sites of such actions and have been proposed to be the location at which ECM transduces signals across plasma membranes (Burridge et al., 1988). The cytoplasmic domain of integrin tl1 has been shown to bind to talin and a-actinin and recruit them to focal contacts when cells were plated on FN Dejana et aI., 1988;Singer et al., 1988;Tapley et al., 1 989;Otey etal., 1 990). In this respect, it is worth noting that the platelet integrin GPIlbllla (integrin allb13) also induces tyrosine phosphorylation of several intracellular proteins concomitant with its induction of platelet aggregation (Ferrell and Martin, 1989;Golden et al., 1990). Therefore, aggregation of cell surface receptors might be a general phenomenon required for integrinmediated signal transductions. Aggregation of receptors might be required for several reasons. First, the affinity of interactions between the integrin cytoplasmic domain and the putative kinases or substrates might be low so that aggregation of multiple receptors is necessary to produce high avidity interactions to bring kinases and substrates together. Second, multiple proteins might be involved in a series of events that lead to activation of a kinase to phosphorylate pp120. Receptor aggregation may be necessary to induce interactions among multiple proteins. Consistent with this idea is the requirement for several domains of FN for induction of full phosphorylation of ppl 20. The role of multiple receptors in assuring stable cellcell interactions was demonstrated previously for T lymphocytes with their target cells (Springer, 1990). Last, aggregation of integrin receptors with their interacting components might be necessary to generate regions of plasma membrane that segregate phosphorylated ppl 20 from cellular protein tyrosine phosphatases. This is in agreement with the sustained phosphorylation of ppl 20 in growing cells and its rapid dephosphorylation on cell detachment from substrates by a number of different methods (Fig. 6 and our unpublished observation). Finally, it should be pointed out that the above three possibilities were not mutually exclusive and could all be necessary for pp120 phosphorylation induced by FN/integrin interactions. Testing of these hypotheses requires identification of ppl 20 and preparation of specific reagents to it. Although the identity of ppl 20 is unknown at present, our experiments suggest that it is a major tyrosine-phosphorylated protein in nontransformed fibroblasts and is possibly localized in the inner membrane and focal contacts in cells plated on FN. Maher and Pasquale (1988) have reported a 120-kDa tyrosine-phosphorylated protein in many embryonic tissues, and Pasquale et al. (1988) have described a tyrosinephosphorylated protein of similar molecular weight in Balbc3T3 cells and showed that tyrosine phosphorylation of the protein was stimulated by a variety of soluble growth factors. This protein was also localized to focal contacts as detected by polyclonal anti-phosphotyrosine serum. If indeed this phosphoprotein is identical with ppl 20 described in this report, that would suggest a role for ppl 20 in integrating signals generated by both soluble growth factors and insoluble ECM proteins. Others have reported a protein of similar size as a substrate for the insulin receptor protein tyrosine kinase (Rees-Jones and Taylor, 1985;Sadoul et al., 1985). Cooperation between soluble growth factors and ECM exists in many biological systems, and study of regulation of pp120 phosphorylation may allow examination of the molecular mechanisms underlying this cooperativity. In particular, it could shed light on our understanding of growth regulation of both normal and malignant cells. For example, the following hypothesis could be offered to explain the anchorage-independent growth of transformed cells. In nor-mal cells stimulated by high levels of growth factors or in transformed cells where cytoplasmic kinases are constantly turned on, critical substrates including ppl 20 might be highly phosphorylated independent of cell attachment to matrix. Indeed Linder and Burr (1988) and Reynolds et al. (1989) have reported elevated phosphorylation of several proteins of around 120 kDa correlating with oncogenic transformation in chicken embryo fibroblasts infected with viruses encoding membrane-associated pp6Osrc, a known tyrosine protein kinase. These phosphoproteins could be related to the ppl 20 described here. Preliminary results using antibodies provided by Dr. J.T. Parsons (Kanner et al., 1990) do suggest some cross-reactivity (J.-L. Guan, unpublished data). Thus one could postulate that elevated levels of phosphorylation of ppl 20 could occur constitutively in transformed cells but that normal cells require either high levels of growth factors or cooperation between growth factors and matrix adhesion (anchorage dependence) to achieve sufficient levels of phosphorylation. Alternatively, adequate phosphorylation of ppl 20 and other substrates in normal cells might require clustering of kinases and their substrates together and perhaps also away from cellular phosphatases. These processes might be controlled by ECM molecules through their integrin receptors. These models are testable and will be examined.
In conclusion, we have demonstrated that integrin/FN interactions can regulate tyrosine phosphorylation in the absence of any added soluble growth factors. This may provide a mechanism by which integrins convey information across membranes, which is responsible for triggering the subsequent events necessary for ECM to influence cellular behavior. Identification of ppl 20 will allow us to study the biological consequences of its phosphorylation and its role in integrin-mediated signal transduction. Use of cytoplasmic domain mutants of the integrin fl subunit in transfected cells will allow us to dissect in detail the mode of regulation of ppl 20 phosphorylation and its relationships with other integrin $1-mediated events in focal contacts.

Materials and methods Cells and reagents
NIH3T3 cells were maintained in DMEM plus 1 0% calf serum (CS, GIBCO Laboratories, Grand Island, NY). NIH3T3 cells expressing the chicken integrin #I subunit and its various mutations were described previously (Marcantonio et al., 1990;Solowska et al., 1989), and they were maintained in DMEM plus 10% CS and 0.5 mg/ml G418 (GIBCO Laboratories). The mutations are Al lacking 40, A2 lacking 37, A4 lacking 13, A5 lacking 4 amino acids from the carboxyl terminus, and YF in which tyr 788 is changed to phe. Rat pFN, peptite-2000, 1 20-kDa chymotryptic fragment of human pFN, and mouse laminin were purchased from Telios (San Diego, CA). PLL and concanavalin A were obtained from Sigma (St. Louis, MO) and type IV collagen was purchased from Collaborative Research (Lexington, MA). Recombinant FN fragments 7-15.0 and 7-1 5.BAV produced in CHO cells grown in serum-free medium and purified by affinity chromatography on columns of a monoclonal antibody specific for rat fibronectins were generous gifts of Drs. Paul Johnson and John Peters of our Laboratory. Monoclonal antibody py2O (Glenney et al., 1988) was purchased from ICN (Costa Mesa, CA) and also kindly provided by Dr. Glenney, Jr. (University of Kentucky). CSAT monoclonal antibody was prepared by affinity chromatography on protein A Sepharose from hybridomas (Neff et al., 1982) kindly provided by Dr. Clayton Buck (Wistar Institute, Philadelphia, PA). Polyclonal antiserum 363 was described previously (Marcantonio and Hynes, 1988) and monoclonal antibody against vinculin was purchased from Sigma.
Cells were plated in DMEM without serum for 2 h on coverslips previously coated with rat pFN (10 ,ug/ml). They were rinsed twice in phosphate-buffered saline (PBS) and fixed for 15 min in a freshly prepared 40/o solution of paraformaldehyde (Fluka Chemical Co., Buchs, Switzerland) in PBS, rinsed, and permeabilized with 0.50/o NP40 in PBS for 15 min. Cells were stained with primary antibody in 1 0%/o normal goat serum in PBS for 30 min at 370C. After three washes with PBS, the secondary antibody mixture (rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated goat anti-mouse IgG in 1 0% normal goat serum in PBS; Organon Teknika-Cappel, Malvern, PA) was added and incubated for 30 min at 37°C. After three washes, coverslips were mounted in Gelvatol and examined using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY) and photographed (Tri-X film; Eastman Kodak Co., Rochester, NY).

Preparation of detergent lysates from cells plated on various substrates
Tissue culture plates were coated overnight with various substrates at 4°C, washed twice with PBS, and then incubated with 2 mg/ml heat-inactivated (1 h at 700C) BSA in PBS for 2 h at 37°C. Cells were harvested by brief trypsinization and then washed twice with PBS containing 0.5 mg/ ml soybean trypsin inhibitor (Sigma). The cells were resuspended in DMEM without serum and added to coated plates at 106/ml (1 ml for 35-mm plate or 2.5 ml for 60-mm plate). After various times of incubation at 37°C, cells were washed twice with cold PBS and lysed in RIPA buffer (150 mM NaCI, 50 mM tris(hydroxymethyl)aminomethane [Tris], pH7.5, 1 % Triton X-1 00, 1 % deoxycholate, 0.10% SDS, and 2 mM EDTA) containing a cocktail of protease and phosphatase inhibitors (2 mM phenylmethylsulfonyl fluoride [PMSF], 0.2 TIU/mI aprotinin, 1 ,g/ml leupeptin, 0.5 mM vanadate, 50 mM NaF, and 30 mM sodium phosphate). The cell lysates were centrifuged for 10 min at 40C in a microfuge to remove nuclei.
The protein concentrations in the supernatants were determined using BioRad Protein Assay (Richmond, CA). In some experiments, adherent cells were photographed just before lysis using a Nikon inverted phase-contrast microscope.

Western immunoblotting
Equal amounts of protein lysates prepared from cells on various substrates were electrophoresed on 70%o SDS-polyacrylamide gel electrophoresis by the method of Laemmli (1970). Proteins were electrophoretically transferred to nitrocellulose filters (Schleicher and Schuell, Inc., Keene, NH) for 2 h at 250 mA (Towbin et al., 1979). Filters were stained with India ink to confirm the presence of equal amounts of cellular proteins in all lanes. They were then blocked with blocking buffer (5% BSA in TBS; 150 mM NaCI, 50 mM Tris, pH 7.4) for 2 h and incubated with py2O at 1 gg/ml in blocking buffer overnight. After washing with TBS containing 0.50/% Tween-20, the filters were incubated for 2 h with 1251-labeled sheep anti-mouse IgG (0.15 jsCi/ml; Amersham Corp., Arlington Heights, IL). The filters were then washed, dried, and exposed to XAR-5 film (Eastman Kodak Co.) with an enhancing screen. Quantitation of the autoradiographs was carried out by computing densitometer (Molecular Dynamics, Sunnyvale, CA). In some experiments, monoclonal antivinculin (1:500 dilution in blocking buffer) or antiserum 363 (1:500 dilution in blocking buffer) was used as the primary antibodies, and '251-labeled protein A (0.15 ,Ci/ml; Amersham Corp.) was used as the secondary reagent.

Immunoprecipitation and subcellular fractionation
Immunoprecipitation was carried out by incubating detergent lysates with py2O-agarose (ICN) at 40C for 1 h. The unbound fraction was collected by centrifugation, and, after washing in RIPA buffer, the bound fraction was obtained by boiling the py2O-agarose beads for 3 min in SDS-sample buffer. For subcellular fractionation, cells were homogenized in hypotonic buffers (10 mM Tris, pH 7.4, 1 mM MgCI2) containing 2 mM PMSF, 0.15 TIU/ml aprotinin, 1 ,Ag/ml leupeptin, and 0.5 mM vanadate. After removing nuclei by lowspeed centrifugation, cellular membrane and cytoplasmic fractions were separated by ultracentrifugation at 100 000 x g for 1 h. The pellet (membrane) fraction was dissolved in RIPA buffer to equal the volume of the cytosolic fraction.