A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

Experimental Cell Research 302 (2005) 108 – 114 www.elsevier.com/locate/yexcr

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization Sami Aifaa, Jan Aydina,b, Gunnar Nordvallc, Ingemar Lundstrfmd, Samuel P.S. Svenssona,c,*, Ola Hermansone a Department of Pharmacology, Linko¨ping University, SE-58185 Linko¨ping, Sweden Department of Physiology and Pharmacology, Karolinska Institute, SE-17177 Stockholm, Sweden c AstraZeneca R&D So¨dert7lje, SE-15165 So¨derta¨lje, Sweden d Department of Applied Physics and Measurement Technology, Linko¨ping University, SE-58185 Linko¨ping, Sweden e Department of Cell and Molecular Biology (CMB), Karolinska Institute, SE-17177 Stockholm, Sweden b

Received 8 March 2004, revised version received 9 August 2004 Available online 21 September 2004

Abstract The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645–R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SKN-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function. D 2004 Elsevier Inc. All rights reserved. Keywords: Epidermal growth factor; Signal transduction; Tyrosine kinase activity; SK-N-MC

Introduction Epidermal growth factor receptor (EGFR) belongs to the Erb receptor tyrosine kinase family, which includes ErbB1 (EGFR), ErbB2 (c-Neu), ErbB3, and ErbB4 subtypes [1]. The receptor consists of an extracellular ligand-binding domain, a single a-helical transmembrane domain, and an intracellular domain containing a tyrosine kinase domain flanked by regulatory sequences [2].

* Corresponding author. Fax: +46 13 149106. E-mail address: [email protected] (S.P.S. Svensson). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.08.032

The EGFR has been shown to be fundamental for normal cell growth control and development but is also highly implicated in a variety of pathophysiological conditions such as cancers of the breast, lung, and neck [3]. As a consequence, much research has been carried out in the field of EGFR-selective antagonists and inhibitors of EGFR tyrosine kinase [4]. Many models place the EGFR at the convergence point for several signaling transduction pathways [5]. For example, the proliferative effects of different G-protein-coupled receptors (GPCR) have been shown to be dependent on EGFR [6]. Following ligand binding, the EGFR dimerizes followed by autophosphorylation of specific tyrosine residues within

S. Aifa et al. / Experimental Cell Research 302 (2005) 108–114

the intracellular domain of the receptor, providing docking sites for signaling proteins. These events lead to the formation of a multimeric signal transduction complex and the subsequent activation of signaling pathways such as the MAP kinase and JNK kinase cascades [1,7]. X-ray crystallography studies of the extracellular domain of the EGFR, bound EGF or TNF-a, have suggested that the dimerization process is driven by a ligand-induced receptor-dependent mechanism, that is, ligand binding induces or stabilizes a conformation change of the receptor allowing for the formation of dimers [8,9]. How the conformational change induced by ligand binding causes activation of the intracellular kinase domain remains unclear. The tyrosine kinase domain and the C-terminal tail of the receptor are critical in a variety of EGFR-dependent signaling events [1]. However, the juxtamembrane domain (JM) seems to play an important role for receptor regulation and signaling specificity, such as phosphorylation by protein kinase C and MAP kinase [10]. We and others have previously demonstrated that the 13 amino acids within the JM region, named P13 (R645–R657), are involved in the interaction with calmodulin [11,12]. The P13 peptide has also been shown to stimulate GTP binding and GTPase activity of Gs-proteins [13,14]. Furthermore, this peptide has been suggested to induce a conformational change in the EGFR and thereby modulate the receptor tyrosine kinase activity [15]. The peptide has homology to known nuclear localization signals (NLS) and has in addition been considered to function as an NLS for the full-length EGFR [16]. In this article, we have investigated the role for the P13 peptide in EGFR function by studying the characteristics of the intracellular domain and full-length EGFR lacking P13. We have demonstrated that EGFR lacking P13 fails to dimerize and autophosphorylate. Based on our results and in silico modeling, we suggest a structural model for EGFR dimerization and autophosphorylation.

Materials and methods Strains and reagents Escherichia coli JM109-competent cells (Promega) were used for plasmid construction and maintenance. Vector pLXSN, containing the full-length human EGFR, was a gift from Dr. Axel Ullrich (Max-Planck-Institute, Martinsried, Germany). Vector pCDNA3.1+ from Invitrogen was used for EGFR expression in mammalian cells. SK-N-MC and COS-7 cell lines, both from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, were the main hosts of this expression. Anti-EGFR antibodies (sc-03, sc-03-agarose conjugate, sc-120) and anti-phosphotyrosine (PY99/sc-7020) were obtained from Santa Cruz Biotechnology. Donkey anti-

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rabbit antibody was purchased from Amersham Pharmacia Biotech. Constructions The strategy was based on PCR amplification from pLXSN-HER, restriction digestion of the amplified fragment with the same restriction enzymes as the vector pcDNA3+ and ligation. DNA amplification was performed with Pfu (Stratagene). We used S-300 columns (Amersham Pharmacia Biotech) in order to remove primers and nucleotides from PCR products. We adopted a Qiagen kit to remove restriction enzymes from digested DNA before ligation. Ligation was done by the bready to goQ T4 DNA ligase from Amersham Pharmacia Biotech. The following primers were used in our PCR amplifications (Fig. 1): (1) (2) (3) (4) (5) (6) (7)

5V-GA GCT AGC AGC GAT GCG ACC CTC CGG3V SENSE 5V-GA CTC GAG CCT CCG TGG TCA TGC TCC A-3V ANTI-SENSE 5V-CG GCT AGC ATG CGA AGG CGC CAC ATC GTT C-3V SENSE 5V-TG GGT ACC CTG CTG CAG GAG AGG GAG C-3V SENSE 5V-GA TCT AGA CCT CCG TGG TCA TGC TCC A3V ANTI-SENSE 5V-GA GCT AGC AGC GAT GCG ACC CTC CGG3V SENSE 5V-AC GGT ACC CAT GAA GAG GCC GAT CCC CAG-3V ANTI-SENSE

Primers (1) and (2) were served for the full-length EGFR fragment amplification. The PCR fragment was digested by NheI–XhoI and inserted in pCDNA3+, linearized by the same restriction enzymes. The obtained vector was called pCDNA-HER (Human EGF Receptor). Primers (2) and (3) were used in the amplification of the intracellular part of EGFR (TKJM; Tyrosine Kinase Juxta Membrane domain). The obtained DNA fragment was digested with NheI–XhoI and inserted in pCDNA3+. This construct was named pCDNA-TKJM. Primers (4) and (5) served in the amplification of DTKJM DNA corresponding to the intracellular EGFR domain lacking P13. The obtained fragment was digested with KpnI–XbaI and inserted in pCDNA3. The obtained vector was called pCDNA-DTKJM. Primers (6) and (7) were used in amplifying Ext-TM DNA fragment, corresponding to HER without its intracellular domain. The amplified DNA fragment was inserted in pCDNA3+ using NheI and KpnI sites. The obtained construction was called pCDNA-Ext-TM. The PCR DNA product DTKJM was inserted into pCDNA-Ext-TM using KpnI–XbaI restriction sites. The obtained vector was called pCDNA-DHER. Restriction enzymes and DNA sequencing were used to verify all constructions.

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Fig. 1. Pfu-amplified products of EGFR encoding DNA (right) and corresponding constructions in pCDNA3 vector (left). HER: full-length EGFR encoding DNA; DHER: P13-deleted HER; TKJM: intracellular EGFR encoding DNA; DTKJM: P13-deleted TKJM; Ext-TM: DNA fragment corresponding to the extracellular and trans-membrane domains of HER. These PCR fragments were cloned in pCDNA3 vector and used in SK-N-MC and COS-7 cell transfections.

Cell culturing and transfection SK-N-MC and COS-7 cells were incubated at 378C with 5% CO2 in DMEM-medium supplemented with 10% and 5% heat-inactivated fetal bovine serum, respectively. When reaching 40–50% confluence, the cells were transfected using JetPEI (Polyplus-transfection SAS, Strasbourg, France) according to the manufacturers instructions. Stable transfected cell lines were obtained by G418 selection (700 mg/l) during 1 month of cell culturing. Western blotting and immunoprecipitation Following stimulation with EGF (100 ng/ml) for 15 min at 378C, cells were starved of serum overnight and cellular extracts were prepared and immunoprecipitated using AntiEGFR sc-03-agarose conjugate antibodies as described by Santa Cruz Biotechnology. Membranes were extracted in a nondenaturating buffer as described by Monstein et al. [17] and membranous proteins were immunoprecipitated with anti-EGFR sc-120 antibodies according to the supplier’s protocol. Immunoblotting was performed with anti-EGFR (sc-03) or anti-phosphotyrosine (PY99) antibodies. Primary antibodies were detected by horseradish-peroxidase-conjugated secondary antibodies and chemoluminescence (ECL, Amersham or SuperSignal, Pierce) was recorded by a CCD camera (Fuji). Immunocytochemical staining Cells were grown on Lab-TekII tissue culture chamber/ slides system (Nalge Nunc International, USA) and fixed with 10% formalin. Cells were incubated with sc-120 primary antibody followed by Texas red-conjugated antimouse secondary antibody in 1 PBS with 1% bovine serum albumin and 0.1% Triton X (both Sigma). Immuno-

cytochemistry was performed according to Hermanson et al. [18]. Nontransfected SK-N-MC cells served as negative control for EGFR staining.

Results and discussion Immunoprecipitation of the intracellular domain of EGFR with or without P13 deletion The intracellular domains of wild-type EGFR and of EGFR lacking the P13 (R645–R657) were transiently transfected in COS-7 cells expressing endogenous EGFR. The corresponding proteins were called tyrosine kinase juxta membrane domain (TKJM) and DTKJM, respectively (Fig. 1). Immunoblotting of whole cell extracts from COS-7 cells with an antibody directed against the intracellular portion of EGFR (EGFR-IC) revealed prominent expression of proteins at around 70 kDa, the predicted sizes of TKJM and DTKJM (Fig. 2). By using an antibody specifically against phosphorylated tyrosine (pTyr), we found that TKJM but not DTKJM was phosphorylated after EGF stimulation of COS-7 cells (Fig. 2). The same approach revealed a basal tyrosine phosphorylation of endogenous EGFR that was significantly increased after EGF stimulation (Fig. 2). Immunoprecipitation using anti-EGFR-IC confirmed that TKJM and DTKJM was expressed to a similar extent and that only TKJM was phosphorylated after EGF stimulation (Fig. 2, lower panel). There was no significant difference in subcellular localization of TKJM and DTKJM as revealed by immunoblotting on cell extracts after subcellular fractionation (data not shown). We then performed co-immunoprecipitation of the membrane fraction of transfected COS-7 cells with an antibody directed against the extracellular domain of EGFR (EGFR-EC). Subsequent immunoblotting against EGFR-IC

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Fig. 2. TKJM and DTKJM expression and tyrosine phosphorylation in COS-7 cells. TKJM and DTKJM were expressed in COS-7 cells. An antibody against the intracellular portion of EGFR (EGFR-IC) revealed a band migrating at 70 kDa, the predicted size of TKJM and DTKJM. Immunoblotting of antiphosphotyrosine (pTyr) revealed a band only in cells stimulated with EGF and transfected with TKJM. Endogenous EGFR is basally phosphorylated but a significant increase in tyrosine phosphorylation is seen after EGF stimulation. Immunoprecipitation using an anti-EGFR-IC antibody and subsequent immunoblotting using the same antibody or pTyr antibody revealed that TKJM and DTKJM were expressed at roughly similar levels but that only TKJM was phosphorylated after EGF stimulation. Nontransfected, sham-stimulated, or EGF-stimulated COS-7 cells were used as controls (Ctrl) in respective experiments.

and pTyr revealed that TKJM, but not DTKJM, dimerized with endogenous EGFR (Fig. 3) and that TKJM, but not DTKJM, showed increased tyrosine phosphorylation after EGF stimulation (Fig. 3). Our results demonstrate that P13 is required for (i) the dimerization of the intracellular domain of EGFR with full-length EGFR and (ii) for

Fig. 3. TKJM, but not DTKJM, can be co-immunoprecipitated with wildtype EGFR. TKJM and DTKJM were transfected in COS-7 cells and the cells were subsequently either sham stimulated or stimulated with 100 ng/ ml EGF for 15 min. Cell lysates were prepared and immunoprecipitated with anti-extracellular EGFR antibody (EGFR-EC) followed by immunoblotting using EGFR-IC or pTyr antibodies as indicated. Only TKJM could be co-immunoprecipitated with endogenous EGFR after EGF stimulation. This result indicates that the interaction between TKJM and endogenous wild-type EGFR requires EGF stimulation and the P13 region.

phosphorylation of tyrosine residues within the intracellular domain of EGFR. Consequences of the P13 peptide deletion within the juxtamembrane region of EGFR To further investigate the role for P13 in the context of full-length EGFR, we utilized SK-N-MC cells that lack endogenous EGFR expression. A mutant EGFR lacking P13 (R645–R657) from the intracellular juxtamembrane region called DHER was stably transfected in SK-N-MC cells. In parallel experiments, SK-N-MC cells were stably transfected with wild-type human EGFR (HER; human EGF receptor). We then analyzed the tyrosine phosphorylation of HER and DHER in SK-N-MC cells stimulated with EGF by immunoblotting with pTyr. This experiment revealed a phosphorylated protein migrating at around 170 kDa in cells transfected with HER after EGF stimulation, whereas no such phosphorylated protein could be detected in cells transfected with DHER (Fig. 4). To investigate if this result was secondary to a lower expression of DHER, we immunoprecipitated HER and DHER with anti-EGFR-EC followed by immunoblotting using anti-EGFR-IC and antipTyr. This experiment revealed that although both receptors could be immunoprecipitated from SK-N-MC cell extracts to a similar extent, only the wild-type HER was tyrosine phosphorylated (Fig. 4, lower panels). Immunocytochemistry and subsequent confocal microscopy revealed that the subcellular distribution of the transfected HER and DHER was identical (Fig. 5).

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Fig. 4. HER, but not DHER, is phosphorylated after stimulation of SK-NMC cells. SK-N-MC cells stably transfected with HER or DHER were either sham stimulated or stimulated with EGF (100 ng/ml, 15 min). Immunoblotting of whole cell lysates with pTyr antibody revealed a phosphorylated protein migrating at 170 kDa, the predicted size of EGFR, only in SK-N-MC cells transfected with HER and stimulated with EGF. Immunoprecipitation using anti-EGFR-EC antibody and subsequent immunoblotting using EGFR-IC or pTyr antibody revealed that although HER and DHER were expressed at a similar level, only HER was phosphorylated after EGF stimulation. Nontransfected SK-N-MC cells stimulated with EGF were used as control (Ctrl).

Immunoblotting of plasma membrane fractions confirmed this result (data not shown). The lack of phosphorylation of DHER was thus not the result of inappropriate cellular localization of the protein (Fig. 5). Altogether, results from SK-N-MC cells further strengthen the hypothesis that the P13 peptide (R645–R657) plays a key role in EGFR dimerization and autophosphorylation. A model for EGFR dimerization and autophosphorylation Previous models for the mechanisms underlying EGFR dimerization have included roles for the trans-membrane region of EGFR [19] as well as ligand-independent dimerization of EGFR [20]. Analogous crystal structure

analysis using TGFa as a ligand revealed a dimerization mediated by the extracellular domain of EGFR [9]. However, the question of how receptor dimerization might lead to increased receptor activation remains unsolved. Since a highly acidic peptide sequence of 13 residues is present downstream of the tyrosine kinase domain (corresponding to 979 DEEDMDDVVDADE991, denoted the MIG domain), we speculated in a P13/MIG domain dimerization model within the EGFR intracellular domains. The Mig-6 protein is a functional antagonist to EGFR that has been shown to bind to residues R985–R995 of EGFR. It has been suggested that Mig-6 thereby interferes with the dimerization process and thus causes functional antagonism of EGFR [21]. In order to test if an interaction between the P13 and the MIG domain could be possible, we generated an in silico model using the recently published X-ray structure of the truncated intracellular domain of EGFR [22]. The model showed that the negatively charged MIG domain in one EGFR monomer and the positively charged P13 region of another EGFR monomer are well positioned to be able to interact via electrostatic interactions (Fig. 6). The core site of interaction in the proposed EGFR dimer has respective betasheet subdomains in close proximity. This could further stabilize the interaction between individual monomeric units in a dimeric structure. Such a mechanism could not only explain the ligand-independent intracellular dimerization of EGFR [22], but also the auto-phosphorylation of tyrosine residues. We speculate, in accordance with other models of G protein-coupled receptor (GPCR) activation, that conformational changes of the extracellular domain of the EGFR could be propagated through the transmembrane domain, for example, by rotation of the transmembrane helix, to change the conformation of the juxtamembrane domain leading to receptor dimerization and kinase activation. In the present study, we show that the initial 13 amino acids (P13) within the intracellular juxtamembrane ( 645RRRHIVRKRTLRR657) of the human EGFR are involved in the dimerization process. Intracellular domain of EGFR lacking the P13 region could not be immunoprecipitated with antibodies against extracellular EGFR and

Fig. 5. HER and DHER show similar subcellular localization in SK-N-MC cells. Stably transfected SK-N-MC cells with HER and DHER were analyzed by confocal microscopy. Immunoreactivity was detected with EGFR-EC antibody (red). There is no aberrant subcellular localization of DHER as compared to HER. DAPI was used to stain for nuclei (blue).

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Fig. 6. Modeling of intracellular EGFR dimerization. A model of how intracellular domains of EGFR could dimerize as seen from the plane of the membrane (A) or as seen perpendicular to the plane of the membrane (B). The published X-ray structure of the intracellular domain of EGFR [22] does not contain the juxtamembrane amino acids. An N-terminal extended model was therefore constructed using the following considerations and assumptions: (i) the secondary structure of the majority of this region (R644–E666) is assumed to be alpha-helical. (Secondary structure prediction in Cameleon (Accelrys Inc) using GORII method suggesting that this may be the case (unpublished data).) (ii) The two proline residues (Pro667/Pro670) at the end of this region are assumed to induce a turn that connects the helix to the crystallized domain. (iii) The missing fragment was generated within Sybyl 6.9 (Tripos Inc.) using standard parameters for alpha-helix and turn conformations. (iv) The two EGFR monomers (green and purple) were manually aligned to form a proposed dimer structure. The negatively charged MIG domain (red) and positively charged P13 region (yellow) are well positioned to interact via electrostatic interactions. The site of interaction in the proposed EGFR dimer has respective beta sheet subdomains in close proximity, which could further stabilize the interaction between individual monomers. The ATP binding pocket is not part of the binding interface but is facing the solvent. The P13 domain thus connects to the hypothetically positioned membrane-spanning region of the EGFR.

does not become tyrosine phosphorylated by the full-length wild-type EGFR. To address the question concerning the role of P13 in the context of the full-length receptor, we also made a deletion construct of the P13 region from the fulllength EGFR. Compared to the wild-type HER, the mutant receptor (DHER) does not show any tyrosine kinase activity upon EGF stimulation (Fig. 4). A potential explanation for this could be that DHER has lost the ability to bind EGF. However, several recently published studies have shown that truncated versions of EGFR, lacking the entire intracellular domain of the receptor, still bind EGF ligands [8,9]. The lack of function of DHER is thus most probably related to its inability to dimerize and thereby activate the

intracellular kinase domain. Taken together, our results demonstrate that the 13 amino acid, basic residue-rich, peptide sequence plays a key role in the EGFR dimerization process.

Acknowledgments We thank Drs. Joshua Duckworth and Ana Teixeira for critical reading of the manuscript. This study was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research (SSF), and the Swedish Children’s Cancer Society.

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