Glycosylation-induced Conformational Modification Positively Regulates Receptor-Receptor Association

A STUDY WITH AN ABERRANT EPIDERMAL GROWTH FACTOR RECEPTOR (EGFRvIII/Delta EGFR) EXPRESSED IN CANCER CELLS*

Helen Fernandes, Stanley Cohen, and Subal BishayeeDagger

From the Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103

Received for publication, June 26, 2000, and in revised form, November 3, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The epidermal growth factor receptor (EGFR) is a multisited and multifunctional transmembrane glycoprotein with intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric receptor undergoes dimerization resulting in kinase activation. The consequences of kinase stimulation are the phosphorylation of its own tyrosine residues (autophosphorylation) followed by association with and activation of signal transducers. Deregulation of signaling resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant epidermal growth factor (EGF) receptor missing 267 amino acids from the exoplasmic domain is common in human glioblastomas. The truncated receptor (EGFRvIII/Delta EGFR) lacks EGF binding activity; however, the kinase is constitutively active, and cells expressing the receptor are tumorigenic. Our studies revealed that the high kinase activity of the Delta EGFR is due to self-dimerization, and contrary to earlier reports, the kinase activity per molecule of the dimeric Delta EGFR is comparable to that of the EGF-stimulated wild-type receptor. Furthermore, the phosphorylation patterns of both receptors are similar as determined by interaction with a conformation-specific antibody and by phosphopeptide analysis. This eliminates the possibility that the defective down-regulation of the Delta EGFR is due to its altered phosphorylation pattern as has been suggested previously. Interestingly, the receptor-receptor self-association is highly dependent on a conformation induced by N-linked glycosylation. We have identified four potential sites that might participate in self-dimerization; these sites are located in a domain that plays an important role in EGFR functioning.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human epidermal growth factor receptor (EGFR)1 is a transmembrane glycoprotein with a cysteine-rich extracellular region and an intracellular domain containing uninterrupted kinase site and multiple autophosphorylation sites clustered at the C-terminal tail (see Ref. 1 and reviewed in Ref. 2) (Fig. 1). On the basis of internal sequence identity, the extracellular portion of the EGFR has been subdivided into four domains. Domains I (amino acids 1-165) and III (aa 310-481) have 37% sequence identity, whereas domains II (aa 166-309) and IV (aa 482-621) are rich in cysteines (3) (see Fig. 1). These cysteines are linked by intra-chain disulfide bonding (4). Domain III has been shown to bind directly with EGF, and then two molecules of the monomeric receptor-ligand complex interact to form a dimeric complex. Domain I is believed to be involved in the second interaction (3, 4). The receptor dimerization results in kinase activation. The earliest consequence of kinase activation is the phosphorylation of its own tyrosine residues (autophosphorylation), and this is followed by its association with and activation/phosphorylation of signal transducers leading to mitogenesis. In addition, we have demonstrated a phosphorylation-induced conformational alteration of the EGFR (5). Such conformational change agrees well with the finding that autophosphorylation also results in unmasking of cryptic cytoplasmic domain(s) needed for receptor internalization (6).



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Fig. 1.   Structural features of the WtEGFR and Delta EGFR. The 621 aa in the extracellular region of the EGFR are divided into four domains; domains I (aa 1-165) and III (aa 310-481) have 37% sequence identity. Domains II (aa 166-309) and IV (aa 482-621) are rich in cysteine. Domain III is involved in ligand binding, whereas domain I is important in ligand-induced dimerization. There are 12 potential N-linked glycosylation sites in the wild-type receptor; these sites are marked by the letter N. Delta EGFR lacks aa 6-273 and the missing part is denoted by dashes. Delta EGFR also lacks 4 of the 12 N-linked glycosylation sites. The scheme also shows the locations of transmembrane domain (TM; aa 622-644), tyrosine kinase domain (TK; aa 683-958), and the C-terminal tail (C-ter, aa 959-1186) which contains all five autophosphorylation sites.

Deregulation of signaling due to the aberrant expression of the EGFR has been implicated in oncogenesis. Nearly 50% of grade IV gliomas (glioblastoma multiforme) have amplified EGFR genes. In the majority of such cases, the EGFR gene amplification is correlated with structural rearrangement of the gene, resulting in in-frame deletions that preserve the reading frame of the receptor message. To date, three truncated forms of EGFR have been identified (7-9). The type III deletion mutant occurs in 17% of the glioblastomas and is characterized by an 801-base pair in-frame deletion resulting in the removal of N-terminal amino acid residues 6-273 from the extracellular domain of the intact 170-kDa EGFR (9) (see Fig. 1). Although there is a consensus that this truncated receptor (EGFRvIII/Delta EGFR) lacks ligand binding activity and the kinase is constitutively active (10-12), the subcellular localization of the receptor has not yet been conclusively established. In some transfectants, a significant fraction of the receptor population was reported to be intracellular (10, 11), whereas studies from another laboratory appear to suggest that the receptors are predominantly on the cell surface (12). In addition, the molecular mechanism by which the transfectants acquire transforming activity is not clear. Studies from Cavenee and co-workers (13) suggest that constitutive activation of Ras-mitogen-activated protein kinase pathway contributes to the transforming activity of the Delta EGFR, and the studies from Wong and co-workers (14, 15) have demonstrated that the transformation is mediated through constitutive activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase and not through Ras-mitogen-activated protein kinase pathway.

Although the Delta EGFR undergoes ligand-independent kinase activation, the extent of autophosphorylation of the Delta EGFR is significantly less compared with that of the ligand-stimulated WtEGFR (11, 16). Since phosphorylation-induced conformational change results in exposure of sequence motifs involved in endocytic and lysosomal sorting and such unmasking is thought to be obligatory for receptor down-regulation (6), this may explain the persistent presence of the Delta EGFR on the cell surface. It is possible that the endocytic codes are cryptic in the receptors that are in partially active conformation. However, it is an open question why the Delta EGFRs are not fully active although the receptor is capable of self-dimerization. Is it due to the fact that receptor dimerization is necessary but not sufficient for full kinase activity? It is also not known why the Delta EGFR undergoes self-association, whereas the WtEGFR does not. Such information will be useful in developing strategies to control the activity of the aberrant receptor.

Contrary to earlier reports, our studies reported here suggest that there is no difference between the dimeric Delta EGFR and the ligand-stimulated WtEGFR with respect to kinase activity as determined by the extent of autophosphorylation. We also demonstrate by using a conformation-specific antibody that detects an epitope exposed only upon phosphorylation of tyrosines 992, 1068, and 1086 (17) that the antibody recognizes the Delta EGFR as fully active receptor. This is further confirmed by phosphopeptide analysis. This suggests that in addition to its persistent presence on the cell surface, the high kinase activity of the Delta EGFR also contributes to its tumorigenic activity. Finally, we report that the ligand-independent dimerization of the Delta EGFR is contingent upon core glycosylation. Out of 12 potential N-linked glycosylation sites in the receptor, the four sites located in domain III are likely to be involved in inducing a stable conformation needed for receptor-receptor association. Based on these and related studies, we also propose models for self- and ligand-induced receptor dimerization and how subdomains within the extracellular region might influence such interactions.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The cross-linking reagent 3,3'-bis(sulfosuccinimido)suberate (BS3) was purchased from Pierce, and tunicamycin and swainsonine were obtained from Sigma. Endoglycosidase H was purchased from Glyco (Novato, CA). A substrate for the EGFR (RRKGSTEEEAEYLRV) with an Km of 21 µM (18) containing the tyrosine 1173 autophosphorylation site was purchased from Infinity Biotech Research and Resourse (Upland, PA). EGF was purified from mouse submaxillary glands and radiolabeled with 125I by the chloramine-T procedure (17). Solid-phase EGF was prepared by coupling EGF to Affi-Gel 15 (Bio-Rad) as described (17). Labeled ATP was prepared with 32Pi and gamma -Prep A kit (Promega, Madison, WI) according to the manufacturer's directions. Specific radioactivity of [gamma -32P]ATP was adjusted by adding unlabeled ATP (17). All radioisotopes were purchased from ICN Biomedicals (Costa Mesa, CA).

Iodination of mAb 425-- The monoclonal antibody directed to an extracellular peptide epitope of the human EGFR (19) was iodinated by the chloramine-T method. 5 µg of protein A-purified mAb 425 in a total volume of 20 µl containing 0.15 M sodium phosphate, pH 7.5, and 0.25 mCi of Na125I was incubated at 20 °C with 30 µg of chloramine T. After 1 min, the iodination reaction was terminated by the addition of sodium metabisulfite followed by NaI and BSA. Under these conditions nearly 50% of 125I was incorporated into the protein as determined by trichloroacetic acid precipitation. The labeled antibody was separated from the free radioactivity by Sephadex G-10 chromatography.

EGFR Mutants and Cell Culture-- Human glioblastoma cell line, U87MG, as well as cells expressing the truncated receptor lacking the EGF binding activity and the wild-type receptor were obtained from Drs. W. K. Cavenee and H.-J. Su Huang. The generation and characterization of the mutant have previously been described (16). The transfected cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated cosmic bovine serum (HyClone, Logan, UT) and 0.4 mg/ml G418; parental U87MG cells were grown in the absence of G418. Plasma membranes from these cells were prepared as described (17).

Receptor sites/cell were determined by 125I-EGF binding assay as described previously (17). Briefly, 2.25 ng of 125I-EGF (5.6 × 105 cpm) in a total volume of 200 µl of Earle's balanced salt solution containing 20 mM HEPES, pH 7.5, 2.5 mg/ml BSA, and unlabeled EGF (25 ng/ml for cells expressing less than 1 × 105 receptor sites/cell and 100 ng/ml for cells expressing more than 1 × 105 receptor sites/cell) were incubated at 20 °C for 2 h with cells grown in 2-cm2 24-well plates. Nonspecific binding, which was 5-10% of the total binding, was measured by incubating the cells with labeled EGF in the presence of 200 nM unlabeled EGF. The EGF binding sites/cell in different mutants are shown in Table I.

For antibody binding, 13 ng of 125I-labeled mAb 425 (2 × 106 cpm) mixed with 30 ng of unlabeled antibody was incubated with cells under conditions described above. This represents the saturating concentration of the antibody for the receptor binding. Nonspecific binding that was 2-3% of the total binding was determined by incubating cells with labeled antibody in the presence of 50-fold excess of unlabeled antibody.

Biosynthetic Labeling of the EGFR-- This was done as described (17) except that incubation with Tran35S-label (100 µCi/ml; 1190 Ci/mmol) was carried out in methionine- and cysteine-free Dulbecco's modified Eagle's medium containing 2% cosmic bovine serum.

Antibodies-- Anti-peptide antibody Ab P2 was directed to amino acid residues 964-979 (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the human beta -type platelet-derived growth factor receptor. It was generated in rabbits using HPLC-purified peptide according to the method described previously (20). Monoclonal antibody, mAb 425, raised against human A431 carcinoma cells and polyclonal 170-kDa antibody to denatured EGFR were gifts from Dr. M. Das and were developed as described (19, 21). mAb 425 is directed to a peptide epitope in the extracellular domain of the human EGFR, and it recognizes only the native human receptor (19). The anti-EGFR monoclonal antibody, Ab-15 (clone H9B4), directed to a cytoplasmic domain, close to the C-terminal tail of the receptor was purchased from NeoMarkers (Union City, CA). This antibody, like the polyclonal anti-170-kDa antibody, is highly suitable for Western blot analysis. A rabbit polyclonal anti-EGFR antibody coupled to agarose (catalog number Sc-03AC) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); this antibody that is suitable for kinase assay is directed to the C terminus of the receptor. The mouse monoclonal anti-phosphotyrosine antibody, 1G2, used for purification of the tyrosine-phosphorylated proteins, was generated as described and coupled to activated Sepharose (22).

Quantification of the 32P-Labeled EGFR-- This was carried out as described (17) with some modifications. Briefly, isolated membranes from cells expressing the wild-type or truncated EGFRs were phosphorylated with [gamma -32P]ATP either in the presence (wild-type) or absence (truncated) of EGF under autophosphorylation conditions (5). An aliquot of the labeled proteins purified by anti-phosphotyrosine monoclonal antibody (1G2) was subjected to immunoprecipitation with an antibody directed to a cytoplasmic domain of the EGFR, analyzed by SDS-PAGE, and quantified as described (17). The quantification of the receptor protein is based on the assumption that there is no incorporation of 32P into Ser/Thr residues of the EGFR. This assumption was validated in an earlier report that no 32P-labeled human EGFR could be detected in MI41, a murine NIH 3T3 cell line expressing a human EGFR mutant in which all five acceptor tyrosine residues have been substituted with phenylalanine (17).

Chemical Cross-linking of the EGFR-- The detergent-solubilized plasma membranes or 32P-labeled receptor preparation was incubated with BS3 at 4 °C for 10 min (23, 24). Excess cross-linker was inactivated by the addition of Tris, pH 7.5, and the samples were processed as described in the figure legends.

Immunoprecipitation Technique and Electrophoresis-- These were carried out as described (17). Briefly, the labeled receptor preparation was incubated with the indicated antibody at 4 °C overnight in 15 µl (unless otherwise indicated) of 20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.2% Nonidet P-40, 2.5 mg/ml BSA, 1 mM vanadate, and protease inhibitors. After isolation of the immune complexes by protein A-Sepharose, the receptors were analyzed by SDS-PAGE (7% gel unless otherwise indicated), and the labeled bands were visualized either by autoradiography (for 32P-labeled receptors) or by fluorography (for 35S-labeled proteins) as described (17). The molecular weight markers used were myosin (205,000), beta -galactosidase (116,000), phosphorylase b (97,000), albumin (66,000), and ovalbumin (45,000).

Western Blot Analysis-- This was carried out as described (25). Briefly, the electrophoretically separated proteins were transferred to poly(vinylidene difluoride) membranes (Millipore Corp., Bedford, MA) by electrophoresis at 4 °C overnight at 0.1 mA followed by 1.5 h at 0.2 mA. After incubation with an antibody as specified in figure legends, the antigen-antibody complex was visualized either by ECL Plus reagents from Amersham Pharmacia Biotech or by 125I-protein A. The intensity of the band was quantified using the ImageQuant program. The pre-stained molecular weight markers used (with apparent molecular weight) were alpha 2-macroglobulin (200,000), beta -galactosidase (123,000), fructose-6-phosphate kinase (84,000), and pyruvate kinase (63,000).

Phosphopeptide Analysis-- For phosphopeptide mapping, 32P-labeled wild-type EGFR was purified by EGF-Affi-Gel chromatography as described (17), whereas the truncated receptor was purified by an EGFR-specific antibody, mAb 425. Briefly, isolated membranes from cells expressing the intact EGFRs were solubilized with 0.5% Nonidet P-40 in 20 mM HEPES, pH 7.4, 0.15 M NaCl, 10% glycerol, and protease inhibitors (aprotinin, leupeptin, and phenylmethanesulfonyl fluoride). After centrifugation, the clarified supernatant was incubated at 4 °C for 2 h with EGF-Affi-Gel (1 mg/ml), and the gel beads were washed three times with the binding buffer to remove the unbound proteins. For the truncated receptor, the detergent-solubilized membrane extracts were incubated at 4 °C for 2 h with mAb 425 antibody, and the immune complex was isolated by protein A-Sepharose. The gel beads containing either the wild-type or truncated receptors were incubated with [32P]ATP under autophosphorylation conditions (5). After washing the beads to remove free ATP, the bound receptor was dissociated by heating with SDS-sample buffer and subjected to SDS-PAGE. After overnight in fixing solution (25% methanol, 10% acetic acid in water), the wet gels were processed as described (17) with some modifications. Briefly, the region of the dried gel corresponding to the receptor band was digested with 0.5 ml of sequencing grade trypsin (20 µg/ml; Roche Molecular Biochemicals) in 50 mM ammonium bicarbonate, pH 7.8, for 40 h at 37 °C with an addition of fresh trypsin after 20 h. After centrifugation, an aliquot of the clear supernatant was redigested with trypsin for 20 h at 37 °C, dried in vacuum, and dissolved in 0.1% trifluoroacetic acid in water. Equal counts of phosphopeptides derived from both receptor types were then subjected to reverse-phase HPLC analysis using a DeltaPak 6-µm C18 column (Waters; column size 3.9 × 150 mm) as described (17). Briefly, following injection, the column was washed with 10 ml of 0.1% trifluoroacetic acid in water, and then the phosphopeptides were eluted with a 0-60% acetonitrile gradient containing 0.1% trifluoroacetic acid with a flow rate of 1 ml/min. 0.5 ml fractions were collected, and the Cerenkov counts were determined.

Kinase Assay-- This was performed as described (18) with some modifications. Briefly, the EGFR was immunoisolated using a polyclonal antibody coupled to agarose; this antibody is directed to a cytoplasmic domain of the receptor. After washing three times, the gel beads were incubated in a total volume of 50 µl containing 20 mM HEPES, pH 7.5, 5% glycerol, 0.1% Nonidet P-40, 5 mM MnCl2, 1 mM vanadate, 10 µM [gamma -32P]ATP (5 × 104 cpm/pmol), 200 µM peptide substrate, and protease inhibitors (leupeptin, phenylmethanesulfonyl fluoride, and aprotinin). After 10 min at 20 °C, the reaction was terminated by the addition of EDTA (final concentration 20 mM) and centrifuged. Trichloroacetic acid was added to the supernatant to a final concentration of 5%. BSA was added as carrier protein to facilitate the precipitation of the EGFR that might have dissociated from the antibody during the incubation. After 10 min at 4 °C, the reaction mixture was centrifuged at 4 °C and the supernatant was processed as described to determine the substrate phosphorylation (18).

Sucrose Density Gradient Ultracentrifugation-- This was carried out as described (23) with some modifications. Briefly, the WGA-purified EGFRs were centrifuged at 50,000 rpm at 4 °C for 8.5 h in a SW Ti55 rotor through a linear gradient of 5-20% (w/v) sucrose in 20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.1% Nonidet P-40, 1 mM sodium vanadate, and 1 mM MnCl2. At the end of the run, fractions (150 µl) were collected and processed as described in legend for Fig. 3.

Isolation of Endoplasmic Reticulum from Tunicamycin-treated Cells-- This was carried out as described (26). Briefly, confluent cultures of cells grown in p150 plates were incubated at 37 °C for 20 h in growth medium containing 1 µg/ml tunicamycin. Under these conditions, the newly synthesized EGFRs lacking N-linked oligosaccharides are associated with the endoplasmic reticulum. The microsomal fraction that also contains endoplasmic reticulum was isolated from these cells as described (26).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutation of the EGFR resulting in type III truncation occurs spontaneously in vivo in glioma cells. This led us to use a human glioma cell line, U87MG, as the host for the expression of the Delta EGFR/EGFRvIII and, as a control, the wild-type receptor. The Delta EGFR maintains its transforming activity in this host cell. Another advantage in selecting this cell line is that it is deficient in endogenous EGFR (see below).

Receptor Numbers per Cell in U87MG.Delta EGFR-- Earlier reports suggested that the Delta EGFR lacks ligand binding activity (10, 11, 16). Hence we used 125I-labeled mAb 425 antibody to determine the number of receptor molecules per cell with an objective to investigate whether both receptor types are expressed in comparable numbers. Such studies should also allow us to determine the endogenous receptor level in U87MG.Delta EGFR cells. This information is also important for the studies described in this paper. mAb 425 is a monoclonal antibody directed to a peptide epitope in the extracellular domain of the EGFR, and it binds to the receptor with very high affinity (19). In addition, earlier studies have demonstrated the specific and saturable binding of this antibody to membranes from a human carcinoma cell line, A431(19). Thus, mAb 425 is suitable for determining the receptor numbers under circumstances where EGF cannot be used. The concentration of the antibody used for receptor assay (215 ng/ml) leads to maximum binding to both U87MG.Delta EGFR and U87MG.WtEGFR cells since there was no further increase in the specific binding of the antibody to either cell type when mAb 425 concentration was increased by 3-fold (data not shown). Different laboratories including ours have reported the use of saturating concentration of a ligand for the estimation of receptor numbers (17, 27). Based on the antibody binding, U87MG.Delta EGFR cells express 4.4 × 105 receptors per cell (see Table I). To investigate whether the estimated numbers of antibody-binding sites reflect the actual receptor sites, we also determined the receptor numbers in U87MG.WtEGFR using 125I-EGF binding and compared it with the antibody-binding sites. As shown in Table I, the receptor sites calculated based on ligand binding (5.1 × 105) were similar to the receptor numbers determined by antibody binding (5.3 × 105) indicating the validity of this method. This suggests that (i) the expression levels of both the receptors are comparable (4.4 × 105 receptor sites/cell for Delta EGFR versus ~5 × 105 receptor sites/cell for WtEGFR) and (ii) the bulky size of the antibody of molecular mass 150-kDa relative to EGF of 6-kDa did not adversely influence its binding to the receptor. We also determined the endogenous EGFRs in U87MG.Delta EGFR by 125I-EGF binding (0.2 × 105 sites/cell), and it represents less than 5% of the truncated receptor population. This is further confirmed by Western blot analysis followed by densitometric scanning of the 170-kDa wild-type receptor band and the 145-150-kDa truncated receptor band (Fig. 2).


                              
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Table I
EGFR sites in U87MG cells transfected with WtEGFR or Delta EGFR cDNA clone
The number of EGF- and antibody-binding sites were determined by incubating 1.0 × 105 cells in 24-well plates with saturating concentrations of 125I-EGF and 125I-mAb 425, respectively, according to methods described under "Experimental Procedures." In calculating antibody binding sites, it was assumed that each antibody binds two receptor molecules.



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Fig. 2.   Western blot analysis to quantify endogenous EGFR in U87MG.Delta EGFR. Plasma membranes from U87MG.Delta EGFR were subjected to Western blot analysis using a polyclonal anti-170-kDa antibody directed to denatured human EGFR as described under "Experimental Procedures." The antigen-antibody complexes were visualized by ECL Plus reagents. The receptor bands are marked.

Isolation of the Dimeric Delta EGFR in Biologically Active State and Its Kinase Activity-- Based on chemical cross-linking studies in which intact cells were reacted with a cross-linker, it was concluded that the autokinase activity of the Delta EGFR is 20% that of the EGF-stimulated wild-type receptor (11, 16). Since the Delta EGFR is capable of self-dimerization, it implies that receptor dimerization is obligatory but not sufficient for full kinase activation. Hence to examine the extent of dimerization and more importantly to investigate the kinase activity of the dimeric receptor, sedimentation studies were performed to isolate the monomeric and dimeric forms of the Delta EGFR in the biologically active state. For this purpose the WGA-agarose-purified receptors from U87MG.Delta EGFR cell membranes were subjected to sucrose sedimentation analysis, and the kinase activity of the gradient fractions was assayed by autophosphorylation. As control, the WtEGFR was also subjected to sedimentation analysis under similar conditions; however, the gradient fractions were assayed by autophosphorylation in the presence of EGF. The identity of the EGFR in the gradient fractions was confirmed by immunoprecipitation with mAb 425 antibody. As shown in Fig. 3, the Delta EGFR exists in two forms, a slow moving form and a fast moving form; however, under similar conditions the WtEGFR moves as a single entity with a mobility similar to that of the slow moving component of the Delta EGFR. To determine the molecular weights of the different components, we analyzed the peak fractions by chemical cross-linking. For this purpose, the 32P-labeled receptor preparations were subjected to chemical cross-linking with 50 µM BS3, immunoprecipitated with mAb 425, and then analyzed by SDS-PAGE. As shown in Fig. 3 (insets), following BS3 treatment of the faster moving peak from the Delta EGFR, a cross-linked complex corresponding to a molecular weight of ~300,000 could be detected; the receptor population that escaped cross-linking is present as an 145-150-kDa band. The 145-150-kDa band and not the 300-kDa band was seen when cross-linking was carried out with the slower moving peak. These suggest that the fast and the slow moving components of the Delta EGFR represent the dimeric and the monomeric forms of the receptor, respectively. No cross-linked complex could be seen when the peak fraction from the WtEGFR was subjected to BS3 treatment and the 32P-labeled receptor moved as a 170-kDa protein, a size consistent with the monomeric form of the receptor. For the experiment described in Fig. 3, the truncated receptor has undergone a series of treatments that not only took more than 10 h to complete but also resulted in significant receptor dilution. Despite these treatments, a significant portion of the Delta EGFR exists in the dimeric state.



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Fig. 3.   Sucrose gradient analysis of the WtEGFR and Delta EGFR. WGA-purified receptor preparations were subjected to 5-20% sucrose gradient centrifugation (50,000 rpm for 8.5 h in SW55Ti) as described under "Experimental Procedures." The distribution of the receptor in gradient fractions was determined by autokinase assay either in the absence (for Delta EGFR and WtEGFR) or presence (for WtEGFR) of EGF followed by SDS-PAGE autoradiography and densitometric scanning of the labeled band. Insets, the peak gradient fractions were phosphorylated with [gamma -32P]ATP and chemically cross-linked with 50 µM BS3 as described under "Experimental Procedures." Following immunoprecipitation by mAb 425, the labeled receptors were analyzed by 3.5-10% SDS-PAGE. The 300-kDa band was absent when no cross-linker was used.

We also determined the specific activity of monomeric Delta EGFR kinase. For this purpose we quantified both the wild-type and the truncated receptors in the gradient fractions by Western blot analysis using a polyclonal antibody directed to the denatured 170-kDa EGFR (see Fig. 4). Both the receptors were also quantified by probing the blot with a monoclonal antibody directed to an epitope located in the C-terminal tail of the EGFR. The relative intensities of the bands are similar to that of the blot probed with the anti-170-kDa antibody. This strengthens the possibility that the wild-type and truncated receptors react equally well in Western blot with anti-170-kDa antibody as well as with the C-terminal antibody, and hence the Western blot result reflects the actual amount of these two proteins in the gradient fractions. As shown in Fig. 4, the kinase activity per unit receptor protein for the dimeric receptor is 5-7-fold higher relative to the monomeric receptor. Furthermore, the extent of autophosphorylation per molecule of the dimeric Delta EGFR (fractions 15-18) is similar to that of the ligand-activated WtEGFR (fractions 19-22) (see Fig. 4). This suggests that receptor dimerization, at least with respect to the Delta EGFR, is sufficient for full kinase activation.



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Fig. 4.   Quantification of kinase activity per unit of protein. Protein in the gradient fractions in Fig. 3 was quantified by Western blot using a polyclonal antibody directed to denatured human EGFR, and the immune complexes were visualized by 125I-protein A. The relative intensities of the bands were the same when the blot was probed with a monoclonal antibody directed to an epitope in the C-terminal tail of the receptor. Plots of autokinase activity determined either in the absence (for Delta EGFR) or presence (for WtEGFR) of EGF in the gradient fractions per unit of protein are shown. The positions of the monomeric and dimeric receptor are marked by arrows.

Interaction of the Delta EGFR with an Antibody That Recognizes Only the Activated Receptor-- The studies described above revealed that there is no difference with respect to kinase activity between the Delta EGFR which undergoes dimerization in the absence of the ligand and the EGF-stimulated wild-type receptor. This led us to investigate whether the phosphorylation pattern and phosphorylation-mediated conformational change of the receptors are also similar. Such information is important since it has been proposed that the slow endocytic rate of the Delta EGFR is due to its altered phosphorylation (16). For this purpose we studied the interaction of a conformation-specific antibody, Ab P2, with the Delta EGFR and compared it with that of the WtEGFR. This anti-peptide antibody Ab P2 recognizes the phosphorylated and hence activated EGFR and not the unphosphorylated receptor; however, Ab P2 is not directed to phosphotyrosine (5, 20). The receptor conformation recognized by the antibody is positively regulated by phosphorylation of three tyrosine residues at 992, 1068, and 1086. The phosphorylation of the other two acceptor sites, namely tyrosines 1148 and 1173, does not play any role in antibody binding (17, 28). It should be mentioned in this context that the interaction between Ab P2 and the activated receptor is independent of EGF (5). To study the interaction between Ab P2 and the EGFR, the detergent-solubilized membranes from the wild-type receptor stimulated by EGF and the truncated receptor (both expressed in U87MG cells) were phosphorylated by labeled ATP, and the tyrosine-phosphorylated receptors were isolated by 1G2-Sepharose. Equal amounts of the receptors were then subjected to immunoprecipitation by Ab P2, and the receptor bands following SDS-PAGE were quantified by densitometric scanning. The extent of precipitation of the Delta EGFR at each of the antibody concentrations was similar to that of the WtEGFR at the corresponding antibody concentration (Fig. 5). This indicates that the affinity of the antibody for both receptor types is similar and the phosphorylation pattern, at least with respect to Tyr 992, 1068, and 1086, of the Delta EGFR is comparable to that of the WtEGFR. This is further confirmed by phosphopeptide analysis of the receptors. The HPLC elution profiles of the phosphopeptides from the Delta EGFR were similar to that of the WtEGFR (Fig. 6). This suggests that the inefficient internalization of the Delta EGFR is not due to its altered phosphorylation.



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Fig. 5.   Immunoprecipitation of the WtEGFR and Delta EGFR by a conformation-specific antibody, Ab P2. 1.25 pmol of 1G2-purified 32P-labeled WtEGFR and Delta EGFR (quantified according to the method described under "Experimental Procedures") were immunoprecipitated by indicated amounts of Ab P2 and then analyzed by SDS-PAGE autoradiography (A). The intensity of the receptor band was quantified by densitometric scanning (B).



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Fig. 6.   Phosphopeptide maps of the WtEGFR and Delta EGFR after trypsin digestion. WtEGFR and Delta EGFR bound to EGF-Affi-Gel 15 and mAb 425, respectively, were phosphorylated with labeled ATP under autophosphorylation conditions. After dissociation from gel beads by heating with SDS sample buffer, the 32P-labeled receptors were subjected to SDS-PAGE, digested with trypsin, and then 100,000 cpm were analyzed by reverse-phase HPLC as described under "Experimental Procedures." black-square, WtEGFR; black-triangle, Delta EGFR.

Glycosylation-induced Conformational Modification and Kinase Activation of the Delta EGFR-- The EGFR is a glycoprotein, and it has 12 potential N-linked glycosylation site (1) (see Fig. 1). Earlier studies have demonstrated the importance of core glycosylation in EGF binding and hence for kinase activation (29, 30). Since four of the 12 sites are missing from the Delta EGFR and it lacks ligand binding activity, we investigated whether the conformation of the truncated receptor is influenced by glycosylation and if so whether such conformational modification plays any role in kinase activation. Like the aglyco-WtEGFR which does not interact with mAb 425 (19), aglyco-Delta EGFR synthesized in the presence of tunicamycin is also not capable of binding with the antibody (data not shown). As discussed above (see Table I), this monoclonal antibody is directed to an extracellular peptide epitope of the EGFR (19), and it also recognizes the Delta EGFR. This suggests that the aglyco-Delta EGFR, like the aglyco-WtEGFR, fails to attain certain conformation needed for mAb 425 binding. We also investigated if there is any relationship between the conformational alteration induced by glycosylation and kinase activation of the Delta EGFR. Cells expressing the Delta EGFR were metabolically labeled with [35S]methionine in the absence or presence of tunicamycin, and then the labeled receptors were quantified by immunoprecipitation with a polyclonal anti-EGFR antibody coupled to agarose. Based on this quantification, equal amounts of the labeled receptors from each cell lysate were phosphorylated with unlabeled ATP. The autophosphorylated receptors were immunoisolated by 1G2 and then analyzed by SDS-PAGE. Upon tunicamycin treatment, the intensity of the truncated receptor band is drastically reduced to the same extent as that of the wild-type receptor (Fig. 7). Similar results were also seen when 1G2-purified receptors were immunoprecipitated by a receptor-specific antibody (data not shown). It should be mentioned in this connection that although anti-phosphotyrosine antibody binding is independent of the number of tyrosine residues in a receptor molecule being phosphorylated, nevertheless any phosphorylation (one or five per molecule) is a reflection on its kinase activity. Thus the drastic reduction in the intensity of the bands from Tu-treated cells suggests the lower kinase activity of the aglyco-receptor compared with that of the glycosylated receptor. We have also assayed the kinase activity of the Tu-treated/untreated cells by exogenous substrate assay (Table II). In this assay, equal amounts of the glycosylated and aglyco-receptors (quantified by Western blot using an C-terminal anti-EGFR antibody) were immunoisolated by a polyclonal anti-EGFR antibody coupled to agarose, and then the kinase activity in the gel beads was assayed by phosphorylation of an EGFR substrate (18). As with the autokinase assay, the extent of phosphorylation of the substrate by the aglyco-receptor was much less compared with that of the control receptor (Table II). Thus, as with the EGFR, core glycosylation of the Delta EGFR is needed to generate a conformation which eventually leads to its kinase activation.



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Fig. 7.   Role of core glycosylation in EGFR kinase activation. Cells expressing the WtEGFR or Delta EGFR were labeled with [35S]methionine either in the absence (-) or presence (+) of 1 µg/ml tunicamycin under conditions described under "Experimental Procedures." Following quantification of the labeled receptors by immunoprecipitation with an immobilized polyclonal antibody directed to the C-terminal of the EGFR, equal amounts of labeled receptors from each cell lysate were incubated with ATP either the absence (for Delta EGFR) or presence (for WtEGFR) of EGF. Following immunoisolation of phosphorylated receptor by 1G2 antibody, the labeled proteins were analyzed by SDS-PAGE autoradiography, and the intensity of the receptor band was quantified by densitometric scanning. The molecular weights of the receptor under different experimental conditions are marked. The identity of the marked band as EGFR was established by immunoprecipitation with a receptor-specific antibody.


                              
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Table II
In vitro kinase activity of the control and tunicamycin-treated Delta EGFR
The detergent-solubilized microsomal fraction from tunicamycin-treated U87MG.Delta EGFR cells was incubated with WGA-agarose, and the flow-through was used as the source for the tunicamycin-treated receptor. Isolated plasma membranes from untreated cells were used as the source for control receptor. Equal amounts of the tunicamycin-treated receptor and the control receptor (quantified by Western blot analysis using a monoclonal antibody, Ab-15, directed to a cytoplasmic epitope) were immunoisolated using a polyclonal antibody coupled to agarose. This antibody is directed to an intracellular domain of the receptor. The kinase activity of the washed gel beads was measured by determining the incorporation of 32P into the peptide substrate under conditions described under "Experimental Procedures."

We also investigated the mechanism by which the carbohydrate chains are involved in kinase activation. One possibility is that the oligosaccharide chains participate directly in kinase stimulation, and removal of the carbohydrate chains reverts the receptor back to its kinase-inactive state. Alternatively, the function of the N-linked glycosylation is to impart a stable kinase-active conformation to the receptor, and once the receptor attains such a conformation, the carbohydrate chains are dispensable. To distinguish between these two possibilities, we studied the effect of enzymatic removal of the carbohydrate chains from the mature Delta EGFR on its kinase activity. One such enzyme is endoglycosidase H that cleaves between the N-acetylglucosamine residues of the chitobiose unit of N-glycans that are linked to asparagine. The high mannose form of the truncated receptor which is a substrate for the enzyme was synthesized by growing cells with [35S]methionine in the presence of swainsonine, an inhibitor of mannosidase II. The receptor synthesized in the presence of swainsonine and not the control receptor is susceptible to endoglycosidase H cleavage (data not shown). When equal numbers of acid-insoluble counts from control- and swainsonine-treated cell lysates were phosphorylated with unlabeled ATP and then the tyrosine-phosphorylated receptors were purified by 1G2 antibody, the intensity of the treated band was similar to that of the control band. Thus, the modified receptor of Mr ~140,000 is as active as the control receptor (Fig. 8A). Similar results were also seen by exogenous substrate assay (data not shown). These suggest that terminal processing blocked by swainsonine is not required for kinase activation. Deglycosylation of the high mannose receptor by treatment with endoglycosidase H results in a protein of Mr ~115,000 (see Fig. 8B). The intensities of both the 115-kDa and 140-kDa bands after phosphorylation with unlabeled ATP and immunoisolation by 1G2 are similar suggesting that the autokinase activity of the deglycosylated receptor is similar to that of the untreated receptor. Similar results have also been reported earlier with the wild-type receptor in which complete removal of the carbohydrate chains from the glycosylated receptor neither abolished the ligand binding activity nor the kinase activation (29, 30). Our results suggest that (i) the initial N-linked glycosylation but not its terminal processing is sufficient for kinase activation; and (ii) the function of the glycosylation is to impart a kinase-active conformation to the Delta EGFR, and it does not revert back to its kinase-inactive conformation upon deglycosylation.



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Fig. 8.   Effect of endoglycosidase H on autokinase activity of the Delta EGFR synthesized in the presence of swainsonine. A, cells expressing the Delta EGFR were labeled with [35S]methionine either in the absence (control) or presence of 1 µg/ml swainsonine (swainsonine-treated) under conditions described under "Experimental Procedures." Equal numbers of trichloroacetic acid-insoluble radioactive counts from each cell lysate were then incubated with ATP, and then tyrosine-phosphorylated receptor was isolated by anti-phosphotyrosine antibody (1G2). B, lysates from cells labeled with [35S]methionine in the presence of swainsonine were incubated in a total volume of 15 µl either without (-) or with (+) 10 milliunits of endoglycosidase H (Endo H) and 15 µg of BSA at pH 6.0 for 15 min at 20 °C followed by 4 h at 4 °C. After adjusting the pH to 7.4 with 5 µl of 250 mM HEPES, pH 7.4, the lysates were incubated with ATP for 30 min at 4 °C. Following termination of the reaction with 20 mM EDTA, the receptor preparations were incubated for 30 min at 4 °C with solid-phase 1G2 either in the absence (-) or presence (+) of 40 mM phenyl phosphate (Phe phos). The bound radioactivity was eluted by incubating the gel beads with 40 mM phenyl phosphate. Samples from both A and B were analyzed by SDS-PAGE autoradiography, and the intensity of the receptor band was quantified by densitometric scanning, and the relative values are shown. The molecular weights of the receptor under various experimental conditions are marked. The identity of the labeled band as EGFR was established by immunoprecipitation with a receptor-specific antibody.

Aglyco-Delta EGFR Synthesized in the Presence of Tunicamycin Lacks Dimer Forming Ability-- Since self-dimerization/oligomerization is obligatory for Delta EGFR kinase stimulation, we investigated whether the reduced kinase activity of the aglyco-receptor is due to its loss of dimer forming ability. It should be mentioned in this connection that based on quantifying receptor protein by Western blot, the autokinase activity of the aglyco-Delta EGFR is 10-20% of the glycosylated receptor (data not shown). This agrees well with our finding that the kinase activity of the dimeric Delta EGFR is 5-7-fold higher relative to the monomeric receptor (see Fig. 4). To test whether the reduced kinase activity of the aglyco-receptor is indeed due to its loss of dimer forming ability, chemical cross-linking studies were carried out. The microsomal fraction from Tu-treated cells contains the aglyco-receptors as well as the glycosylated receptors synthesized prior to tunicamycin treatment. The detergent-solubilized microsomal fraction was passed through WGA-agarose column, and the flow-through (source of aglyco-receptor) was phosphorylated with [gamma -32P]ATP. Following purification of the labeled receptor with 1G2, it was subjected to cross-linking with BS3 and then immunoprecipitated with a receptor-specific antibody. The glycosylated receptor bound to WGA-agarose was eluted by N-acetylglucosamine and incubated with labeled ATP. After purification of the receptor with 1G2, it was incubated with BS3 and then subjected to immunoprecipitation with an anti-receptor antibody. As shown in Fig. 9A, lane 2 (left-hand panel), following BS3 treatment, a cross-linked complex corresponding to Mr ~300,000 could be detected in the glycosylated receptor in addition to the 145-150-kDa band. The ~300-kDa band was absent when no cross-linker was used (Fig. 9A, lane 1). However, under similar conditions, no cross-linked complex could be seen when the aglyco-receptor was treated with BS3 (see Fig. 9A, lane 4), although the 115-kDa band corresponding to the monomeric form of the receptor was present. Increasing the concentration of BS3 to 250 µM had no effect on the mobility of the aglyco-receptor (data not shown). This suggests that lower kinase activity of the aglyco-receptor is due to its lack of dimer forming ability. This was further confirmed by Western blot analysis (Fig. 9B). In this experiment, detergent-solubilized plasma membranes (source of the glycosylated receptor) and the WGA-agarose flow-through from the Tu-treated microsomal fraction (source of the aglyco-receptor) were subjected to cross-linking with 150 µM BS3, and following immunoprecipitation by a receptor-specific antibody, the cross-linked receptors were subjected to Western blot analysis. In glycosylated receptor, a 300-kDa band as well as a band at ~145-150-kDa could be seen following cross-linking (Fig. 9B, lane 2). The 300-kDa band could not be seen when no cross-linker was used (Fig. 9B, lane 1). However, under similar conditions, no cross-linked complex could be seen when the aglyco-receptor was treated with BS3 (see Fig. 9B, lane 4), although the 115-kDa band corresponding to the monomeric form of the receptor was present. No cross-linked complex could be seen when BS3 concentration was increased to 300 µM (data not shown).



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Fig. 9.   Glycosylated Delta EGFR and not the aglyco-receptor undergo self-dimerization. A, the detergent-solubilized microsomal fraction from U87MG.Delta EGFR cells grown in the presence of tunicamycin was incubated with WGA-agarose. Both the flow-through (source of aglyco-receptor) (marked as Tu-treated Delta EGFR) and N-acetylglucosamine eluate (source of mature receptor) (marked as Control Delta EGFR) were phosphorylated with [gamma -32P]ATP, and the tyrosine-phosphorylated receptors were isolated by 1G2. Following incubation without (-) or with (+) 50 µM BS3 under conditions described under "Experimental Procedures," the labeled proteins were immunoprecipitated with a receptor-specific antibody and then analyzed by 3.5-10% SDS-PAGE. B, the detergent-solubilized microsomal fraction from tunicamycin-treated cells was incubated with WGA-agarose. The flow-through (Tu-treated Delta EGFR) and the plasma membranes from cells not treated with tunicamycin (Control Delta EGFR) were incubated without (-) or with (+) 150 µM BS3. The cross-linked complexes were immunoisolated by a solid-phase polyclonal antibody directed to the C terminus of the EGFR and subjected to 3.5-15% SDS-PAGE. The proteins were electrophoretically transferred onto membranes as described under "Experimental Procedures" and probed with a monoclonal antibody directed to an epitope in the C-terminal tail of the EGFR. The immune complexes were visualized by ECL Plus reagents. The monomeric and dimeric receptors in both the experiments are marked.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major findings of our studies are as follows. (i) The hyperactivation of the Delta EGFR is due to its stable receptor-receptor self-association. (ii) The extent of kinase activation and the phosphorylation pattern of the Delta EGFR resulting from its self-dimerization are the same as that of the ligand-induced dimerization of the WtEGFR. (iii) Most interestingly, the self-dimerization of the Delta EGFR leading to kinase activation is highly dependent on a conformation induced by core glycosylation. These studies were conducted by expressing both receptor types in U87MG, a glioblastoma cell line. Our binding studies with 125I-labeled mAb 425 revealed that the expression levels of both receptor types are comparable. In addition, the endogenous receptor level in host cell is also low (~5% of the truncated receptor) (see Table I). It should be mentioned in this connection that the Delta EGFR binding sites/cell determined in the present study differ significantly from the one reported by Huang et al. (16) although both groups used the same cell line. By using fluorescence-activated cell sorter analysis and comparing the intensity of the stained cells with that of A431 cells, Huang et al. (16) estimated that the expression level of Delta EGFR was 1-3 × 106 receptors/cell. Such drastic differences in receptor number might be due to the differences in receptor assay (125I-antibody binding versus flow cytometry which is an indirect method) and/or difference in cell culture including different passage number.

During the isolation of the dimeric form of the Delta EGFR by sucrose gradient centrifugation, the receptor preparation has undergone a series of experimental manipulations that not only took considerable time to complete but also resulted in significant dilution of the receptor. Despite these, nearly 40% of kinase activity could be recovered in a dimeric state suggesting that the receptor-receptor interaction is highly stable. Thus, it is likely that under in vivo conditions in which the receptor concentration is very high (due to amplification), the Delta EGFR will be predominantly in dimeric form. Our studies also revealed that on a molar basis, the kinase activity of the dimeric receptor is 5-7-fold higher compared with that of the monomeric receptor. We also showed that the kinase activity per molecule of the dimeric Delta EGFR is similar to that of the EGF-stimulated WtEGFR as determined by autophosphorylation assay. This is in contrast to the earlier studies that reported that the kinase activity as determined by phosphotyrosine content per molecule of the Delta EGFR is only 13-20% of the WtEGFR stimulated by EGF (13, 16). Those studies were further supported by the finding that only 20% of the receptor population could be chemically cross-linked. However, it should be noted that chemical cross-linking is neither a quantitative reaction nor does it provide any direct information as to the activation state of the receptor. Another reason for differences between our results and those of others (13, 16) might lie in the way the studies were performed. Unlike the present study in which the dimeric receptor complex was separated from the monomeric and hence dormant receptor kinase to determine its activity, the earlier works (13, 16) were conducted on a receptor preparation that contained a mixture of monomeric and dimeric receptors. Our Western blot analysis of the sucrose gradient fractions revealed that only 10-15% of the total receptor protein is present in the dimeric form. Thus the ~10-fold lower kinase activity per molecule of the Delta EGFR relative to the WtEGFR as reported by Huang et al. (16) could be attributed to the presence of a significantly large population of the monomeric receptor with very low kinase activity along with a small population of the dimeric receptor and hence does not reflect the actual kinase activity of the dimeric receptor.

The facts that the affinity and the extent of binding of the conformation-specific antibody, Ab P2, with the Delta EGFR are similar to that of the EGF-activated wild-type receptor (Fig. 5) suggest a similar phosphorylation-induced conformational change of both the receptors. It should be mentioned in this context that Ab P2 recognizes an epitope in the intracellular domain of the EGFR which is unmasked only upon phosphorylation of tyrosines 992, 1068, and 1086, located in the C-terminal tail of the receptor, as a unit. This suggests that the Delta EGFR is recognized by the antibody as an "active" molecule. This is further supported by the phosphopeptide analysis which revealed that there is no difference in the phosphorylation pattern of the Delta EGFR with that of the wild-type receptor stimulated by EGF (Fig. 6). Thus whether the receptor undergoes self-dimerization (as is the case with the Delta EGFR) or ligand-induced dimerization, the outcome is the same. Our results and hence the conclusion are in contrast to earlier reports that correlated the inefficient down-regulation of the Delta EGFR with its low autokinase activity and hence altered phosphorylation pattern. Since phosphorylation-induced exposure of the otherwise cryptic endocytic sequence motifs is needed for receptor internalization and down-regulation (6), Huang et al. (16) suggested that the cryptic internalization codes are not exposed in the Delta EGFR. Since we could not detect any altered phosphorylation pattern of the Delta EGFR in relation to the wild-type receptor, it suggests that the defective endocytosis is not due to low or altered phosphorylation but due to a mechanism that is not yet clear. Thus the high tumorigenic activity of the Delta EGFR is probably not only due to the persistent presence of the receptor on the cell surface as has been suggested (16) but is also possibly due to the higher kinase activity of the dimeric receptor generated by self-association. It should be mentioned that due to high stability of the dimeric receptor, most of the receptor population under in vivo situation will be present in dimeric state.

The importance of the carbohydrate chains on the conformation of the Delta EGFR is underscored by our finding that like the aglyco-WtEGFR, Delta EGFR synthesized in the presence of tunicamycin fails to bind a monoclonal antibody, mAb 425, directed to a peptide epitope in the extracellular domain of the EGFR (data not shown). This antibody, however, binds to the wild-type as well as truncated EGFR (see Table I). These results suggest that glycosylation-mediated post-translational modification confers a conformation needed for mAb 425 binding to the wild-type receptor as well as to the truncated receptor. The conformation induced by core glycosylation and recognized by mAb 425 results in proper folding of the full-length receptor to generate an EGF-binding active conformation, and once such a conformation is attained, the carbohydrate chains are dispensable (29, 30). Although the Delta EGFR undergoes dimerization and kinase activation in the absence of EGF binding, interestingly, our studies revealed that core glycosylation is also needed for receptor-receptor self-association and hence for kinase activation. In addition, as with the wild-type receptor, the glycosylation-induced conformation of the truncated receptor is stable since removal of the carbohydrate chains from the glycosylated receptor does not have any negative effect on receptor dimerization and kinase activation (see Figs. 8 and 9).

There are 12 potential N-linked glycosylation sites in the EGFR-2 in each of domains I and II and four in each of domains III and IV ((2) see Fig. 1). Eight of the 12 chains are present in the Delta EGFR. It should be mentioned in this connection that the type II-truncated EGFR expressed in certain glioblastomas lacks a major portion of domain IV (amino acids 520-603) (9). Despite the fact that three of the four glycosylation sites located in domain IV are also missing from the receptor, type II receptor has EGF binding activity. Thus it is likely that the oligosaccharide chains in domain IV have no influence on ligand binding. This result together with the observations that mAb 425 (i) inhibits EGF binding to the wild-type receptor (19), (ii) mimics EGF in its receptor binding profile (19), and (iii) interacts only with the glycosylated Delta EGFR (lacking domain I and a major portion of domain II and missing four carbohydrate chains) suggest that the four N-linked oligosaccharide chains in domain III are probably sufficient to induce receptor-active conformation, i.e. a conformation needed for EGF binding (for WtEGFR) or for self-dimerization (for Delta EGFR).

The similar characteristics with respect to sugar requirements for self-dimerization and for EGF binding appear to suggest that glycosylation positively regulates receptor-receptor association, and this is probably a prerequisite step for ligand binding. The fact that unliganded intact receptor pre-exists in a monomeric form whereas the truncated receptor undergoes self-dimerization suggests that there are two opposing interactions in the EGFR. One interaction is involved in receptor-receptor self-association and is dependent on glycosylation. This site is probably located in domain III (see above). The other exerts negative influence on protein-protein interaction, and this site is located in domain I. In the full-length receptor, the protein is in a monomeric state since the negative influence of domain I is much stronger compared with the positive influence exerted by domain III. However, EGF binding to domain III somehow overcomes the inhibitory effect of domain I. Thus, although we think of activation by ligand as a positive step, it may, instead, simply reflect the ability of a ligand to remove a negative constraint. Future studies with the receptor in which different domains are swapped will allow us to understand the role of individual domains in inter-receptor association and dissociation. Furthermore, studies with the receptor in which potential glycosylation sites are mutated will enable us to understand the influence of individual oligosaccharide chains located in domain III in receptor structure and function. More importantly, such information will be useful in developing antibodies to block receptor self-dimerization as a way to control aberrant EGFR.


    ACKNOWLEDGEMENTS

We thank Drs. Webster K. Cavenee and H.-J. Su Huang of Ludwig Institute of Cancer Research, La Jolla, CA, for providing us with U87MG.WtEGFR and U87MG.Delta EGFR cells.


    FOOTNOTES

* This work was supported in part by a grant from the Foundation of the University of Medicine and Dentistry of New Jersey (to S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Medical Science Bldg., Rm. C-567, Dept. of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714. Tel.: 973-972-2623; Fax: 973-972-5909; E-mail: bishayee@umdnj.edu.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005599200


    ABBREVIATIONS

The abbreviations used are: EGFR, epidermal growth factor receptor; Ab, antibody; BSA, bovine serum albumin; BS3, 3,3'-bis(sulfosuccinimido)suberate; EGF, epidermal growth factor; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin; WtEGFR, wild-type EGFR; aa, amino acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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