©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
SHC and GRB-2 Are Constitutively Activated by an Epidermal Growth Factor Receptor with a Point Mutation in the Transmembrane Domain (*)

(Received for publication, February 1, 1995; and in revised form, May 8, 1995)

Mariarosaria Miloso (1)(§) Maria Mazzotti (1) William C. Vass (3) Laura Beguinot (1) (2)(¶)

From the  (1)Laboratorio di Oncologia Molecolare, DIBIT, (2)Istituto di Neuroscienze e Bioimmagini del CNR, HS Raffaele, Milano, Italy and the (3)Laboratory of Molecular Oncology, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A single point mutation, Glu Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence of EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu-EGF receptor showed a transformed phenotype and were not arrested in G(0) upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2, was constitutively associated with the GluEGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.


INTRODUCTION

Oncogenes have been proposed to derive from surface receptors by three types of mutations, resulting in a constitutive activation of tyrosine kinase activity and cell transformation. In the first type, v-ErbB oncogene arose from the avian EGF (^1)receptor by deletion of the extracellular ligand binding domain(1) , suggesting that the extracellular domain imposes a negative constraint that is relieved by EGF binding. In the second type, a point mutation in the colony-stimulating factor receptor, which corresponds to the major activating mutation in v-Fms is again located in the extracellular, ligand binding domain and induces a constitutive activation of its intrinsic kinase activity without affecting colony-stimulating factor binding(2, 24) . The third example is the single substitution of a hydrophobic residue in the transmembrane domain of Neu, LH, FGF3, and insulin receptors(3, 4, 5, 18) . The diverse ways through which constitutive activation of growth factor and hormone receptors occurs indicate that ligand-induced conformational changes can be mimicked by structural changes in several positions of the receptor.

The receptor for EGF, a 170-kDa glycoprotein, is a member of the protein tyrosine kinase family(6, 7) . Like other growth factor receptors, the EGF receptor is composed of three domains: the extracellular, ligand binding domain, a single transmembrane domain, and a cytoplasmic portion containing the kinase domain with a C-terminal tail including the autophosphorylation sites. EGF binding to the receptor triggers its tyrosine kinase activity, which is essential to induce all responses to EGF(8, 9) , leading to autophosphorylation of the receptor and tyrosine phosphorylation of specific cellular substrates (for review, see (10) and (11) ). Tyrosine autophosphorylation regulates the biological activity of the EGF-R by influencing its own kinase activity (12, 13) and by creating binding sites for physiologically important substrates containing sequence motifs called Src homology (SH2) domains.

The role of the transmembrane domain in various growth factor and hormone receptors has been studied for the past several years. The activating mutation in Neu/c-ErbB2, a substitution of a hydrophobic with a charged residue (Val to Glu), is located in the transmembrane domain(14, 15) . This mutant has a constitutively activated tyrosine kinase, increased level of autophosphorylation, and constitutive coupling and activation of phospholipase C (PLC)(16, 17) . Modulation of receptor activity through the transmembrane domain region has been described also in other cases. A similar mutation has been shown to constitutively activate the insulin receptor kinase activity and its metabolic effects (3, 4) , the luteinizing receptor (LH), which gives rise to a familiar precocious male puberty(5) , and the FGF3 receptor causing the most common genetic form of dwarfism(18) . In contrast, the identical point mutation in the EGF receptor has been previously reported not to affect at all its biological and kinase activity(19, 20) .

To further address the role of the transmembrane domain in regulating EGF receptor function, we have created the same point mutation present in the transmembrane domain of Neu/ErbB2 by exchanging Val for Glu. In this report, we show that this mutation constitutively activates EGF receptor biological and transforming ability and constitutively activates the Ras kinase cascade without affecting ligand-dependent activation.


EXPERIMENTAL PROCEDURES

Materials

EGF was from Promega, Sepharose-conjugated m-anti-pY, I-EGF, I-protein A, and I-sheep anti-mouse IgG were from Amersham Corp. Aprotinin, phenylmethylsulfonyl fluoride, and glutathione-agarose were from Sigma; calpain inhibitor I was from Boehringer Mannheim, propidium iodide was from Calbiochem, GammaBind-Sepharose was from Pharmacia Biotech Inc., and nitrocellulose membrane was from Schleicher and Schuell. G418, transferrin, cell culture medium, and serum were from Life Technologies, Inc. Polyclonal against GRB-2 and monoclonal antibodies against phosphotyrosine and against the extracellular domain of the EGF-R were from Upstate Biotechnology Inc. and Oncogene Science; monoclonal anti-GRB-2 was from Transduction Laboratories, and polyclonal anti-MAP kinase antibodies were from Santa Cruz Biotechnology. Polyclonal antibodies against intracellular domain of EGF-R were previously described(21) ; anti PLC (37) and anti-SHC sera, anti-SHC-CH, and anti-SHC-SH2 (28) were kindly provided by Graham Carpenter (Vanderbilt University, Nashville, TN) and PierGiuseppe Pelicci (University of Perugia, Italy), respectively.

EGF Receptor Mutant and Tissue Culture

Kinase-negative EGF-R mutant was previously described(37) . Human EGF-R transmembrane mutant was obtained by site-directed mutagenesis of Val with Glu, using the following oligonucleotide: 5`-CGCCACTGGGATGGAAGGGGCCCCTCCTCTTG-3`(2124-2154). Mutagenesis was performed in M13 mp19 encoding the NaeI-BstEII EGF-R cDNA fragment(2014-2889). A single-stranded template was prepared, and mutagenesis was performed as previously described (12) and confirmed by dideoxy-sequencing(23) . The mutated fragment was cloned back into pMMTV-EGF-R vector, and the mutated EGF-R cDNA (SacII-XhoI) was subcloned into the pCO 11 vector (27) to give rise to pMI 70.

NIH3T3 cells, which contain about 5,000 receptor/cell, were used for transfections. Transfections, G418 selection, foci formations, growth in low serum, and growth in agar were performed as previously described (27) . As determined by I-EGF binding and Scatchard analysis, the mutant receptors were expressed at 3.7 10^5 receptor/cell. As control, a NIH3T3 line (Cl 17) expressing 4 10^5 human wild type EGF-R/cell was used(27) . Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf serum (NCS) with penicillin, streptomycin, and glutamine.

Cell Lysates, Immunoprecipitation, and Western Blot Analysis

Cells grown to about 90% confluence in 100-150-mm dishes with 10% NCS were incubated overnight in DMEM with 10M Na(2)SeO(3) and 5 µg/ml transferrin without serum. The cells were incubated without or with 100 ng/ml EGF for 30 min at 4 °C or for 2 min at 37 °C, washed three times with ice-cold phosphate-buffered saline, and solubilized in lysis buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 10% glycerol, 1% Triton X-100) containing freshly added protease and phosphatase inhibitors (4 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 µg/ml leupeptin, 10 mM sodium orthovanadate, 20 mM sodium pyrophosphate) for 15 min at 4 °C. When required, 100 µM calpain inhibitor I was added. The lysates were clarified by centrifugation at 4 °C. Total proteins were measured by Bio-Rad.

For immunoprecipitation experiments, total cellular lysates (3 mg of protein) were incubated with appropriate antibodies for 4 h at 4 °C. For non-conjugated antibodies (SHC and PLC antibodies), immunocomplexes were collected by binding to GammaBind G-Sepharose 4B. Immunocomplexes were washed five times with an ice-cold buffer (HNTG) containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 0.1% Triton X-100; immunoprecipitates solubilized in 1 Laemmli buffer (38) were boiled and run on SDS-polyacrylamide gels.

For immunoblots, analysis proteins were transferred to nitrocellulose filters. Membranes were then incubated at room temperature for 2 h in 5% bovine serum albumin-TBS buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). Blots were incubated with primary antibodies for 2 h, washed in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), and incubated with I-protein A for 1 h. For mGRB-2 Ab, blots were incubated with I-goat anti-mouse for 2 h.

Blots were exposed to Kodak X-OMAT x-ray film at -80 °C and developed.

Cross-linking Experiments

Cells were grown to confluence in 100-mm dishes. Monolayers were washed with cold DMEM, incubated in binding medium (DMEM, 20 mM Hepes, 0.1% bovine serum albumin) with or without 100 mg/ml EGF for 1 h at 4 °C. Cells were then washed three times with ice-cold phosphate-buffered saline and solubilized in lysis buffer containing 3 mM bis-sulfosuccimidyl suberate for 20 min at 4 °C with gentle shaking. 250 mM glycine was added to terminate the cross-linking reaction, and the extract was incubated for an additional 5 min at 4 °C(41) . The cell lysates were then immunoprecipitated with a polyclonal anti-EGF-R Ab, separated by a 6% SDS-polyacrylamide gel, transferred to nitrocellulose filters, and reacted with an anti-EGF-R Ab.

Preparation of GST-GRB2 Fusion Protein and in Vitro Binding Studies

Bacterial cultures expressing pGex-GST-GRB-2-FL cDNA (a generous gift of PierGiuseppe Pelicci, University of Perugia) were grown in LB medium with 100 µg/ml ampicillin, induced with 1 mM isopropyl beta-D-thiogalactopyranoside for 5 h. Bacteria were lysed by sonication in phosphate-buffered saline solution. The GST fusion protein was purified using glutathione-agarose beads(28) .

Cell lysates (2 mg of proteins) were incubated with 5 µg of immobilized GST-GRB-2 protein at 4 °C for 90 min. The protein complexes were washed three times with HNTG buffer and boiled in Laemmli buffer. Protein complexes were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were washed and incubated with polyclonal anti-EGF-R Ab, as described above.

Cell Cycle Analysis

Cells were made quiescent by serum starvation in DMEM with 0.5% NCS for 48 h, and then incubated with 10% NCS to induce synchronous entry into cell cycle. For flow cytometric analysis, cells were trypsinized, and DNA was stained with propidium iodide solution containing RNase A and Nonidet P-40 according to Becton & Dickinson's protocol. Immediately before analysis, each sample was filtered through a Filcons filter (30 µm). Fluorescence intensity was measured with FACSCAN analysis (Becton & Dickinson). The percentages of cells in G(1), S, or G(2)/M phases of cell cycle were determined by analysis with the computer program CELLFIT (Becton & Dickinson).


RESULTS AND DISCUSSION

To test whether the point mutation in the transmembrane domain of the EGF receptor was able to induce transformation, as in the case of the the Neu oncogene, Val (corresponding to Val in the Neu/c-ErbB2 proto-oncogene) was converted into a glutamic acid, and the mutant receptor transfected into NIH3T3 cells.

As shown in Table 1, the transforming activity of Glu-EGF receptor was evident both in the presence and in the absence of EGF, while transformation by the normal receptor is strictly dependent on the presence of the ligand. Mutant receptor-induced transformation in the absence of the ligand was lower, only 20% of that of wild type in presence of EGF, but was clearly evident and reproducible in foci formation, growth in agar, and growth in low serum. In the presence of EGF, transformation was similar in mutant and wild type receptor. In addition, the mutant receptor showed a higher sensitivity to EGF for transformation. Indeed at 5 ng/ml EGF, maximum transforming activity was achieved in the mutant while only 25% of maximum with wild type receptor (Table 1, lowerpanel). The higher sensitivity to EGF is an intrinsic property of the mutant receptor not due to a difference in affinity for EGF. Low affinity mutant receptor had a K of 3.6 nM for EGF, very similar to that of wild type receptor (3.2 nM) ( (12) and data not shown). In addition, NIH3T3 cells transfected with the mutant receptor clearly showed a morphological transformed phenotype, could not be arrested in G(0) by serum deprivation, but were always actively proliferating, as indicated in Table 2. These findings demonstrate that the single point mutation in the transmembrane domain of the EGF receptor is sufficient to stimulate its oncogenic potential, rendering it, at least partially, ligand independent. This result is important because in the case of Neu/ErbB2, there have been contrasting reports on whether the same point mutation is really essential for transformation (14) or whether the proto-oncogene itself is able to transform, depending on the level of expression(26) . In the case of wild type EGF receptor, transformation is only and strictly ligand-dependent and not dependent on the level of expression(25, 27) . In addition, in our experiments, both mutant and wild type receptors are expressed at similar levels, at about 4 10^5 receptors/cells as determined by Scatchard analysis (data not shown and Fig. 1). Our findings are different from those by Kasles et al.(19) and Carpenter et al.(20) , which have previously reported that the same point mutation in the EGF receptor was not transforming. However, Carpenter (20) has used B82 fibroblasts instead of NIH3T3 cells, and in their successive experiments with NR6 cells even wild type receptor was not transforming in the presence of physiological concentrations of EGF (22) . It is therefore possible that the difference is dependent on the higher sensitivity and reproducibility of the NIH3T3 transformation assays(25, 26, 27) .






Figure 1: Autophosphorylation and tyrosine phosphorylation of cellular proteins by Glu-EGF receptor in the absence of EGF. Panel A, NIH3T3 expressing wild type (lanes3, 4) and mutant EGF-R (lanes1, 2) were incubated for 30 min with and without 100 ng/ml EGF and lysates prepared. Total cellular extracts (150 µg of proteins) were run on a 7.5% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted with anti-phosphotyrosine Ab. Lanes1 and 3, extracts from untreated cells; lanes2 and 4, extracts from cells preincubated with EGF. Arrow indicates EGF-R. Exposure of the autoradiogram was for 18 h. Panel B, total cellular extracts prepared from untreated (lanes1, 3) or EGF-treated cells (lanes2, 4) were run on a 10% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted with anti-phosphotyrosine Ab. Lanes1 and 2, extracts from cells expressing mutant EGF-R; lanes3 and 4, from wild type (W.T.) expressing cells. Exposure of the autoradiogram was for 48 h.



To elucidate the mechanism by which the point mutation in the transmembrane domain of the receptor is able to induce transformation, we performed a biochemical characterization of the Glu-EGF receptor. As shown in Fig. 1(panelA), the mutant receptor was autophosphorylated in the absence of EGF, albeit at a lower level than in the presence of EGF. Quantitation of the band intensity indicated that, in the absence of EGF, the mutant receptor was phosphorylated 4-fold less than in the presence of EGF. This finding correlates well with the 5-fold biological activity observed in the absence of EGF (Table 1). In addition, tyrosine-kinase activity of the receptor toward cellular substrates, assayed with anti-pY antibodies, was higher than that of wild type receptor stimulated with EGF and was again evident in the absence of the ligand (Fig. 1, panelB).

A band of approximately 52 kDa appeared to be phosphorylated specifically in the absence of EGF in Glu cell extracts (Fig. 1, panelB). To test whether part of this 52-kDa band corresponded to the SHC adaptor protein, extracts were immunoprecipitated with anti-pY antibody and then blotted with anti-SCH antibody. As shown in Fig. 2(panelA), SHC was indeed phosphorylated by the mutant receptor in the absence of EGF, while phosphorylation by the wild type receptor occurred only in the presence of EGF. In agreement with the finding reported by Pelicci et al.(28) , low tyrosine phosphorylation of the 52-kDa form was evident in NIH3T3 cells expressing wild type receptors even without EGF (13% of EGF-dependent phosphorylation). However, basal phosphorylation was much more intense in the mutant-expressing cells (85% of EGF-induced). In addition, in mutant-expressing cells also the 46-kDa form of SHC was phosphorylated in the absence of EGF at levels comparable with those observed in the presence of EGF. In contrast, another SH2-containing substrate, PLC, was not constitutively phosphorylated in cells expressing the mutant receptor, while in the presence of EGF, PLC was 3-fold more phosphorylated by mutant than wild type receptor (Fig. 2, panelB). On the contrary, the point-mutated Neu induces a constitutive PLC phosphorylation, while SHC and GRB-2 were not analyzed(17) . Interestingly, the only other EGF receptor mutant that possesses transforming activity in absence of EGF, Dc 214, was obtained by deletion of the whole receptor cytoplasmic tail. Dc 214 is able to phosphorylate SHC and GAP and to stimulate Ras in the GTP-bound state activating the MAP kinase pathway, while it does not phosphorylate PLC(29, 30) . These findings indicate that phosphorylation and activation of SH2-containing substrates is specific and may suggest a priority in the pathway of signal transduction by the EGF receptor. Indeed, SHC is the only EGF-R substrate found associated in high amounts with EGF receptor; 10% of the total SHC is associated with the activated EGF receptor, compared with only 1% of total PLC(30) . In addition, SHC is also phosphorylated by physiological levels of receptor, as in parental NIH3T3 cells(31) . As shown in Fig. 2, panelC, in mutant-expressing cells GRB-2 is associated with SHC in the absence of EGF (55% of EGF-induced), whereas in wild type receptor-expressing cells GRB-2 is associated with SHC only in the presence of EGF.


Figure 2: Phosphorylation of SHC, PLC, and GRB-2 association with SHC by wild type (W.T.) and mutant EGF-R. Panel A, top total cellular lysates (3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated by anti-phosphotyrosine Ab, blotted on nitrocellulose filters, and reacted with anti-SHC-SH2 Ab. Lanes1 and 3, untreated cells; lanes2 and 4, cells pretreated with EGF. The arrows indicate SHC proteins. Exposure of the autoradiogram was for 20 h. Bottom, 150 µg of total lysates were run on a 10% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted with anti-SHC-SH2 Ab. Arrow indicates SHC bands. Exposure of the autoradiogram was for 16 h. In Panel B, top, lysates (3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with anti-PLC Ab, transferred to nitrocellulose filters, and reacted with antiphosphotyrosine Ab. Lanes1 and 3, untreated cells; lanes2 and 4, cells pretreated with EGF. The arrow indicates PLC. Exposure of the autoradiogram was for 20 h. Bottom, 150 µg of lysates were run on a 7.5% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted with anti-PLC Ab. Arrow indicates PLC. Exposure of the autoradiogram was for 78 h. In panel C, top, lysates (3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with anti-SHC-CH Ab, transferred to nitrocellulose filters, and reacted with monoclonal anti-GRB-2 Ab. Lanes1 and 3, untreated cells; lanes2 and 4, treated with EGF. The arrow indicates GRB-2. Exposure of the autoradiogram was for 72 h. Bottom, 100 µg of total cell lysates were run on a 12.5% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted with polyclonal anti-GRB-2 Ab. Arrow indicates GRB-2. Exposure of the autoradiogram was for 16 h.



We then tested whether GRB-2 was also constitutively associated with the mutant EGF receptor. In Fig. 3, panelA, GRB-2 was coimmunoprecipitated with the mutant receptor both in absence and presence of EGF while only in the presence of the ligand with wild type receptor. Quantitation indicated that constitutive association amounted to 60% of that in the presence of EGF. Furthermore, in vitro the GST-GRB-2 fusion protein was able to interact with the mutant receptor both when stimulated or not with EGF (Fig. 3, panelB). These data indicate that GRB-2 is able to associate constitutively with mutant receptor.


Figure 3: GRB-2 association with wild type (W.T.) and mutant EGF receptors in vivo and in vitro. Panel A, top, lysates (3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with polyclonal anti-EGF-R Ab, transferred to nitrocellulose filters, and reacted with polyclonal anti-GRB-2 Ab. Lanes1 and 3, untreated cells; lanes2 and 4, treated with EGF. The arrow indicates GRB-2. Exposure of the autoradiogram was for 72 h. Bottom, 100 µg of total cell lysates were run on a 12.5% SDS-PAGE gel, transferred on to a nitrocellulose filter, and reacted with polyclonal anti-GRB-2 Ab. Arrow indicates GRB-2. Exposure of the autoradiogram was for 72 h. Panel B, lysates (2 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were incubated with 5 µg of GST-GRB-2 fusion protein run on 7.5% gels, transferred to nitrocellulose filters, and reacted with anti-EGF-R Ab. Lanes1 and 3, extracts from untreated cells; lanes2 and 4, treated with EGF. EGF-R is indicated. Exposure of the autoradiogram was for 16 h.



Since binding of the transmembrane mutant receptor to GRB-2 should bring Sos in contact with Ras and stimulate Ras in the GTP-bound state, we tested whether the downstream Ras-activated kinase pathway was constitutively activated in mutant-expressing cells. As shown in Fig. 4, MAP kinase was constitutively phosphorylated in mutant-expressing cells (50% of EGF-induced), indicating that the mitogenic pathway of Ras was activated and hence might be responsible for the transformed phenotype induced by Glu-EGF receptor. Constitutive MAP kinase activation has been found so far only in cell lines expressing the oncogenic form of Ras and Raf(32, 33) . Interestingly, MAP kinase activation has been implicated in the G(0)-G(1) transition phase(34, 35) , and a constitutive MAP kinase activity could explain why EGF receptor mutant-expressing cells could not be arrested in G(0) (Table 1).


Figure 4: Phosphorylation of MAP kinase in cells expressing wild type (W.T.) and mutant EGF receptors. Lysates (50 µg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were run on a 10% gel, transferred to a nitrocellulose filter, and reacted with anti-MAP kinase Ab. Lanes1 and 3, extracts from untreated cells; lanes2 and 4, treated with EGF. Phosphorylated MAP kinase (pp42) and MAP kinase (p42) are indicated. Exposure of the autoradiogram was for 16 h.



As evident from Fig. 1, the mutant EGF receptor was present in two forms of 170 and 150 kDa, both in the absence and presence of EGF, and both forms were tyrosine phosphorylated. The 150-kDa form was previously described to be a proteolytic digest of the intact receptor due to a calcium-dependent protease, calpain, which cleaves the cytoplasmic tail of the EGF receptor(36) . This finding, consistently observed with mutant receptor in immunoprecipitates by anti-pY and anti-EGF-R antibodies, suggests that Glu receptor is more susceptible to cleavage by calpain. We tested six different clones and two pools of transfectants derived from independent transfections with the mutant receptor, and we always observed the 170- and 150-kDa forms (data not shown), indicating that the presence of two EGF-R bands is not a clonal artifact. To confirm that the 150-kDa form was due to the proteolytic activity of calpain, we prepared extracts using a specific calpain inhibitor in addition to the other protease inhibitors always present in the lysis buffer. As shown in Fig. 5, in the presence of the calpain inhibitor, the mutant receptor appeared as a 170-kDa form, indicating that the smaller band is really due to calpain cleavage. Indeed, with the wild type EGF-R, we never observed the 150-kDa form in extracts untreated with EGF. In some cases, the 150-kDa form was evident upon EGF treatment (Fig. 5, lane4) but never as intense as with the mutant receptor. Thus, possibly the cleavage site is exposed upon EGF treatment in the wild type receptor and by the Glu mutation in the mutant receptor. This suggests that the mutation may induce a conformational change in the cytoplasmic domain of the receptor, which mimicks ligand activation. Quantitation of the 170- and 150-kDa forms indicated that they were present almost at a 1:1 ratio (60 and 40%, respectively). The cleavage site of calpain is Lys, which is equivalent to removal of the last 150 amino acids of the receptor cytoplasmic tail. The cleavage into the 150-kDa form cannot be responsible for the transforming phenotype of the mutant receptor. We have previously shown that removal of the last 123 or 165 amino acids of the EGF receptor dramatically decreases its biological and transforming ability to less than 10% of the wild type and abolishes substrate phosphorylation(37, 38) . Therefore, the transmembrane point mutation and not the deletion of part of the C-terminal receptor tail is responsible for constitutive receptor activity.


Figure 5: Calpain cleaves Glu-EGF receptor in cells untreated and treated with EGF. Lysates from mutant (lanes3, 4) and wild type (W.T.) EGF-R-expressing cells (lanes1-4) were prepared in the absence (left panel) or presence (right panel) of a specific calpain inhibitor I, run on a 10% SDS-PAGE gel, transferred on to a nitrocellulose filter, and reacted with anti-phosphotyrosine Ab. Lanes1 and 2, extracts from untreated cells; lanes3 and 4, extracts from cells treated with EGF. Arrow indicates the EGF-R. Exposure of the autoradiogram was for 18 h.



Dimerization is considered the first event occurring after ligand binding able to bring together the two kinase domains that phosphorylate each other and start the chain of tyrosine phosphorylation(39) . We therefore tested whether the point mutation in the transmembrane domain of the EGF-R was able to induce dimerization and whether dimer formation in the absence of EGF could account for the increased kinase activity and the transforming ability. However, in agreement with other findings(19, 20) , cross-linking experiments showed that Glu mutant receptor was able to dimerize in the presence of EGF but not in its absence (Fig. 6). Therefore, formation of dimer receptors is not an essential step for the constitutive functional activity of the transmembrane mutant receptor, while it may contribute to its full activity in the presence of EGF.


Figure 6: In vivo cross-linking of wild type and mutant EGF receptors. NIH3T3 cells expressing wild type (lanes1, 2) and mutant EGF receptors (lanes3, 4) were incubated in the presence (lanes2, 4) or absence of EGF (lanes1, 3). Lysates were prepared with 3 mM bis-sulfosuccimidyl suberate as cross-linking reagent and immunoprecipitated with anti-EGF-R Ab, run on a 6% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and reacted with anti-EGF-R Ab. Arrows indicate the monomer and dimer forms of EGF-R. Exposure of the gel was for 18 h.



In conclusion, substitution of a hydrophobic with a negatively charged residue constitutively activates the EGF receptor as well as Neu, LH, FGF3, and insulin receptors. The mechanism by which this single substitution is able to activate different receptors is still not resolved. Three-dimensional structure studies performed on peptides corresponding to the transmembrane domain of Neu indicate that there is no significant difference in the conformation of wild type and mutated sequences, but the results are consistent with alternative models involving receptor-packing interactions(40) . Further studies are necessary to address this point.


FOOTNOTES

*
This work was supported by grants from Associazione Italiana Ricerca sul Cancro and from Consiglio nazionale delle Ricerche (PFACRO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from Fondazione H S Raffaele, Italy.

To whom correspondence should be addressed: Laboratory of Molecular Oncology, DIBIT, H S Raffaele, Via Olgettina 60, 20132 Milano Italy. Tel.: 39-2-2643-4747; Fax: 39-2-2743-4844.

(^1)
The abbreviations used are: EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; PLC, phospholipase C ; DMEM, Dulbecco's modified Eagle's medium; NCS, newborn calf serum; Ab, antibody; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase.


ACKNOWLEDGEMENTS

We thank Dr. Graham Carpenter (Vanderbilt University, Nashville, TN) for helpful discussions and for providing anti-PLC Ab, Dr. PierGiuseppe Pelicci (University of Perugia, Italy) for anti-SHC-SH2 and -CH Ab and pGEXSH2SHC plasmid, and Kjoung-Jin Shon and Kristian Helin (Fibiger Institute, Copenhagen) for help in mutant construction.


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