David K Moscatelloand Renato V Iozzo 1 Introduction

The control of cell proliferation depends on the interactions between growth factors and their specific receptor-activated signaling pathways. It is well accepted that the local extracellular matrix can modulate cellular responses to a given signal in several ways, such as by modulating the affinity of the ligand for its cognate receptor (1), by binding and limiting availability of a growth factor (1-3), or by influencing proteolytic processing and internalization (3). However, it has only recently been shown that "structural" components of the extracellular matrix can interact directly with, and activate, receptor tyrosine kinases (RTKs). This was first shown by Vogel et al. and Shrivastava et al., who demonstrated that the "orphan" receptor tyrosine kinases DDR1 and DDR2 in fact bind fibrillar collagen (4,5). This binding required the native triple-helical structure of collagen and showed much slower kinetics than observed with other ligand-receptor interactions (5). Decorin (6-8), a member of a family of small leucine-rich proteoglycans (3), binds to fibrillar collagen and is an important regulator of matrix assembly (9-11). Decorin content is elevated in the tumor stroma of colon cancer (11), and ectopic expression of decorin inhibits cell growth (11-13). The growth-suppressive properties of decorin are independent of p53 or retinoblastoma proteins but require functional p21 protein (Waf1/Cip1/Sdi1) (13-16).

We recently reported that decorin activated the epidermal growth factor receptor (EGFR) in A431 squamous carcinoma cells and other transformed cell lines. This signaling was mediated by the protein core of decorin and induced MAP kinase activation and a protracted upregulation of endogenous p21, thereby leading to growth suppression (17). This activation occurred as a result of a direct interaction of decorin with the EGF receptor (17,18). However, even "small" proteoglycans such as decorin (~100 kDa, with a ~42-kDa core protein) are quite large in comparison with most ligands of receptor tyrosine kinases, which are typically 6-25 kDa, a fact that complicates analysis of crosslinking studies. Thus, in combination with their relatively

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

promiscuous binding properties, establishing a direct interaction of proteoglycans with receptor tyrosine kinases requires a number of methodological approaches. Examples of a few such techniques are presented here, but the reader is encouraged to consider other chapters in this volume as well, since other approaches may be useful in the context of different ligand-receptor systems. While the sample protocols presented all involve the EGFR and decorin, the same approaches should prove useful for other receptor-proteoglycan interactions.

2. Materials

2.1. Cells, Culture Media, and Growth Factors

1. A431, HT-1080, and a wide variety of other human tumor cell lines can be obtained from the American Type Culture Collection (Rockville, MD), as can CHO and NIH-3T3 cells. The latter two cell lines are widely used for transfection and overexpression of proteins of interest.

2. Fetal bovine serum (FBS) is used at 10% (v/v) to culture A431 and most human tumor cell lines. Ten percent calf serum (CS) is used to grow NIH-3T3 cells and transfectants. Both can be obtained from Hyclone (Logan, UT) or Life Technologies (Rockville, MD). A431 and 3T3 cells are grown in Dulbecco's modified Eagle medium (Life Technologies or Mediatech) (Herndon, VA). RPMI 1640, D-PBS, epidermal growth factor, and trypsin (0.25% for A431, 0.05% for 3T3) are all obtained from Life Technologies.

3. Conditioned media for soluble receptor ectodomain or recombinant proteoglycan production are readily concentrated using Amicon Ultrafree-15 centrifugal filter units with Biomax-50 membranes (50,000-dalton nominal molecular weight limit) and filter-sterilized with 0.45-^m pore vacuum filter units (Millipore, Bedford, MA).

2.2 Antibodies and Biochemical Reagents

1. Anti-EGFR and anti-phosphotyrosine antibodies can be obtained from Promega (Madison, WI) and Upstate Biotechnology (Lake Placid, NY), respectively. Antibodies to a variety of EGFR epitopes are also available from Calbiochem-Novabiochem Corp. (San Diego, CA).

2. Bovine serum albumin (BSA, Cohn fraction V) and all other reagents not specified are from Sigma Chemical (St. Louis, MO).

a. For blocking cells, a 0.2% BSA (w/v) solution in RPMI 1640 is filter sterilized and stored at 4°C.

b. Membranes are blocked with 2% BSA in TBST (TBS [100 mMTris-HCl, pH 7.5, 150 mM NaCl] plus 0.1% Tween 20) or 5% nonfat dry milk in TBST.

c. Kits for labeling by the Iodo-Gen method are available from Pierce Chemical (Rockford, IL). [125I]NaI is from Amersham (Arlington Heights, IL).

d. Active, affinity-purified EGFR was purchased from Sigma Chemical (cat. no. E2645). [y-32P] ATP (6000 Ci/mmol), enhanced chemiluminescence (ECL) reagents and Hybond ECL membranes were from Amersham. Nitrocellulose membranes from Schleicher & Schuell (Keene, NH) also work well.

e. Immulon 4HXB wells are from Dynex Technologies (Chantilly, VA).

f. The membrane-impermeable crosslinker bis[sulfosuccinimidyl]-suberate (BS3) is from Pierce. Nickel-nitrilo-triacetic acid (Ni-NTA) columns for purification of His6-tagged recombinant proteins are from Qiagen (Valencia, CA).

3. PBS/TDS: 10 mMNa2HPO4, 150 mMNaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.02% sodium azide, 0.004% sodium fluoride, and

1 mM sodium orthovanadate, pH 7.25. The stock is stored at 4°C, and phenylmethylsulfonyl fluoride (made as a 100x stock in isopropanol and stored at -20°C) is added to 100 ^g/mL immediately before use.

4. In vitro reactions with EGFR kinase are performed in 20 mM HEPES, pH 7.4, 2 mM MnCl2, 10 mM^-nitrophenyl phosphate, 40 mM Na3VO4, 0.01% BSA with 15 mM ATP (kinase buffer). For blocking wells in radioligand binding assays, 0.1% BSA in TBS with

3. Methods

3.1. Binding Competition with Known Receptor Ligands

1. Growth of cells. Cells are seeded into 24-well (16-mm) plates and grown for 2 d or until just confluent. Sufficient wells must be plated for triplicate determinations of all conditions. A431 cells are commonly used to analyze binding to the EGFR.

2. Recombinant human decorin (see Note 1) is labeled to high specific activity (~8 x 106 cpm/mg) with [125I]NaI (Amersham) by the Iodo-Gen method (Pierce Chemical).

3. The cells are washed twice with D-PBS, then blocked with 1 mL of RPMI-1640 containing 0.2% BSA at 4°C or on ice for 1 h (see Note 2).

4. The blocking medium is removed, and the cells are then incubated with 0.5 mL of 0.2% BSA/ RPMI-1640 containing ca. 200 ng (1.5 x 106 cpm) 125I-decorin with or without the appropriate concentrations of specific ligand. In this example, EGF is added at concentrations from 10 to 300 ng/mL. A 100-fold excess of unlabeled test ligand is added to triplicate wells to correct for background binding.

5. The binding media are removed, and the wells are washed three times with cold PBS. The cells are then dissolved in 1 mL of 1 M NaOH and the radioactivity measured in a gamma counter. The means ± SD of the triplicate determinations are then calculated.

3.2. Chemical Crosslinking to Receptors

1. A431 cells (or other cells of interest) are plated in 6-well (35-mm) plates and grown until nearly confluent. The cells are incubated in serum-free medium (e.g., DMEM only) for 24-36 h.

2. The cells are washed twice with D-PBS, then blocked with 1 mL of RPMI-1640 containing 0.2% BSA at 4°C or on ice for 1 h.

3. The cells are incubated with decorin (DCN), decorin protein core (ADCN)(100 ^g/mL), or EGF (16 nM), or combinations thereof, at 4°C or on ice for 1 h.

4. The cells are washed three times with 2 mL of cold PBS per well and incubated for 10 min at room temperature in PBS with 15 mM crosslinker BS3. If the lysates are to be subjected to immunoprecipitation, the cells are scraped into 0.5 mL/well ice-cold lysis buffer, centri-fuged at 12,000g in microcentrifuge tubes for 10 min at 4°C, and the supernatants are transferred to clean tubes. If the lysates are only to be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the cells may be lysed directly in SDS sample buffer. The cell lysates are separated by SDS-PAGE in thin (0.75-mm), 3-15% acrylamide gels, transferred to nitrocellulose membranes, and subjected to Western immunoblotting with anti-EGFR antibody (18).

5. After exposing the blot to film, the autoradiogram is analyzed for the presence of receptor monomers and dimers; the latter should be present only in lanes with crosslinker. In the case of studies involving the EGFR, the monomeric receptor migrates at an Mr of 170 kDa, and the dimers at an Mr of 340 kDa. Slower-migrating species may also be observed if the ligand is also crosslinked; in the present instance, a single decorin molecule bound to a EGFR dimer would be expected to migrate at an Mr of ~440 kDa, and two decorin molecules per dimer would be expected to migrate at an Mr of ~540 kDa (see Note 3).

3.3. Interaction with Soluble Receptor Ectodomain

1. A431 cells synthesize a secreted form of EGFR of ~105 kDa lacking the transmembrane and intracytoplasmic domains (19). A431 cells are grown to near confluence in standard medium, washed twice with D-PBS, and incubated with serum-free medium for 24-48 h (see Note 4).

2. Medium conditioned by confluent A431 cells is concentrated by centrifugation in Ultrafree-15 (50,000-nmwl) units at 4°C according to the manufacturer's protocol. The conditioned medium is then filter-sterilized (0.45-^m-pore filters) or centrifuged at high speed to remove insoluble material. NaN3 (0.02% final concentration) may be added to inhibit microbial growth. Such preparations are ideally used fresh, but may be stored for a few days at 4°C, or mixed with an equal volume of glycerol and stored at -20°C.

3. Serial dilutions of BSA, decorin or its protein core are slot- or dot-blotted onto nitrocellulose membranes using a vacuum manifold and blocked overnight at 4°C (or for 1 h at room temperature) with 5% FBS and 5% nonfat milk (see Note 5).

4. The blots are washed several times in TBST and incubated with the serum-free medium conditioned by the A431 cells. The blots are again washed three times, then incubated with gentle agitation using an antibody against the ectodomain of the human EGFR (e.g., anti-LEEKK of the human EGFR N-terminal sequence, affinity purified on peptide linked to Sepharose) at 1 ^g/mL in BSA/TBST for 1-2 h at room temperature. The antibody used should not interfere with the binding, in this case because it recognizes the N-terminal end of the EGF receptor, distant from the known ligand-binding site. It is desirable, however, to use an antibody that does recognize the ligand-binding domain as an additional control (e.g., monoclonal antibody 225 raised against the EGF-binding domain of the EGFR, ref. 20).

5. The membranes are washed three times with TBST and incubated with the appropriate secondary antibody (against either rabbit polyclonal or mouse monoclonal antibody) for 1 h at room temperature. Alternatively, radiolabeled secondary antibodies may be used.

6. Finally, the membranes are incubated with anti-rabbit or anti-mouse IgG coupled to horseradish peroxidase (1/5000) in TBS-T, washed three times, and incubated with enhanced chemiluminescence reagent. Multiple exposures are performed to guarantee linearity, and the intensities of the bands are quantitated by laser scanning densitometry.

3.4. In Vitro Phosphorylation of the Receptor Tyrosine Kinase

1. Constant amounts (~300 ng) of immunopurified EGFR are incubated with kinase buffer alone or containing various concentrations (5-20 ^g) of decorin, decorin protein core, or collagen type I. A control tube should include 100 ng of EGF.

2. After 15 min of incubation in kinase buffer, 1 ^Ci [y-32P]ATP and 0.2% NP40 are added in a final volume of 60 ^L. The mixtures are incubated for an additional 10 min, and the reactions are terminated by boiling in SDS sample buffer.

3. The samples are separated by SDS-PAGE, and the gel is either dried, or the proteins are transferred to nitrocellulose. The latter technique enables one to use immunoblotting to confirm the identities of the labeled proteins. Phosphorylated proteins are visualized by autoradiography. Control samples omitting either [y-32P]ATP or EGFR should show no activity (see Fig. 1).

EGFR

Protein C(we

Fig. 1. In vitro phosphorylation assay using immunopurified EGFR and decorin, its protein core, or collagen. Constant amounts (~300 ng) of immunopurified EGFR were preincubated with buffer alone (lane 1) or containing decorin (lanes 2-4; at 5, 10, and 20 ^g, respectively), decorin core protein (lanes 5-7; at 5, 10, and 20 ^g, respectively), or collagen (lanes 8-10; at 5, 10, and 20 ^g, respectively). After 15 min in kinase buffer, 1 ^Ci of [y-32P]ATP and 0.2% Nonidet P-40 were added. The mixtures were incubated for an additional 10 min, stopped by boiling in SDS buffer, and analyzed by SDS-PAGE and autoradiography. Note the strong EGFR autophosphorylation induced by decorin and decorin core protein relative to the collagen control.

3.5. Interaction of Proteoglycan with Purified Receptor Kinase

1. Column protocol: Recombinant decorin with an N-terminal tag of six histidine residues (6 His) allows for a rapid and efficient purification via Ni-NTA affinity chromatography (21).

2. Purified EGFR is phosphorylated to a high specific activity using the EGF control reaction in vitro phosphorylation procedure described in Subheading 3.4., steps 1 and 2, except that the reactions are not boiled in sample buffer and labeled receptor is separated from unincorporated label by Sephadex G-50 chromatography (Roche Quick-spin columns).

3. Constant amounts of 32P-labeled EGFR are incubated with increasing concentrations of decorin, decorin core protein, or control proteins for 30 min at 4°C under gentle agitation. The spin columns are equilibrated with three column volumes of binding buffer (300 mM NaH2PO4, pH 8.0, 300 mM NaCl), and then the samples are applied and spun at 750 g for 5 min.

4. Following two consecutive washes, the bound decorin/EGFR complexes are eluted with buffer containing 250 mM imidazole. Aliquots of the fractions are counted in a scintillation counter and the remainder are analyzed by SDS-PAGE and autoradiography.

5. Solid-phase binding protocol: Immulon 4HXB wells are coated with 1 ^g (~22 pmol) of recombinant decorin in 100 ^L of PBS for 2 h at 37°C or overnight at 4°C. The wells are washed three times with PBS and blocked with 0.1% BSA in TBS supplemented with 2mM CaCl2, 2 mM MgCl2, and 0.02% NaN3 (blocking buffer) for 1 h.

6. Purified EGFR is labeled as described in step 2, 0.1-10 pmol 32P-EGFR is added in blocking buffer, and the wells are incubated under gentle shaking (60 rpm) for 4-14 h at 4°C.

Background binding is corrected for by incubation with at least 100-fold molar excess unlabeled EGFR. All samples should be done in triplicate. After incubation the wells are washed three times with ice-cold TBST and measured using a scintillation counter. Data are analyzed by Scatchard analysis, e.g., using the Ligand program (18).

4. Notes

1. It is imperative that the proteoglycans in question be purified by techniques that permit retention of a "native" conformation (21). Many commercially available extracellular matrix proteins and proteoglycans, particularly those from tissues such as skin or tendon, are prepared by harsh extraction methods that result in denatured products. Not surprisingly, such preparations have greatly reduced or no biological activity (18,22), and of course are unsuitable for use in studies of signaling interactions. Recombinant or endogenous materials isolated from cell cultures are generally suitable, as they can be purified by relatively gentle procedures (see Chaps. 1, 4, 20, 21; and ref. 21 for relevant protocols). Wherever possible, it is also desirable to use a closely related proteoglycan as a control for nonspecific binding. For example, we use biglycan (17,21) as a control for decorin in experiments with EGFR; biglycan does not bind or activate the EGFR (17,22). We also use a deglycosylated proteoglycan, or mutated recombinant protein lacking the glycosaminoglycan chain (17,21,23), to ascertain the importance of the glycosaminogly-can chain in the interaction.

2. Only purified proteins such as bovine serum albumin should be used as blocking agents in binding experiments. Gelatin cannot be used, as it is composed of collagen, with which many proteoglycans can interact. Other commonly used blocking agents such as nonfat milk or sera contain growth factors or matrix components that may interact with either the receptor or proteoglycan of interest, respectively.

3. Very large complexes (>400 kDa) do not penetrate standard polyacrylamide gels, and are not efficiently transferred to membranes for blotting. A possible alternative approach would be the use of cleavable crosslinkers such as 3,3'-dithiobis[sulfosuccinimidyl] propionate (Pierce). Crosslinked receptor-proteoglycan complexes are immunoprecipitated from lysates, the cleaving reagent is added, the samples are boiled in SDS sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting against both the receptor and the proteoglycan (or labeled ligand may be used).

4. Alternatively, constructs encoding ectodomains of other receptors of interest may be trans-fected into NIH-3T3 cells by standard methods. Conditioned media would be produced and collected in the same fashion as for EGFR ectodomain from A431, although the kinetics of expression would have to be determined empirically. Expression of the soluble receptor would be confirmed by immunoblotting, and an epitope tag such as 6 « His would provide a convenient "handle" for purification and isolation of receptor-ligand complexes.

5. Alternatively, the reciprocal experiment may be performed; that is, the soluble receptor preparation may be slot-blotted and incubated with proteoglycan solution. The blots would then be probed with antibody to the proteoglycan, or labeled proteoglycan could be used.

References

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2. Somasundaram, R. and Schuppan, D. (1996) Type I, II, III, IV, V, and VI collagens serve as extracelular ligands for the isoforms of platelet-derived growth factor (AA, BB, and AB). J. Biol. Chem. 271(43), 26884-26891.

3. Iozzo, R. V. (1997) The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth. Crit. Rev. Biochem. Mol. Biol. 32, 141-174.

4. Vogel, W., Gish, G. D., Alves, F., and Pawson T. (1997) The discoidin domain receptor tyrosine kinases are activated by collagen. Mol. Cell. 1,13-23, 1997.

5. Shrivastava, A., Radziejewski, C., Campbell, E., Kovac, L., McGlynn, M., Ryan, T. E., Davis, S., Goldfarb, M. P., Glass, D. J., Lemke, G., and Yancopoulos G. D. (1997) An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors. Mol. Cell. 1, 25-34.

6. Krusius, T. and Ruoslahti, E. (1986) Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc. Natl. Acad. Sci. (USA). 83, 7683-7687.

7. Day, A. A., McQuillan, C. I., Termine, J. D., and Young, M. R. (1987) Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone. Biochem. J. 248, 801-805.

8. Fisher, L. W., Termine, J. D., and Young, M. F. (1989) Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J. Biol. Chem. 264, 4571-4576.

9. Toole, B. P. and Lowther, D. A. (1968) The effect of chondroitin sulphate-protein in the formation of collagen fibrils in vitro. Biochem. J. 109, 857-866.

10. Vogel, K. G., Paulsson, M., and Heinegard, D. (1984) Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem. J. 223, 587-597.

11. Adany, R., Heimer, R., Caterson, B., Sorrell, J. M., and Iozzo. R. V. (1990) Altered expression of chondroitin sulfate proteoglycan in the stroma of human colon carcinoma. Hypomethylation of PG-40 gene correlates with increased PG-40 content and mRNA levels. J. Biol. Chem. 265, 11389-11396.

12. Santra, M., Skorski, T., Calabretta, B., Lattime, E. C., and Iozzo, R. V. (1995) De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells. Proc. Natl. Acad. Sci. (USA) 92, 7016-7020.

13. Santra, M., Mann, D. M., Mercer, E. W., Skorski, T., Calabretta, B., and Iozzo, R. V. (1997) Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases. J. Clin. Invest. 100, 149-157.

14. De Luca, A., Santra, M., Baldi, A., Giordano, A., and Iozzo, R. V. (1996) Decorin-induced growth suppression is associated with upregulation of p21, an inhibitor of cyclin-depen-dent kinases. J. Biol. Chem. 271, 18961-18965.

15. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 75, 805-816.

16. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell. 75, 817-825.

17. Moscatello, D. K., Santra, M., Mann, D. M., McQuillan, D. J., Wong, A. J., and Iozzo, R. V. (1998) Decorin suppresses tumor cell growth by activating the epidermal growth factor receptor. J. Clin. Invest. 101, 406-412.

18. Iozzo, R. V., Moscatello, D. K., McQuillan, D. J., and Eichstetter, I. (1999) Decorin is a biological ligand for the epidermal growth factor receptor. J. Biol. Chem. 274, 4489-4492.

19. Weber, W., Gill, G. G., and Spiess, J. (1984) Production of an epidermal growth factor receptor-related protein. Science 224, 294-297.

20. Fan, Z., Lu, Y., Wu, X., and Mendelsohn, J. (1994) Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squa-mous carcinoma cells J. Biol. Chem. 269, 27595-27602.

21. Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) Recombinant decorin glycoforms. Purification and structure. J. Biol. Chem. 271, 19578-19584.

22. Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) Eukary-otic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity. J. Biol. Chem. 271, 19571-19577.

23. Mann, D. M., Yamaguchi, Y., Bourdon, M. A., and Ruoslahti, E. (1990) Analysis of gly-cosaminoglycan substitution in decorin by site-directed mutagenesis. J. Biol. Chem. 265, 5317-5323.

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