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, migra- tion, 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]phos- phatidylcholine (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