A 125/115-Kda Cell Surface Receptor Specific for Vitronectin Interacts With

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Proc. Nati. Acad. Sci. USA Vol. 82, pp. 5766-5770, September 1985 Cell Biology A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin (cell-substrate adhesion/affinity chromatography/liposomes/synthetic peptides) ROBERT PYTELA, MICHAEL D. PIERSCHBACHER, AND ERKKI RUOSLAHTI Cancer Research Center, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Leroy Hood, May 13, 1985

ABSTRACT Affinity chromatography was used to identify inhibited in the presence of millimolar concentrations of the a cell surface receptor for the adhesive protein vitronectin. same synthetic peptides (17, 18). Detergent extracts of human osteosarcoma (MG-63) cells were We have recently found that adhesion of cells to chromatographed on either vitronectin-Sepharose or Sepha- vitronectin is also inhibited by the Arg-Gly-Asp-containing rose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, peptides (20). We have also found, by deducing the amino which includes the flbronectin cell attachment sequence Arg- acid sequence of vitronectin from cloned cDNA, that Gly-Asp. Two cell surface proteins with apparent molecular vitronectin contains an -Arg-Gly-Asp- sequence (unpub- mass of 125 and 115 kDa bound to both columns and were lished data). These results suggested to us that the same specifically eluted with a solution containing the Gly-Arg-Gly- receptor might be responsible for the adhesion ofcells to both Asp-Ser-Pro peptide. These proteins could be incorporated into fibronectin and vitronectin. However, we have found and phosphatidylcholine liposomies and mediated the specific bind- report here the existence of a surface receptor that is specific ing of these liposomes to vitronectin but not to fibronectin. In for vitronectin. This receptor differs from the fibronectin contrast, liposomes containing a previously identified 140-kDa receptor in molecular weight and binding specificity toward fibronectin receptor, which interacts with the Arg-Gly-Asp fibronectin and vitronectin, but it shares with the fibronectin sequence in fibronectin, did not bind to vitronectin. Thus, the receptor the ability to recognize the Arg-Gly-Asp-containing fibronectin and vitronectin receptors each recognize the Gly- peptides; Based on these results and the fact that some other Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive proteins with the -Arg-Gly-Asp- sequence are active in reactivities toward fibronectin and vitronectin. These receptors cellular recognition phenomena (18, 21), we envision a appear to belong to a family of proteins that mediate cell recognition system consisting of a family of cell surface substratum adhesion via related but subtly different specific- receptors each of which interacts with the -Arg-Gly-Asp- ities. sequence in the unique context of an individual extracellular protein. Cell surface-recognition mechanisms that allow cells to interact with one another and with intercellular substances MATERIALS AND METHODS play an important regulatory role in cellular growth, migration, and differentiation (1, 2). Cell-cell interactions (1, 3) and Proteins and Peptides. Vitronectin was purified from hu- adhesion of cells to extracellular matrices (4, 5) appear to be man plasma by affinity chromatography on monoclonal mediated by separate sets of molecules. The extracellular antibody-Sepharose and heparin-Sepharose (11) and then matrix molecules to which cells are known to adhere include was coupled to cyanogen bromide-activated Sepharose (Sig- fibronectin (6, 7), collagens (5, 8), laminin (9-11), vitronectin ma) (22). The resulting matrix contained 2 mg of vitro- (12, 13), and possibly proteoglycans (14, 15). Although some nectin/ml of settled gel. of these proteins have a restricted tissue distribution, no Synthetic peptides were prepared by Peninsula Laborato- clear-cut cell-type specificity has emerged for any of them ries (San Carlos, CA) according to our specifications. Gly- when the attachment of different types of cells to defined Arg-Gly-Asp-Ser-Pro (GRGDSP)-Sepharose was prepared substrata has been tested. This suggests that if such speci- by coupling 100 mg of the peptide to 2 ml of cyanogen ficities exist, they may be more subtle than can be detected bromide-activated Sepharose. with the adhesion assays used. Fibronectin was isolated from human plasma (23), and To gain insight into cell-substratum adhesion, we have laminin from a rat yolk sac tumor (24). The fibronectin studied the molecular mechanisms of adhesion of cells to fragments used have been described (22). The fibronectin fibronectin and other adhesive proteins. We have shown that receptor was isolated from MG-63 human osteosarcoma cells the short amino acid sequence -arginine-glycine-aspartic (25) as described (19). acid- (-Arg-Gly-Asp-) is responsible for the attachment of Cell Culture and Surface Labeling. The MG-63 osteo- cells to fibronectin (16-18), and we have isolated a cell sarcoma cells were grown on 175-cm2 tissue culture dishes in surface receptor for fibronectin by using an Affinity column Dulbecco's modified Eagle's medium supplemented with 5% carrying a large, cell attachment-promoting fragment of the fetal calf serum, glutamine, and penicillin/streptomycin. For molecule. That this receptor specifically recognizes the subculturing and harvesting, confluent layers of cells were -Arg-Gly-Asp- sequence in fibronectin is indicated by the fact incubated in 1 mM EDTA for 15 min. For surface iodination, that it is specifically eluted from the column by small, cells were harvested from confluent cultures, collected by Arg-Gly-Asp-containing, synthetic peptides (19). Further- centrifugation, and resuspended in phosphate-buffered saline more, the adhesion of cells to a fibronectin substratum is (150 mM NaCl/10 mM sodium phosphate, pH 7.3/1 mM CaCl2/1 mM MgCl2) containing 0.2 mM phenylmethylsul- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GRGDSP, Gly-Arg-Gly-Asp-Ser-Pro; GRGESP, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Gly-Arg-Gly-Glu-Ser-Pro. 5766 Downloaded by guest on September 28, 2021 Cell Biology: Pytela et al. Proc. Natl. Acad. Sci. USA 82 (1985) 5767

fonyl fluoride. The suspended cells were radioiodinated with 1 2 3 4 Na125I according to Lebien et al. (26) and lysed in 200 mM octyl f8-D-glucopyranoside (octyl glucoside; Behring Diag- nostics, La Jolla, CA) as described (19). 20() - Affinity Chromatography. The octyl glucoside extract (from 101 cells) in 1 ml was applied to the affinity matrix, which had been equilibrated with column buffer (phosphate- buffered saline containing 50 mM octyl glucoside and 1 mM 116 - phenylmethylsulfonyl fluoride). Elution with the synthetic 94 - cell attachment peptide was carried out by washing the column over a period of 1 hr with 1 volume of column buffer supplemented with 1.5 mM (1 mg/ml) GRGDSP. 67 - Liposome Binding Assay. For preparation ofliposomes, egg yolkphosphatidylcholine (Sigma), [2-palmitoyl-9,10-3H]phosphatidylcholine (New England Nuclear), and appropriate protein fractions were dissolved in column buffer and dia- lyzed against phosphate-buffered saline (19, 27). The binding 43 - of the resulting liposomes was studied as described (19). Gel Electrophoresis. For NaDodSO4/PAGE, samples were boiled for 3 min in the presence of 3% NaDodSO4 and 5% (vol/vol) 2-mercaptoethanol and electrophoresed in 7.5% FIG. 1. Affinity chromatography of surface-iodinated MG-63 cell acrylamide gels according to Laemmli (28). Molecular mass extract on vitronectin-Sepharose: analysis by NaDodSO4/PAGE markers were myosin (200 kDa), /8-galactosidase (116 kDa), followed by autoradiography. An octyl glucoside extract (1 ml) of phosphorylase b (94 kDa), bovine serum albumin (67 kDa), surface-radioiodinated human osteosarcoma cells was applied to the ovalbumin (43 kDa), and carbonic anhydrase (30 kDa). Bands vitronectin column (bed volume 1 ml); elution was with buffer were visualized by autoradiography or by silver staining with containing the cell-attachment peptide GRGDSP. Lanes 1-5: con- a commercial kit (Bio-Rad). secutive fractions (0.5 ml) obtained after the addition of GRGDSP peptide to the elution buffer. The arrow marks the top of the RESULTS separating gel. Size markers (in kDa) are at left. Affinity Chromatography on Sepharose Linked to Vitro- nectin and Cell Attachment Peptides. An affinity chromatog- appropriate peptide from the vitronectin or GRGDSP col- raphy experiment designed to identify a potential cell surface umns, even after longer exposure of the autoradiographs. receptor for vitronectin was performed as follows. Moreover, the fibronectin receptor could be isolated by An octyl glucoside extract of surface-iodinated MG-63 os- chromatography on fibronectin fragment-Sepharose after the teosarcoma cells was applied to an affinity column con- 125/115-kDa protein had been removed on (GRGDSP)- taining purified vitronectin. Elution was with buffer contain- Sepharose (result not shown). ing detergent and the synthetic peptide GRGDSP, which is a Preliminary experiments have shown that different cell attachment-promoting peptide taken from the fibronectin cyanogen bromide fragments are obtained from the 125 kDa sequence (17). This peptide released from the affinity matrix and the 115 kDa polypeptides (not shown), suggesting that two polypeptides having apparent molecular sizes of 125 and the two polypeptides are not related through proteolysis and 115 kDa, respectively, as determined by NaDodSO4/PAGE may instead represent two independent gene products. Fur- under reducing conditions (Fig. 1). ther studies will be necessary to determine whether the two Since the amount of vitronectin available for the preparation polypeptides are present as a complex on the cell surface or of the affinity columns was limited and consequently the yields whether each one of them interacts with the GRGDSP of the 125- and 115-kDa polypeptides were low, we also sequence independently. investigated the possibility that an affinity matrix in which the Binding of Liposomes Made with 125/115-kDa Protein to peptide GRGDSP was coupled to Sepharose could be used Vitronectin and GRGDSP Peptide Substrata. When phospha- instead ofvitronectin-Sepharose. Such a column indeed yielded tidylcholine liposomes were prepared by dialyzing a deter- chemically detectable amounts of the 125- and 115-kDa gent solution containing both the 1251I-labeled 125/115-kDa polypeptides from octyl glucoside extracts of MG-63 cells. protein and the phospholipid against a detergent-free buffer In the experiment represented in Fig. 2, the cell extract was (19, 27), the protein became incorporated into the liposomes. loaded on the GRGDSP column and was eluted sequentially About 90% of the 125/115-kDa protein radioactivity was with two peptide solutions. The first solution contained a found associated with the liposome fraction after the lipo- control peptide, Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), which somes had been floated to the surface of a sucrose gradient does not promote cell attachment (18), and the second con- by ultracentrifugation (data not shown). These liposomes tained the active GRGDSP peptide. After NaDodSO4/PAGE of were used to study the recognition specificity of the 125/115- the column effluent fractions, silver staining (Fig. 2A) revealed kDa protein by assaying the binding of the liposomes to that several proteins were present throughout the effluent, but microtiter wells coated with various extracellular matrix the 125/115-kDa doublet was proteins and peptides. the only one substantially influ- 3H-labeled liposomes containing the 125/115-kDa protein enced by the presence ofGRGDSP peptide in the elution buffer. showed dose-dependent binding to microtiter wells coated As was the case with vitronectin-Sepharose, only the specifi- with vitronectin, whereas they did not bind to fibronectin- cally eluted 125/115-kDa polypeptides were labeled by surface- coated wells (Fig. 3 Left). In contrast, liposomes containing specific radioiodination (Fig. 2B), and the polypeptides from the the 140-kDa fibronectin receptor bound to microtiter wells two columns comigrated, indicating that the same component coated with fibronectin, as demonstrated previously (19), but was isolated by the two procedures. Because the peptide showed no binding to vitronectin (Fig. 3 Right). Specificity column bound significantly more receptor, it was used in controls showed that neither liposome preparation bound to subsequent experiments. laminin, another adhesive extracellular matrix protein (9, 10). No 140-kDa polypeptide corresponding to the fibronectin Moreover, the binding of the 125/115-kDa protein liposomes receptor (19) was detected in the fractions eluted with the to vitronectin could be completely inhibited by the GRGDSP Downloaded by guest on September 28, 2021 5768 Cell Biology: Pytela et al. Proc. Natl. Acad. Sci. USA 82 (1985)

A GRGESP GRGDSP Table 1. Peptide GRGDSP inhibits binding of 125/115-kDa-protein liposomes to vitronectin I 3 4 hX 9 10 11 12 1 14 15 16 1 718 19 Phosphatidylcholine - 200 Coating Inhibitor bound, ng per well Vitronectin None 155 Vitronectin GRGDSP 14 - - - !!r- O." -- - 94 ", 139 W. a. '44k.. .*- Vitronectin GRGESP - 67 11 s. :: Bovine serum albumin None . L. v 1L:X b. ,, 11XI!F ': Plastic microtiter wells were coated with vitronectin or bovine E x f: | iX .,;f:| . ._L__ _ .. serum albumin at 10 ,ug/ml, and the binding ofliposomes to the wells wS .s,.@,...... was assayed in the presence or absence of 1.5 mM synthetic peptides as described in the legend for Fig. 3. il.: .''.. * : .,- :..: :w w: vitronectin. Previous studies have shown that the GRGDSP peptide also inhibits the binding of liposomes containing the B GRGESP GRGDSP fibronectin receptor to a fibronectin-coated surface (19). 1 h Striking contrast in the apparent specificities of the two 1 2 34 56 17 89l01I11?13 14 1 6V:118 19 receptors was also seen when their ability to bind directly to immobilized peptides was examined (Table 2). The 125/115- kDa protein liposomes bound nearly as well to the GRGDSP peptide as they did to vitronectin and somewhat less strongly I to a 30 amino acid peptide containing the GRGDSP and surrounding sequences from fibronectin. These liposomes, however, bound to neither the 120-kDa cell-attachment fragment from fibronectin (22) nor fibronectin itself. The fibronectin-receptor liposomes, on the other hand, showed the opposite specificity; they bound equally well to fibronectin and its large cell-attachment-promoting fragment but did not bind to the synthetic peptides. DISCUSSION FIG. 2. Affinity chromatography of surface-iodinated MG-63 cell The 125/115-kDa protein binds specifically to vitronectin, is extract on (GRGDSP)-Sepharose: analysis by NaDodSO4/PAGE. accessible to lactoperoxidase-catalyzed iodination at the cell The sample applied to the column (bed volume, 2 ml) was eluted surface, and possesses an apparent ability to be incorporated sequentially with buffer (lanes 2-5), buffer plus an inactive analogue of the cell-attachment hexapeptide (GRGESP) (18) (lanes 6-11), and into the lipid bilayer of liposomes. These properties lead us buffer plus the active GRGDSP peptide. (A) Silver staining. (B) to conclude that this protein is a membrane receptor capable Autoradiography. Lanes: 1, flowthrough; 2-18, consecutive frac- of mediating the adhesion of cells to vitronectin. tions (0.5 ml) of the eluate; 19, molecular mass markers (values at The results presented here show that the vitronectin right in kDa). Arrowhead in A and in B points to 115/125-kDa receptor differs from the fibronectin receptor (19) with regard doublet. to molecular weight and binding specificity. Furthermore, preliminary results indicate that antibodies raised against the peptide but not by the control peptide GRGESP (Table 1), vitronectin receptor do not react with the fibronectin receptor indicating that the GRGDSP-binding site of the 125/115-kDa and that these antibodies interfere with adhesion of cells to protein is involved in the binding of the liposomes to vitronectin but not to fibronectin (unpublished results). This

VN U

0 0 FN 200 k 100 ;t 3 a

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'/O LM *9S-no 9--AO-O=O~~~LMFN 10_._ VN 0.1 1 10 100 0.1 1 10 100 Protein in coating solution, ,ug/ml FIG. 3. Binding of liposomes to adhesive proteins. The 125/115-kDa protein (Left) or the fibronectin receptor (Right) was incorporated into phosphatidylcholine liposomes (19, 27), and the binding of the liposomes to microtiter wells coated with various concentrations of vitronectin (VN), fibronectin (FN), or laminin (LM) was assayed. The binding assay was performed precisely as described (19). The amount of phosphatidylcholine (PtdCho) bound to the wells was calculated from the specific activity of the 3H-labeled PtdCho in the liposomes. Downloaded by guest on September 28, 2021 Cell Biology: Pytela et al. Proc. Natl. Acad. Sci. USA 82 (1985) 5769 Table 2. Binding of receptor liposomes to various substrata that additional reinforcing interactions between fibronectin % maximal binding* of and its receptor are required for direct adhesion to occur, liposomes made with even though some interaction with the peptide is clearly apparent from the inhibition results. 125/115-kDa Fibronectin Since the Arg-Gly-Asp-containing peptides interact with Microtiter-well coating protein receptor more than one adhesion receptor, in vivo inhibition experiments Vitronectin 100 0 using these peptides (31) should be interpreted carefully. Like- Fibronectin 0 100 wise, that only the liposomes containing the vitronectin recep- 120-kDa fibronectin tor attached to the immobilized peptides (see Table 2) indicates fragmentt 0 65 that the attachment of cells to these peptides takes place 30-residue synthetic primarily through the vitronectin receptor. cell-attachment peptidet§ 24 0 The recognition mechanism involving the Arg-Gly-Asp GRGDSP(C)§ 80 0 sequence appears to be an ancient one. The bacteriophage X GRGESP(C)§ 0 0 receptor ofEscherichia coli has a five-residue homology with fibronectin including the Arg-Gly-Asp sequence (32). The X *The binding of the 125/115-kDa-protein liposomes to vitronectin mamma- and the binding of fibronectin-receptor liposomes to fibronectin receptor functions as a cell attachment protein for were set at 100%, and binding to other substrata is given as percent lian cells in vitro (unpublished results), and mutations near its of this maximal binding. For the 125/115-kDa-protein liposomes, Arg-Gly-Asp sequence are known to abolish the binding of this was 150 ng of phosphatidylcholine per well, and for the the X phage to the receptor (32). Discoidin, a Dictyostelium fibronectin receptor, 80 ng per well. discoideum protein believed to be involved in the aggregation tRef. 22. of these organisms when they form a fruiting body, also tPeptide IV in ref. 16. contains an Arg-Gly-Asp sequence (33), and it has recently §A cysteine residue (C) was added to these peptides for coupling to been shown that synthetic peptides containing this sequence the substrate, as described (17). inhibit Dictyostelium aggregation (21). In mammalian spe- cies, the Arg-Gly-Asp sequence may also be important for the lends further support to the idea that the fibronectin and function offibrinogen, because Arg-Gly-Asp-containing pep- vitronectin receptors are separate entities at the cell surface. tides inhibit the binding offibrinogen to platelets and prevent The specificities of the vitronectin and fibronectin recep- platelet aggregation (34, 35). Fibrinogen, however, does not tors, however distinct they may be, are nevertheless related promote the adhesion of fibroblasts (17), indicating that a in that both of them interact with the Arg-Gly-Asp sequence platelet-specific fibrinogen receptor may exist. from the cell-attachment domain of fibronectin. The attach- The results discussed above lead us to propose the exist- ment of cells to fibronectin and vitronectin is inhibited by ence of a family of receptors each recognizing the Arg-Gly- peptides containing this sequence (17, 18, 20, 29), as is the Asp sequence within the context of individual proteins. The attachment of liposomes containing the receptors for fibro- receptors for each of these proteins may or may not cross- nectin (19) and vitronectin (this paper). Surprisingly, how- react with one or more of the other proteins containing the ever, even though the peptide is derived from fibronectin, the Arg-Gly-Asp sequence. vitronectin receptor interacted with the peptides more The so-called "area code" hypothesis postulates a series of strongly than did the fibronectin receptor: only the vitronec- cell surface recognition molecules governing differentiation tin receptor bound to GRGDSP-Sepharose and only lipo- and migration of cells during development (36). Perhaps as somes containing the vitronectin receptor attached to plastic extracellular proteins evolved to fulfill the unique structural coated with the hexapeptide GRGDSP, whereas the fibro- requirements of advanced life forms, a set of cell surface nectin receptor lacked these activities. Moreover, the binding receptors co-evolved that each recognize the Arg-Gly-Asp of the vitronectin-receptor liposomes to vitronectin was sequence in a subtly new polypeptide environment, giving completely inhibited by 1.5 mM GRGDSP peptide, whereas rise to what appears to be one family of cell surface the binding of the fibronectin-receptor liposomes to fibro- recognition molecules. nectin is only partially inhibited at this concentration of the peptide (19). We thank Dr. Eva Engvall for a gift of laminin. This work was Based upon these results, one might almost conclude that supported by Grants P01 CA28896 (to E.R.) and CA38352 (to the Arg-Gly-Asp-containing peptides could have two differ- M.D.P.) and Cancer Center Support Grant CA30199, all from the National Cancer Institute. R.P. is the recipient of Fogarty Inter- ent specificities, one that makes them capable of interfering national Fellowship 1FO5 TWO 3303-01 from the Department of with the function of the fibronectin receptor and another that Health and Human Services. enables them to interact with the vitronectin receptor. It is, however, highly unlikely that two different specificities 1. Edelman, G. M. (1983) Science 219, 450-457. would be involved, because the same modifications in the 2. Ekblom, P., Alitalo, K., Vaheri, A., Timpl, R. & Sax~n, L. peptide structure eliminate interaction with both receptors. (1980) Proc. Natl. Acad. Sci. USA 77, 485-489. The smallest modifications that render the Arg-Gly-Asp 3. Yoshida, C. & Takeichi, M. (1982) Cell 28, 217-224. sequence incapable ofinteracting with either receptor are the 4. Grinnell, F. (1978) Int. Rev. Cytol. 53, 65-144. glutamic for aspartic acid and alanine for glycine substitu- 5. Kleinman, H. K., Klebe, R. J. & Martin, G. R. (1981) J. Cell tions. Since these changes only add a single methylene group Biol. 88, 473-485. 6. Hynes, R. 0. & Yamada, K. M. (1982) J. Cell Biol. 95, to the peptide and preserve the general structural features of 369-377. the peptide (such as charge relationships), it is likely that the 7. Ruoslahti, E., Engvall, E. & Hayman, E. G. (1981) Collagen peptides interact with the two receptors through the same Rel. Res. 1, 95-128. mechanism. It may be that the short, synthetic peptides 8. Rubin, K., Hook, M., Obrink, B. & Timpl, R. (1981) Cell 24, assume a conformation that more closely mimics the cell 463-470. attachment site in vitronectin than the one in fibronectin, 9. Terranova, V. P., Rohrbach, D. H. & Martin, G. R. (1980) resulting in a stronger interaction with the vitronectin recep- Cell 22, 719-726. tor. Analysis of shared pentapeptide sequences in proteins of 10. Carlsson, R., Engvall, E., Freeman, A. & Ruoslahti, E. known three-dimensional structure has shown that short (1981)Proc. Natl. Acad. Sci. USA 78, 2403-2406. amino acid sequences can assume entirely different confor- 11. Crouchman, J. R., Hook, M., Rees, D. A. & Timpl, R. (1983) mations in different proteins (30). However, it may also be J. Cell Biol. 96, 117-183. Downloaded by guest on September 28, 2021 5770 Cell Biology: Pytela et al. Proc. Natl. Acad. Sci. USA 82 (1985)

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