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such a procedure is, however, that it is difficult to elute the receptor from these columns with retained activity, since the kinase activity of the receptor is susceptible to low pH and to chaotropic agents. This chapter describes the purification to homogeneity of nanomole quantities of functionally active PDGF/3 receptors, from a readily available source. The method should be particularly useful for the preparation of receptor for kinetic experiments and for experiments aimed at characterizing the substrate specificity of the receptor kinase. Acknowledgments We thank Ingegtird Schiller for valuable help in the preparation of this manuscript.

[31] Affinity P u r i f i c a t i o n of A c t i v e E p i d e r m a l G r o w t h F a c t o r Receptor Using Monoclonal Antibodies

By JUSTIN HSUAN and PNINA YAISH Introduction

Epidermal Growth Factor Receptor The cell surface receptor for epidermal growth factor (EGF) is a transmembrane glycoprotein of apparent molecular weight commonly between 150,000 and 170,000 as estimated by SDS-PAGE. In fact this receptor is able to bind other ligands competitively with EGF, notably including transforming growth factor a, with similar affinity. The receptor is derived from a single gene but structural heterogeneity is derived from differential glycosylation and phosphorylation of the extracellular and intracellular regions, respectively. The former is thought to comprise at least four domains by examination of the primary structure: two homologous, globular (L) domains that may be directly involved in binding ligand and two homologous, cysteine-rich (S) domains, arranged as L1, S1, L2, $2. This region is followed by a single transmembrane helix. The intracellular region comprises a regulatory juxtamembrane domain, a protein-tyrosine kinase domain, and a putatively flexible autophosphorylation domain at the C terminus.l The EGF receptor is expressed in a wide variety of mammalian cell 1 j. j. Hsuan, G. Panayotou, and M. D. Waterfield, Prog. Growth FactorRes. 1, 23 (1989).

METHODS IN ENZYMOLOGY, VOL. 200

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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types, with the important exception of hemopoietic lineages, and structurally similar proteins have been found in avian and insect species.

Purification Schemes Although much has been learned from the many independent studies that have addressed key issues, many important questions still remain unanswered regarding the structure, regulation, and activity of the EGF receptor, such as the tertiary structure of the molecule, the definition of the ligand-binding site, the mechanism of receptor activation by ligand, and the identity of important cellular substrates. As one approach to achieving these ends, a source of pure and active receptor has been established using monoclonal antibody affinity to purify receptor from the A431 human epidermal carcinoma cell line. 2 This particular approach has the great advantage over those purification schemes that use ligand affinity, conventional chromatography, or certain other types of antibody affinity in that it allows a relatively rapid isolation of receptor using extremely mild conditions. The yield is sufficient for many types of functional analysis and we are currently exploring the possibility of scaling the preparation up to a level that allows structural studies. This type of purification has been achieved using an anti-carbohydrate antibody (9A) that is directed against the blood group A antigen, present on the EGF receptor of A431 cells. The receptor can be eluted from this monoclonal antibody (MAb) using competition with a simple, dialyzable monosaccharide. In contrast, while the use of anti-polypeptide antibodies or ligand affinity frequently gives higher yields, more severe physical conditions are required for the recovery of receptor. Anti-phosphotyrosine antibody affinity has been widely used for the analysis and purification of tyrosine kinases under mild conditions, but this method may be inefficient if little tyrosine phosphosphorylation is present on the receptor or if many other proteins are phosphorylated on tyrosine residues. It may also preferentially select highly phosphorylated receptor subpopulations.

Assay Principle Saturation ligand binding is routinely exploited to assay EGF receptor in membranes and in detergent solution. In the latter case a high-affinity monoclonal antibody is used to separate bound from free radiolabeled 2 p. j. Parker, S. Young, W. J. Gullick, E. L. V. Mayes, P. Bennet, and M. D. Waterfield, J. Biol. Chem. 259, 9906 (1984).

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ligand. This procedure has the disadvantage that it does not determine the ligand-dependent kinase activity of the receptor. This is an important point as the kinase activity is first a distinct and second a more labile property relative to ligand-binding ability. Accordingly, an assay for peptide phosphorylation activity is also described here. In contrast to the peptide kinase assay (below), a receptor phosphorylation assay is relatively specific to EGF receptor kinase activity. While this is often used with SDS-PAGE for the detection of active receptor, autophosphorylation does not easily lend itself to quantitative kinetic analysis. Polypeptide phosphorylation is an important complementary assay to peptide phosphorylation. Common substrates include synthetic poly(glutamic acid, tyrosine) polymers and various glycolytic enzymes, which are generally separated from receptor by SDS-PAGE. Conditions for this assay have been described elsewhere) Greater specificity of the peptide kinase assay can be obtained using receptor immunoprecipitated on monoclonal antibody EGFR1 and protein A-Sepharose after the incubation with EGF, essentially as described below.

Procedures 125I-LabeledEpidermal Growth Factor Radioimmunoassay. 4Duplicate aliquots for assay of between 10 and 40/zl are incubated with a saturating concentration of 0.2/~M 125I-labeled EGF (Amersham, Aylesbury, UK) of known specific activity and 67 nM monoclonal antibody EGFR15 (2/zg/ sample; Amersham) and made up to a final volume of 200/xl with 10 mM sodium phosphate, pH 7.4, containing 0.2% (v/v) Triton X-100 and 150 mM NaC1 (Triton buffer). After tumbling for 1 hr at room temperature, 20 /zl of a 50% (v/v) mixture of protein A-Sepharose (Pharmacia, Piscataway, N J) in Triton buffer is added. After a further 30-min tumbling at room temperature, the pellet is washed with three 1-ml aliquots of Triton buffer at 4° and then counted in a y counter. Background, nonspecific binding is estimated by the use of normal mouse serum in place of EGFRI. Peptide Kinase Assay. Samples containing EGF receptor are incubated in 25 mM HEPES, pH 7.4, containing 10 mM M g C I 2 , 0.1 mM N a a V O 4 , 0.5 mM EGTA either with or without 150 nM EGF for 30 min at 20° in a final volume of about 100/xl. Samples are transferred to 30° and [y-a2p]ATP 3 N. Reiss, H. Kanety, and J. Schlessinger, Biochem. J. 239, 691 (1986). 4 W. J. Gullick, D. J. H. Downward, J. J. Marsden, and M. D. Waterfield, Anal. Biochem. 141, 253 (1984). 5 M. D. Waterfield, E. L. Mayes, P. Stroobant, P. L. P. Bennet, S. Young, P. N. Goodfellow, G. S. Banting, and B. Ozanne, J. Cell. Biochem. 20, 149 (1982).

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(specific activity routinely 5000-!0,000 cpm/pmol; Amersham) and peptide RR-src (RRLIEDAEYAARG, synthesized on an Applied Biosystems 430A automated peptide synthesizer using Fmoc chemistry, or available from Sigma, St. Louis, MO) are then added to final concentrations of 100 /xM and 1 mM, respectively. Samples of 10 /zl are removed at 2-min intervals, rapidly spotted onto - 2 - c m 2pieces of P81 cellulose filters (Whatman, Clifton, N J), and immediately quenched in 30% (v/v) acetic acid, 0.5% (v/v) phosphoric acid with gentle stirring. After two further acid washes of 10 min each the filters are washed in ethanol for 10 min and dried before scintillation counting. Samples are assayed in duplicate and the background is estimated by omission of the peptide substrate. It is possible to substitute other peptide substrates for RR-src, such as angiotensin II or peptides derived from the autophosphorylation sites of the EGF receptor. 6 This method has been successfully used with immunoprecipitates (it is important to perform the incubation with ligand prior to immunoprecipitation), purified receptor in the presence of 0.1% (v/v) Triton X-100, and intact membrane preparations. Units. The EGF receptor is thought to stoichiometrically bind a single molecule of EGF, hence the results of ligand-binding assays are expressed as either moles or moles/liter. The unit of kinase activity is commonly expressed as picomoles of phosphate incorporated per minute (pmol/min), and specific activity is expressed as units per picomole of receptor (U/ pmol or pmol/min/pmol) measured at 30°.

Purification Antibody Preparation. The original generation of anti-receptor antibodies used intact A431 cells injected into BALB/c mice. Fusion was with X63 myeloma cells and screening was initially by binding to cultured A431 cells and a panel of I0 monoclonal hybridomas was isolated. Antibody 9A (IgG 3) was chosen for the purification scheme as it can be readily purified from ascites fluid using protein A affinity chromatography and has a relatively high binding affinity.2 Affinity Matrix Preparation. We routinely couple 30 mg 9A to 15 ml Affi-Gel 10 (Bio-Rad, Richmond, CA) in 0.1 M HEPES buffer, pH 7.5. The antibody is dialyzed against pH 7.5 buffer and the matrix is prepared as described by the manufacturer. After tumbling overnight and standing for a further 12 hr at 4°, free reactive groups are blocked by treatment with 15 ml 0.1 M ethanolamine/HCl, pH 8.0 for 1 hr. The product is washed in phosphate-buffered saline (PBSA) and stored in PBSA containing 0.02% azide at 4 °. Coupling efficiency is monitored by the Bradford assay. 6 j. Downward, M. D. Waterfield, and P. J. Parker, J. Biol. Chem. 260, 14538 (1985).

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Cell Culture. A431 cells (clone 7; Imperial Cancer Research Fund, London, England) are cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, penicillin, and streptomycin (GIBCO-BRL, Uxbridge, UK) at 37° in an atmosphere of saturating humidity and 10% (v/v) carbon dioxide. Stock cultures of cells are passaged I : 10 or 1 : 20 at confluence (about every 4 days). Cells should not be allowed to overgrow as they become difficult to detach and less viable. Fresh stocks are used every 2 months. We routinely grow 20 roller bottles for each preparation, 2 of which are used to seed the subsequent culture. Under these conditions cells grow to a density of - 3 . 5 x 105/cm2 and the doubling time is ~24 hr. Using lower concentrations of newborn calf serum or calf serum causes a decrease in the cell density to below 2 x 105/cm2 and an increase in the doubling time by up to 100%. Although there is no apparent change in receptor numbers per cell in different sera as revealed by fluorescence-activated cell sorting (FACS) analysis, largescale roller bottle cultures are best maintained in 10% (v/v) fetal calf serum. Initial attempts to grow A431 cells on microcarriers have met with only limited success due to extensive aggregation of stirred cultures. LysatePreparation. Confluent A431 cells (1-2 x 108 cells/roller bottle) are aspirated and washed twice with calcium- and magnesium-free PBSA containing 5 mM EDTA. Cells are then harvested by incubation at 37° in PBSA containing 5 mM EDTA, pelleted by centrifugation at 200 g for 10 min at 4 °, washed once with PBSA at 4°, and lysed in 50 mM HEPES, pH 7.5 containing 150 mM NaC1, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 25 mM benzamidine, 10% (v/v) glycerol, and 1% Triton X-100 (lysis buffer, 25 ml/roller bottle) with tumbling for 20 min at 4 °. The pH is immediately adjusted to 8.5 with 1 M NaOH in order to limit proteolysis. The lysate is cleared by centrifugation first at 3000 g for 10 min at 4°, and then at 100,000 g for 1 hr at 4°. At this stage the supernatant can be assayed for EGF receptor and stored at - 7 0 ° if necessary for several months with no apparent loss of activity. Monoclonal Antibody Binding. The lysate is tumbled with the 9A matrix at 4° for 2 hr, followed by washing in a filtration unit (Nalgene, Rochester, NY) with 50 ml PBSA containing 0.5 M NaCI, 1 mM EDTA, 0.1% Triton X-100 (buffer A), then 50 ml buffer A containing 0.25 M D-glUCOSe, and finally 500 ml buffer A, all at 4 °. Elution is performed by tumbling the matrix with 15 ml 50 mM HEPES, pH 7.5, containing 0.3 M N-acetylgalactosamine, 0.15 M NaCI, I mM EDTA, 0.05% (v/v) Triton X-100 (elution buffer) for 30 min at 4°. The eluted material is collected by filtration as above. A second volume of elution buffer is added and the procedure repeated. The eluates are pooled, immediately dialyzed against 50 mM HEPES, pH 7.5, containing 50% (v/v) glycerol, 1 mM dithiothreitol

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(DTT), 1 mM EDTA and stored at - 20°. Under these conditions the EGF receptor can be stored for several months with no significant loss of EGFdependent autophosphorylation. The matrix is washed and stored in PBSA containing 0.05% azide at 4°. Mono Q. At this point the preparation should be -70% pure as determined by SDS-PAGE and contain - 3 0 /.~g receptor/roller bottle used. Further purification and/or concentration may be required for many types of study and we have accordingly developed a simple fast protein liquid chromatography (FPLC) (Pharmacia, Uppsala, Sweden) ion-exchange step that is performed throughout at 4 °. The EGF receptor preparation is diluted with 3 vol of 50 mM Tris/HC1, pH 7.4 (4°) containing 1 mM 2-mercaptoethanol and 0.1% Triton X-100 (buffer A). This is applied to a Mono Q column (HR5/5) equilibrated with buffer A at 0.1 ml/min. After washing at 0.2 ml/min to constant absorbance with buffer A, receptor is eluted using a single step to 2 M NaC1 in buffer A. Poor recoveries were obtained using 0.4 M NaC1 washing and using gradient elution, probably due to charge heterogeneity of the receptor preparation. The elution is monitered by measuring the absorbance at 280 nm using buffer A in the reference cell to offset the high background absorbance. A clearer analysis can be obtained using Bradford reagent or by spiking the preparation with 32p-labeled receptor, which shows greater than 60% recovery in the peak fractions (Fig. 1). Fractions containing receptor are finally pooled and immediately dialyzed twice against 50 mM Tris/HCl, pH 7.4 (4°) containing 1 mM 2mercaptoethanol, 0.01% Triton X-100, and 50% (v/v) glycerol for 1 hr each. This further concentrates the preparation, decreases the salt concentration, and allows storage at - 2 0 ° without freezing. Comments on Purification We have found that the yield of receptor decreases with the number of preparations, probably due to loss of 9A viability. In order to improve the efficiency of the antibody affinity step, we have decreased the amount of reducing agent in the buffers and initial experiments have investigated the use of Protein A-Sepharose CL-4B (Pharmacia) in place of Affi-Gel 10. In this case antibody immobilization is only via the Fc region and the antibody can readily be removed and the matrix regenerated. The efficiency of this method is sensitive to lysis volume, EGF receptor concentration, and antibody concentration. 7 Optimal parameters have been determined only on relatively small cultures (150 cm 2) from which 7 p. Yaish, J. J. Hsuan, and M. D. Watertield, unpublished results (1989).

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0.12

0.i0

0.08

0.05

0.04

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0

o I-I

0.00

0.00

N

o

N



6

4b

6b

8'0

i00

Time (min) FIG. 1. Purification and concentration by ion-exchange FPLC. Affinity-purified EGF receptor (0.5 ml) in 50% glycerol buffer was diluted to 2 ml with buffer A (see text) and spiked with 32p-labeled EGF receptor. This was loaded onto a Mono Q FPLC column (HR5/ 5) at 0.1 ml/min. After 20 min the column was washed in buffer A for 20 rain at 0.2 ml/min and then eluted with 2 M NaC1 in buffer A. Fractions (1 min) were collected from 40 min and counted for Cerenkov radiation (solid line) and aliquots (50/zl) were analyzed for protein using the Bradford assay (dotted line). Peak fractions were essentially pure and active EGF receptor as determined by SDS-PAGE and EGF-dependent autophosphorylation.

400/zg MAb 9A can extract at least 70% of the total EGF receptor in 2 ml lysis buffer. The most significant factors that lower the efficiency are decreasing the lysis volume and decreasing the antibody concentration. This may be due to recognition of other factors containing the 9A epitope, such as glycolipids. Not only is the 9A epitope expressed by other cell components in addition to the EGF receptor, but there appears to be considerable variation in both the glycosylation s and expression of the receptor itself. 9 A relatively rapid method for selecting 9A-positive cells is to use FACS, in which cultured cells are suspended, incubated with 9A, and then with fluorescent-labeled second antibody before automated sorting according to fluorescence intensity. A similar method can be used to select against low EGF receptor-expressing mutants using sorting with, for example, MAb EGFRI 5 or fluoresceinated EGF. 9

8 L. H. K. Defize, D. J. Arndt-Jovin, T. M. Jovin, J. Boonstra, J. Meisenhelder, T. Hunter, H. T. de Hey, and S. W. de Laat, J. Cell Biol. 107, 939 (1988). 9 R. C. Chatelier, R. G. Ashcroft, C. J. Lloyd, E. C. Nice, R. H. Whitehead, W. H. Sawyer, and A. W. Burgess, EMBO J. 5, 1181 (1986).

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Properties The K m value of the EGF receptor for ATP measured at 0° is lower for autophosphorylation than for substrate phosphorylation: the former has been estimated at 0.21-0.25 /zM, 1°,1~ although values up to 3 ttM have been reported, ~2 while the latter has been estimated at 2 - 7 / z M ) 2,~3 The variation in these data may be a consequence of factors such as different assay buffer compositions and receptor states. Numerous factors are known to affect receptor kinase activity and these are briefly summarized below. The maximum rate of ligand-stimulated autophosphorylation is similar in the presence of either Mn 2÷ or Mg 2+ , but the basal rate of autophosphorylation is two- to threefold higher in the presence of Mn 2÷ . Furthermore Mn 2÷ stimulates peptide phosphorylation in the presence of MgATP, which has been interpreted in terms of two metal ion-binding sites) 4 The dissociation constants for the binding of both EGF and TGFa to the cellular receptor are approximately 0.1-I .0 nM for high-affinity sites and 2-10 nM for low-affinity sites with a 10- to 20-fold difference between the two classes, except for the avian EGF receptor, which binds murine EGF with 100-fold lower affinity. Following solubilization in the nonionic detergents Nonidet P-40 (NP-40) or Triton X-100 the affinity of both classes decreases by an order of magnitude (see below). Commonly low-affinity sites represent 90% of the total ligand binding, but this fraction is increased by protein kinase C activity. This down modulation of ligand binding appears not to be mediated by phosphorylation at either Thr-654 or Thr669 in the juxtamembrane domain, as was previously suggested. Thapsigargin, a non-12-O-tetradecanoyl phorbol-13-acetate(TPA)-type tumor promoter, also inhibits high-affinity binding, while sphingosine is reported to increase the affinity of low-affinity sites (see below). The mechanism by which these agents act still remains to be defined. The action of ligand on the kinase activity is primarily to increase the Vmax value with little effect on the substrate Km value. The maximum velocity of peptide phosphorylation at 30° in the presence of EGF is extremely sensitive to the state of the receptor and readily decreases during solubilization and purification steps. For example, the Vm~, value l0 p. j. Bertics, W. S. Chen, L. H. Hubler, C. S. Lazar, M. G. Rosenfeld, and G. N. Gill, J. Biol. Chem. 263, 3610 (1988). ii W. Weber, P. J. Bertics, and G. N. Gill, J. Biol. Chem. 259, 14631 (1984). 12 A. Honnegger, T. J. Dull, D. Szapary, A. Komoriya, R. Kris, A. UUrich, and J. Schlessinger, EMBO J. 7, 3053 (1988). 13 C. Erneaux, S. Cohen, and D. L. Garbers, J. Biol. Chem. 258, 4137 (1983). 14 j. G. Koland and R. A. Cerione, J. Biol. Chem. 262, 2230 (1988).

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of angiotensin II phosphorylation has been estimated at 253 pmol/min/ pmol receptor for a plasma membrane preparation, ~° 19.5 pmol/min/pmol for lysates, H and 11 pmol/min/pmol for purified receptor. H The effect of changing receptor concentration may also contribute to changes in specific activity (see below). Prior receptor autophosphorylation does not appear to affect the Vm~, value, but in the absence of ligand the Vmax value decreases by - t w o - to sixfold. 6 The Km value for angiotensin II is 0.8-1.1 mM, ~°-~20.2-1.0 mM for vsrc peptide, 5,~°'12 and 0.16-0.22 mM for a peptide derived from the P1 autophosphorylation site of the receptor.5 The effect of prior phosphorylation or the presence of ligand on the Km value of an exogenous substrate remains unclear, but there is evidence to suggest that the C-terminal autophosphorylation sites of the receptor can competitively inhibit exogenous substrate phosphorylation, l°a2 Four major autophosphorylation sites termed PI to P4 have been identified on the EGF receptor, all of which are found in the C-terminal domain. These are at tyrosine residues 1173, 1148, 1068, and 1086, respectively. 6,~5 P1 is the major site of tyrosine phosphorylation in response to stimulation by EGF both in solution and in intact cells, probably as a result of its low Km value relative to the other sites. 6

Inhibitors and Activators As described above, the E G F receptor is sensitive to magnesium and manganese cations and metal ion-chelating agents such as E D T A are able to totally inhibit kinase activity. Calcium ions do not directly affect the receptor, but the autophosphorylation domain is particularly sensitive to the calcium-activated neutral proteases or calpains. These act rapidly to generate a 150K fragment following cell lysis, which must therefore be performed in the presence of E G T A . Furthermore, there is evidence that C-terminal proteolysis increases protein kinase activity of the receptor by decreasing steric hinderance. 16 The effects of changes in ionic strength on kinase activity are poorly studied, but there is a clear sensitivity to ammonium sulfate in particular. In this case 0.25 M salt has been proposed to stabilize a native, liganddependent conformation of the receptor that exhibits far greater stimulation with EGF and manganese ions than in the absence of salt.14 Both sphingosine and thapsigargin (a non-TPA-type protein kinase C15j. j. Hsuan, N. Totty, and M. D. Waterfield, Biochem. J. 262, 659 (1989). 16 S. Cohen, H. Ushiro, C. Stoscheck, and M. Chinkers, J. Biol. Chem. 257, 1523 (1982).

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independent tumor promoter) are able to indirectly modulate the ligandbinding affinity of the EGF receptor. Sphingosine appears to increase ligand affinity and stimulate kinase activity, ~7 while thapsigargin down modulates receptor activity by decreasing the binding affinity and the ligand-dependent kinase activity.18 Both may act by altering the activity of serine/threonine kinases toward the EGF receptor. Solubilization of the EGF receptor in nonionic detergents increases basal kinase activity and decreases ligand-binding affinities to a single class. The dissociation constant is approximately an order of magnitude below that of the low-affinity membrane sites. The structural basis for these changes remains unclear. The probable basis for the specific subclass of high-affinity sites observed on binding EGF to intact cells is the dimerization of receptor molecules and recently a similar subclass has been identified in highly concentrated solutions of receptor. The dimer form of the receptor is thought to exist in equilibrium with monomers, show highaffinity binding, and elevated kinase activity in the absence of ligand. According to this model the binding of ligand stabilizes dimers relative to monomers. 19 A corollary of the dimerization model is that certain multivalent ligands may enhance kinase activity independent of ligand and indeed this has been demonstrated using bivalent antibodies. Furthermore, prior immobilization of receptors using affinity matrices has been shown to inhibit the ability of ligand to stimulate kinase activity. The role of receptor tyrosine phosphorylation is not clear, but the Cterminal domain is thought to act as a weak competitive inhibitor of exogenous substrate phosphorylation that can be relieved by autophosphorylation, proteolysis, or genetic deletion. Further phosphorylation sites may, however, play a clear regulatory role. These include Thr-654, a major site for protein kinase C, which appears to be involved in down regulating receptor kinase activity. Several other sites have been identified by metabolic labeling studies. The precise function of these sites has yet to be determined, but they may be involved in the regulation of ligand affinity and receptor internalization. Dimethyl sulfoxide is able to directly stimulate EGF receptor kinase activity independent of ligand in either detergent solution or intact membranes. 2° The mechanism of this activation is not known. Unlike the insulin receptor, the EGF receptor kinase activity is not 17R. J. Davis, N. Girones, and M. Faucher, J. Biol. Chem. 2263, 5373 (1988). is K. Takishima, B. Friedman, H. Fujiki, and M. R. Rosner, Biochem. Biophys. Res. Commun. 157, 740 (1988). 19j. Schlessinger, Biochemistry 27, 3119 (1988). 2o R. A. Rubin and H. S. Earp, J. Biol. Chem. 258, 5177 (1983).

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stimulated by disulfide reduction using dithiothreitol. The intraceUular region is, however, sensitive to oxidation and kinase activity is lost in the absence of reducing agents. Concluding Remarks It seems clear that a large number of parameters can affect the enzymatic properties of the EGF receptor such that comparisons of receptor activity between different cell types, using different preparation procedures, and different assay conditions can give misleading and even contradictory results. The protocol described here is rapid, uses extremely mild conditions, and produces a highly purified and active preparation from A431 cells. No other source of EGF receptor has yet been found to carry the 9A determinant. Attempts to overproduce active full-length EGF receptor or fragments in bacteria and in yeast (Schizosaccharomyces pombe) have been unsuccessful. The baculovirus expression system has been shown to give an active, full-length receptor, but with no improvement in yield over A431 cells. 21 Active fragments have proved more successful in this system: fragments comprising the extracellular and intracellular regions have both been expressed and purified, the latter using monoclonal anti-phosphotyrosine affinity chromatography. 22,23 21 M. D. Waterfield and C. Greenfield, this volume [52]. 2z C. Greenfield, I. Hiles, M. D. Waterfield, M. Federwisch, A. Wollmer, T. L. Blundell, and N. McDonald, EMBO J. 8, 4115 (1989). 23 p. B. Wedergaertner and G. N. Gill, J. Biol. Chem. 264, 11346 (1989).

[32] I d e n t i f i c a t i o n o f P h o s p h o h i s t i d i n e in P r o t e i n s a n d Purification of Protein-Histidine Kinases B y Y I N G - F E I W E I a n d HARRY R . M A T T H E W S

This chapter contains a potpourri of methods for working with phosphohistidine and protein-histidine kinase. The phosphate group in phosphohistidine is attached to a side-chain nitrogen, either at the 1- or 3position or both. The nitrogen-phosphate bond is acid labile and the first part of the chapter covers methods for overcoming this and exploiting the alkali stability of phosphohistidine. Because phosphohistidines are not commercially available, they must be synthesized in the laboratory. This METHODS IN ENZYMOLOGY, VOL. 200

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Affinity purification of active epidermal growth factor receptor using monoclonal antibodies.

378 P U R I F I C A T I O N OF P R O T E I N KINASES [31] such a procedure is, however, that it is difficult to elute the receptor from these colum...
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