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EGFR receptor complexes and EGFR monomers, but not to ErbB2
Biochimica et Biophysica Acta 1771 (2007) 873 – 878 www.elsevier.com/locate/bbalip
Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2 Simona Milani, Elena Sottocornola, Stefania Zava, Patrizia Berselli, Bruno Berra, Irma Colombo ⁎ Institute of General Physiology and Biological Chemistry, University of Milan, Via Trentacoste 2-20134, Milan, Italy Received 2 February 2007; received in revised form 10 April 2007; accepted 11 April 2007 Available online 20 April 2007
Abstract Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM3 in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM3 upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors coimmunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM3, but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM3 is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM3 on ErbB2 activation are mediated by EGFR. © 2007 Elsevier B.V. All rights reserved. Keywords: ErbB2; EGFR; GM3; Gangliosides; HC11 cell line
1. Introduction Gangliosides (sialic acid-containing glycosphingolipids, GSLs) are ubiquitous eukaryotic cell membrane components. They have been implicated in the modulation of cell differentiation and proliferation, and in oncogenesis, through their interaction with transmembrane signalling molecules such as growth factor receptor tyrosine-kinases [1–3]. In this view, ganglioside GM31 inhibits EGF-induced cell growth in the human epidermoid carcinoma cell line A431 through inhibition of EGFR auto-phosphorylation [5]. Similar behaviour has been observed for gangliosides GM3 and GM1 upon platelet-derived growth factor receptor (PDGFR) in Swiss 3T3 fibroblastic cells [6] and in neuroblastoma SH-SY5Y ⁎ Corresponding author. Tel.: +39 02 50315781; fax: +39 02 50315775. E-mail address: [email protected] (I. Colombo). 1 The gangliosides cited in the text are abbreviated according to Svennerholm's nomenclature (Ref. [4]). 1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2007.04.008
cells [7]. On the other hand, GM1, by tightly binding to nerve growth factor receptor (NGFR) with consequent dimer formation and its tyrosine auto-phosphorylation, enhances the activity of the receptor in NGF-responsive cells [8]. Other studies have also shown that gangliosides can preferentially alter the signalling of specific growth factors, as in the case of retinal glial cells, where GM3 alters the auto-phosphorylation of fibroblast growth factor receptor (b-FGFR) without affecting EGFR [9]. Altogether, these studies suggest that ganglioside modulation of growth factor receptor function may represent a general phenomenon and that specific gangliosides interact with specific receptors. Many data are available about the modulation of EGFR activation by gangliosides [10–13]; however, very little is known about the effects of gangliosides on other members of the ErbB receptor super-family. The ErbB receptor super-family belongs to the type I family of transmembrane receptor tyrosine-kinases and includes four members: EGFR (ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3) and ErbB4 (HER4) [14,15]. ErbB proteins are
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engaged in extensive network of homo- and hetero-associations resulting in signal diversification and multiple cellular responses [16]. The relative expression levels of the various receptors and the concentration of their respective ligands determine the composition of homo- and heterodimers [17,18]. Among the ErbB receptors, our attention focused on the ligand orphan ErbB2 tyrosine-kinase. ErbB2 plays an important role in growth and differentiation of the mammary gland; its amplification and over-expression is found in many kind of tumours and represents a main feature for a pour prognosis of breast cancer [19]. Furthermore, ErbB2 is the preferred heterodimerization partner of all the other ErbB proteins and enhances ligand binding affinity and signalling power due to its potent latent kinase activity [20]. To investigate the functional influence and molecular interactions between gangliosides and ErbB2, we used the HC11 mouse mammary epithelial cell line. These cells represent a suitable experimental model as they physiologically express ErbB2 in a manner dependent on the degree of cell confluence in culture and the stimulation with EGF: i.e., ErbB2 expression levels increase when cell confluence raises, whereas EGF stimulation produces the EGFR-mediated ErbB2 cross-phosphorylation and an appreciable increment of ErbB2 turn-over [21]. We first related ErbB2 expression levels and activation with the expression of gangliosides, particularly GM3, and hypothesized the important modulating role that GM3 may play [22]. Moreover, scanning confocal microscopy analyses and cell membrane fractionation experiments strongly pointed out the co-localization of ErbB2, EGFR and ganglioside GM3 within the glycosphingolipid enriched microdomains [23], important structural/functional platforms of the cell [24]. In the present study, to better understand the relationship between GM3 and ErbB2, we investigated their association at the molecular level, both before and after EGF stimulation of the cells, by co-immunoprecipitation experiments and subsequent characterization of the immunocomplexes by western blot, high performance-thin layer chromatography (HP-TLC) and TLCimmunostaining analyses. Our results show that ErbB2 is able to form heterodimers with EGFR, both before and after EGF stimulation; however the EGF is essential not only to trigger tyrosine-phosphorylation of the receptors but also to give stability to these high molecular complexes of receptors. Moreover, we demonstrate that ganglioside GM3 but not other gangliosides expressed by HC11 cells (GM2, GD2, GD1a) [20] is strictly and in a SDS-resistant manner associated to the activated ErbB2/EGFR heterodimers and, interestingly, to EGFR rather than to ErbB2 monomers. On the contrary, no ganglioside is associated to either ErbB2 or EGFR, in the absence of EGF stimulation. Our data give further and stronger boost in supporting the important role of GM3 as a regulatory factor in ErbB2 and EGFR activation events and suggest that the possible mechanism of these modulating effects upon ErbB2 may be mediated by a tight and specific molecular interaction with EGFR. 2. Materials and methods
(Institute for Pharmacological Research “Mario Negri”, Milan, Italy). HC11 cells were routinely maintained in RPMI-1640 medium containing 8% heatinactivated newborn calf serum (NCS), 2 mM L-glutamine, 50 μg/ml gentamycin and 5 μg/ml insulin from bovine pancreas (HC11 routine growth medium) (SIGMA, St. Louis, MO, USA). For EGF stimulation, HC11 cells were transferred in HC11 routine growth medium supplemented with 10 nM EGF (SIGMA, St. Louis, MO, USA) and incubated at 37 °C. After 15 min of stimulation, cells were washed twice with ice cold phosphate buffered saline (PBS) and lysed in culture dishes for further experiments.
2.2. Antibodies The C18 rabbit polyclonal anti-ErbB2 IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the Immobilized rProtein A™ (RepliGen Corporation, Cambridge, MA, USA) were employed in the immunoprecipitation experiments. The 1005 rabbit polyclonal anti-EGFR IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C18 anti-ErbB2 antibody were used in the subsequent western blot analyses. The PY20 mouse monoclonal anti-phosphotyrosine IgG2b antibody (Transduction Laboratories, Lexington, KY, USA) was utilized to investigate the tyrosine-phosphorylated form of both ErbB2 and EGFR receptors. Ganglioside GM3 was immunodetected both in TLC-immunostaining and in western blotting analyses by the GMR6 mouse antiGM3 IgM antibody (Seikegaku Corporation, Tokyo, Japan). Bound primary antibodies were visualized by the proper secondary horseradish peroxidase (HRP)linked antibodies (GE Healthcare, Little Chalfont, UK) and immunoreactivity assessed by chemiluminescence.
2.3. Cell lysis, immunoprecipitation and immunoblot analyses HC11 cells, stimulated or not with EGF, were washed twice with ice cold PBS and lysed in culture dishes in complete RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml PMSF, 50 KIU/ml aprotinin, 1 mM Na3VO4 in PBS) for 20 min at 4 °C on a rotary shaker. Collected crude lysates were allowed to stand for another hour on ice and then centrifuged 10 min at 10,000×g at 4 °C to remove nuclei and large cell debris. An aliquot of each sample supernatant (1 mg total proteins in 1 ml complete RIPA buffer) was immunoprecipitated with the anti-ErbB2 antibody. The immunocomplexes, collected by addition of Immobilized rProtein A™, were washed four times with complete RIPA buffer, loaded onto 7.5% SDS-PAGE, transferred onto nitrocellulose membrane (Bio-Rad, Richmond, CA, USA) with the Bio-Rad Transfer Blot Apparatus at 150 mA for 16 h at 4 °C in 25 mM Tris HCl, 190 mM glycine, 20% methanol and 0.05% SDS as transfer buffer and, finally, analysed by western blotting with anti-EGFR, anti-ErbB2, anti-phosphotyrosine, anti-GM3 antibodies. Briefly, nitrocellulose membranes were blocked for 1 h in washing buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20®, T-TBS) added with 5% blocking reagent (ECL System, GE Healthcare, Little Chalfont, UK) for anti-EGFR and anti-ErbB2 antibodies, or with 1% bovine serum albumin (BSA) for anti-GM3 and anti-phosphotyrosine antibodies and then incubated with the specific primary antibody for 1 h at room temperature (2 h for antiGM3) in the appropriate blocking buffer. After six 5-min washes in T-TBS, the blots were incubated for 1 h at room temperature with the proper horseradish peroxidase (HRP)-linked secondary antibodies in the suitable blocking buffer. After six 5-min washes with T-TBS, membranes were finally developed with the ECL western blotting detection reagents (GE Healthcare, Little Chalfont, UK) following manufacturer's instructions. Immunoreactive proteins were visualized by autoradiography on X-OMAT AR film (Kodak, Rochester, NY, USA). Manufacturer's instruction (30 min at 50 °C in 2% SDS, 62.5 mM Tris HCl pH 6.8, 100 mM mercaptoethanol; ECL manual, GE Healthcare, Little Chalfont, UK) were used to strip the nitrocellulose membranes and to re-probe the blots with other antibodies.
2.4. Extraction, purification and analysis of gangliosides from the immunocomplexes
2.1. Cell culture The HC11 mouse mammary epithelial cell line, clonally derived from the COMMA-D mouse mammary cell line [25], was a kind gift of E. Garattini
Gangliosides were extracted and purified from the lyophilised immunoprecipitates obtained with the anti-ErbB2 Ab from control and EGF-stimulated HC11 cell lysates, according to previously described methods [26].
S. Milani et al. / Biochimica et Biophysica Acta 1771 (2007) 873–878 In brief, lyophilised immunoprecipitates were extracted with three different chloroform/methanol mixtures 1:1, 1:2, 2:1 (vol:vol), partitioned with the theoretical upper phase (TUP, chloroform/methanol/water, 47:48:1, vol:vol:vol) and then with water and purified by dialysis against distilled water. Equal volumes of ganglioside fractions from each sample, together with pure reference ganglioside standards (Alexis Biochemicals, San Diego, CA, USA), were loaded onto a high-performance thin-layer chromatography (HP-TLC) plate (Merck GmbH, Darmstadt, Germany) and run in a mixture of chloroform/methanol/ 0.2% aqueous CaCl2 (60:40:9, vol:vol:vol), as solvent system. Gangliosides were colorimetrically visualized by spraying with the resorcinol reagent (200 mg resorcinol, 80 ml 37% HCl, 1 ml 25 mM CuSO4), under carefully controlled conditions (120 °C for 15 min). Each ganglioside was identified by comparing its HP-TLC mobility (Rf) with the Rf of the pure reference standards. For the steady identification of ganglioside GM3, one tenth of the volumes used for the HP-TLC assays were used to perform TLC-immunostaining with the anti-GM3 antibody. Briefly, a known amount of standard ganglioside GM3 (corresponding to 500 ng sialic acid) and equal volumes of the ganglioside fractions from control and EGF-stimulated cells were loaded onto an aluminium-baked TLC plate (Merck GmbH, Darmstadt, Germany) and run in the solvent system described above. The TLC plate was then dried and impermeabilized in 0.1% polyisobuthylmetacrylate in n-exane, sank for 2 h at room temperature in blocking buffer (1% BSA in PBS) and incubated with the antiGM3 Ab in blocking buffer for 2 h at room temperature. After three 5 min washes in blocking buffer, the TLC plate was incubated with the proper HRPlinked secondary antibody in the same buffer for 1 h at room temperature, washed as above described and subjected to the ECL detection reagents. Immunoreactive species were visualized by autoradiography on X-OMAT AR film.
3. Results 3.1. Dimerization and tyrosine-phosphorylation of ErbB2 and EGFR in EGF-stimulated HC11 cells To ensure that ErbB2 and EGFR are dimerization partners in the HC11 cell line, the receptors were co-immunoprecipitated from the whole cell lysates of control and EGF-stimulated cells with the specific anti-ErbB2 Ab and the immunocomplexes subsequently analysed by western blot with the anti-EGFR Ab and, as positive control, with the anti-ErbB2 Ab. Results are presented in Fig. 1, Panels A and B, respectively. As shown in Fig. 1, Panel A, both in control (lane 1) and in EGF-stimulated (lane 2) cells a protein with molecular mass of
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approximately 170 kDa, corresponding to EGFR, is detectable. Similarly, in Fig. 1, Panel B, an immunoreactive species with molecular mass of about 185 kDa, corresponding to ErbB2, is detectable both in control (lane 1) and in EGF-stimulated (lane 2) cells. The 185-kDa molecular species detected in EGF-stimulated cells (Fig. 1 Panel B, lane 2) shows a slower electrophoretic mobility if compared with the species identified in control cells (Fig. 1, Panel B, lane 1). As previously demonstrated [22], the slight decrease of electrophoretic mobility of ErbB2 is due to tyrosine-phosphorylation of the receptor. A species showing molecular mass much higher than those corresponding to EGFR and ErbB2 monomers, but specifically immunoreactive to anti-EGFR and anti-ErbB2 Abs, is evident in samples from EGF stimulated cells (lanes 2 of Fig. 1, Panels A and B, respectively). This molecular species may represent stable SDS-resistant ErbB2/EGFR heterodimers or, possibly, oligomers (see Discussion). To investigate the tyrosine-phosphorylation of ErbB2 and EGFR, western blot analyses were performed with the antiphosphotyrosine Ab using the same blots probed with the antiEGFR and anti-ErbB2 Abs, after careful stripping the nitrocellulose membrane. Results are shown in Fig. 1, Panel C. As expected, no tyrosine-phosphorylated band is evident in control cells (lane 1), whereas three molecular species with molecular masses consistent to those of EGFR, ErbB2 and ErbB2/EGFR high molecular complexes are clearly distinguishable after EGF stimulation (lane 2). Altogether these data indicate that ErbB2 and EGFR tightly, although transiently, interact even in the absence of the specific ligand and that EGF is indispensable to give stability to receptor aggregates and to trigger their tyrosine-phosphorylation. 3.2. Co-immunoprecipitation of ganglioside GM3 with ErbB2 and EGFR after EGF stimulation To assess the molecular association of ganglioside GM3 with ErbB2 and EGFR, the immunocomplexes from control and EGF-stimulated cells were lyophilized and subjected to lipid
Fig. 1. Western blot analyses of the ErbB2 immunocomplexes. Immunocomplexes were obtained by immunoprecipitation with anti-ErbB2 antibody from whole lysates of control and EGF-stimulated cells, separated by a 7.5% SDS-PAGE and analysed by western blotting, as described in Materials and methods. Panels A, B, C, D: western blot analyses with anti-EGFR, anti-ErbB2, anti-phosphotyrosine, anti-GM3 antibodies, respectively. In each Panel: lanes 1: immunocomplexes from control cells; lanes 2: immunocomplexes from EGF-stimulated cells. Molecular masses of EGFR (170 kDa) and ErbB2 (185 kDa) are reported. The asterisk positioned at the right side of Panel B indicates tyrosine-phosphorylated ErbB2. The arrows at the right side of each panel indicate high molecular mass species specifically immunoreactive to the used antibodies.
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extraction and partitioning; the resulting ganglioside fractions were analysed by HP-TLC. As shown in Fig. 2, Panel A, a unique ganglioside species, with retardation factor (Rf) comparable to the pure reference standard GM3 is clearly evident in EGF-stimulated cells (lane 2), whereas neither GM3 nor other ganglioside species are detectable in HC11 control cells (lane 1). To definitely identify the ganglioside detected in EGF-stimulated cells as GM3, one tenth of the same ganglioside fractions were further analysed by TLC-immunostaining with the specific anti-GM3 Ab. As shown in Fig. 2, Panel B, an immunoreactive species is evident in EGF-stimulated cells (lane 3), but not in control cells (lane 2).
No immunoreactive species is detectable in control cells (lane 1); on the contrary, two distinct anti-GM3 reactive species are clearly identifiable following EGF stimulation (lane 2). The slower molecular species displays an electrophoretic mobility almost similar to that of the ErbB2/EGFR aggregates previously detected by western blot with anti-EGFR, anti-ErbB2 and antiphosphotyrosine antibodies (see Fig. 1, lanes 2, Panels A, B and C, respectively). Interestingly, the molecular species having faster electrophoretic mobility seems to move almost like EGFR rather than ErbB2 (compare its electrophoretic mobility with that of tyrosine-phosphorylated EGFR of Fig. 1, Panel C, lane 2), indicating the preferential and SDS-resistant molecular association of GM3 with EGFR rather than ErbB2 monomers.
3.3. Specific association of ganglioside GM3 with EGFR and ErbB2/EGFR complexes
4. Discussion
Our data clearly demonstrate that GM3 co-immunoprecipitates with ErbB2 and EGFR exclusively after EGF stimulation; the specific association of GM3 with both or either the receptors remains to be clarified. To address this question, immunocomplexes from control and EGF-stimulated cells were loaded onto SDS-PAGE and sequentially analysed by western blotting with anti-phosphotyrosine antibody and, after accurate stripping of the membrane, with the specific anti-GM3 antibody. Results are shown in Fig. 1, Panel D.
Fig. 2. Analyses of ganglioside fractions of the ErbB2 immunocomplexes. Ganglioside fractions were extracted and purified from the immunoprecipitates obtained with the anti-ErbB2 antibody from the whole lysates of control and EGF-stimulated cells. Panel A: HP-TLC analysis. The ganglioside fractions of control and EGF-stimulated cells were loaded onto the HP-TLC plate. Ganglioside species were colorimetrically visualized by the resorcinol reagent. Lane 1: ganglioside fraction of control cells; lane 2: ganglioside fraction of EGFstimulated cells; lane 3: pure reference standard gangliosides (from top to bottom: GM3, GM2, GD1a, GD2). Panel B: TLC-immunostaining analysis. One tenth of the ganglioside fraction used in the HP-TLC experiment of Panel A was loaded onto aluminium-baked TLC plate and subjected to the immunostaining procedure with anti-GM3 antibody. Lane 1: pure reference standard GM3; lane 2: ganglioside fraction of control cells; lane 3: ganglioside fraction of EGFstimulated cells.
ErbB2, the ligandless member of the ErbB family of receptor tyrosine-kinases, represents an intriguing research topic. It is well documented that it plays a fundamental role in pathological and physiological processes in many tissues, including the mammary gland [27,28]. Many studies investigated the features of homo- or hetero-dimerization and tyrosine-phosphorylation of ErbB2, the subsequent signalling cascade and the factors capable to modulate these events [29–31]. In this context, we thought it is worth considering the functional relationship between ErbB2 and gangliosides; the latter are amphiphylic molecules that behave as important modulators of many growth factor receptors, such as FGFR, PDGFR, and, especially, EGFR [1,2]. In the HC11 epithelial cell line, we previously demonstrated the involvement of ganglioside GM3 in modulating the expression levels of tyrosine-phosphorylated ErbB2 [22]; the key role of this ganglioside in retaining and targeting the receptor into the so-called glycosphingolipid enriched microdomains was also demonstrated [23]. In the present study we investigated the molecular association of ErbB2 with EGFR (possible and frequent dimerization partner of ErbB2) and with GM3, depending on the activation state of the receptor. To this aim, samples from HC11 cells, stimulated or not with EGF, were subjected to experiments of co-immunoprecipitation by anti-ErbB2 antibody and, subsequently, analysed by western blot with anti-EGFR, anti-phosphotyrosine, and anti-ErbB2 antibodies. Furthermore, we analysed the ganglioside components associated to the receptors after lipid extraction and partitioning of the immunoprecipitates. Our results indicate that ErbB2 co-immunoprecipitates with EGFR, both in control and in EGF-stimulated cells, suggesting the existence of a close relationship between the receptors even in the absence of the proper activating stimuli; however EGF is indispensable to trigger tyrosine-phosphorylation of both ErbB2 and EGFR. Noteworthy, species with higher molecular mass, immunoreactive to anti-EGFR, anti-phosphotyrosine and antiErbB2 antibodies and displaying detergent and reducing agent resistance, are clearly visible in EGF-stimulated cells. These species may represent supra-molecular complexes of phosphorylated ErbB2/EGFR, either heterodimers or oligomers, indicating the strong stabilizing effect of the ligand on the activated receptors.
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Analogous effect, ascribed to the treatment with agonists, has been observed studying the dimerization and oligomerization of G-protein coupled receptors, like the D2 dopamine receptor and the 5-HT1B and 5-HT1D serotonin receptors [32,33]. Moreover, it has been well demonstrated, at least for EGFR, its ability to form not only homo-/hetero-dimers but also higher order association of receptors after stimulation by the ligand EGF [34,35]; time-resolved phosphorescence anisotropy decay experiments performed by Jovin and colleagues [36] established that the EGF-bound EGFR was aggregated in clusters containing 10–50 receptors on the surface of A431 cells. As in our experiments EGFR coimmunoprecipitated with ErbB2, we cannot exclude the possible organization of mixed clusters of receptor populations (i.e. ErbB2 and EGFR) at the HC11 cell surface. Although GM3 accounts for a low content of the various gangliosides expressed by HC11 cells, the HP-TLC analyses of the ganglioside fraction extracted from the immunoprecipitates displayed the presence of a unique ganglioside, with Rf analogous to the standard GM3, exclusively in EGF-stimulated cells. On the contrary, no ganglioside species was revealed in control cells. The identity of this species was assessed by TLC-immunostaining with anti-GM3 antibody. These data are consistent with and enforce our previous findings indicating a tight relationship among ErbB2, EGFR and GM3, from both a functional and a structural point of view [22,23]; moreover these results strongly support the hypothesis that GM3 could modulate ErbB2 and EGFR activation by specific interaction with either or both the receptor molecules. Literature data indicate that EGFR glycosylation is essential for GM3 binding and GM3-mediated suppression of EGFR activation [37]. More recently, the extracellular domain of the human recombinant EGFR was demonstrated to be able to directly bind GM3, in a site that seems different from the EGFbinding site; results indicate that GM3 has the highest affinity among many other ganglioside species, suggesting the importance of the characteristic extracellular saccharidic chain of GM3 molecule [38]. It has also been suggested that gangliosides, like GD1a and GM1, may act by altering the membrane topology and favouring a ligand-independent EGFR dimerization, termed pre-dimerization, that in turn enhances the efficiency of binding and signalling once stimulated by the growth factor [12]. To answer the question concerning the role of ErbB2 and its possible activation by a tight molecular interaction with ganglioside GM3, we analysed the immunocomplexes by western blotting with the specific anti-GM3 antibody; this approach is currently used to investigate the association of co-immunoprecipitated gangliosides and proteins [8,39–41]. Our results indicate that no immuno-reactive species was detectable in control cells, whereas the existence of two distinct immuno-reactive species was evident in EGF-stimulated cells. Comparison of their electrophoretic mobility with those of ErbB2, EGFR and ErbB2/EGFR higher molecular complexes indicated that GM3 joins with the tyrosine-phosphorylated ErbB2, in a stable manner, only within the ErbB2/EGFR complexes; moreover it is preferentially, or perhaps more stably, associated to the phos-
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phorylated EGFR monomer rather than to the phosphorylated ErbB2 monomer. These data are in agreement with our previous results [22] indicating that ganglioside GM3 plays a fundamental role in modulating tyrosine-phosphorylated ErbB2 levels when the receptor is cross-activated by EGF-stimulated EGFR; on the contrary no regulating effect is observed when ErbB2 undergoes constitutive auto-phosphorylation through an homodimerization mechanism. Collected data lead us to conclude that even if ganglioside GM3 plays a main role in modulating ErbB2 activation, its influence is not directly exerted on ErbB2, but it is mediated by molecular interaction with EGFR. Moreover, the formation of a ternary complex between ErbB2/EGFR/GM3 may produce effects that go beyond the modulation of receptor activation and could explain GM3 influence upon internalization of both the receptors, as previously hypothesized [22]. Acknowledgement We sincerely thank Dr. T. Topini for his kind help in drafting and preparation of the manuscript. References [1] A.A. Rampersaud, J.L. Oblinger, R.K. Ponnappan, R.W. Burry, A.J. Yates, Gangliosides and growth factor receptor regulation, Biochem. Soc. Trans. 27 (1999) 415–422. [2] A.J. Yates, A.A. Rampersaud, Sphingolipids as receptor modulators, an overview, Ann. N.Y. Acad. Sci. 845 (1998) 57–71. [3] S. Hakomori, Y. Igarashi, Functional role of glycosphingolipids in cell recognition and signalling, J. Biochem. 118 (1995) 1091–1103. [4] L. Svennerholm, Chromatographic separation of human brain gangliosides, J. Neurochem. 10 (1963) 613–623. [5] E.G. Bremer, J. Schlessinger, S.I. Hakomori, Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor, J. Biol. Chem. 261 (1986) 2434–2440. [6] E.G. Bremer, S.I. Hakomori, D.F. Bowen-Pope, E. Raines, R. Ross, Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation, J. Biol. Chem. 259 (1984) 6818–6825. [7] D.L. Hynds, M. Summers, J. van Brocklyn, M.S. O'Dorisio, A.J. Yates, Gangliosides inhibit platelet-derived growth factor-stimulated growth, receptor phosphorylation, and dimerization in neuroblastoma SH-SY5Y cells, J. Neurochem. 65 (1995) 2251–2258. [8] T. Mutoh, A. Tokuda, T. Miyadai, M. Hamaguchi, N. Fujuki, Ganglioside GM1 binds to the Trk protein and regulates receptor function, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5087–5091. [9] E. Meuillet, G. Cremel, H. Dreyfus, D. Hicks, Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Muller glia, Glia 17 (1996) 206–216. [10] A.R. Zurita, H.J. Maccioni, J.L. Daniotti, Modulation of EGFR phosphorylation by endogenously expressed gangliosides, Biochem. J. 335 (2001) 465–472. [11] X. Wang, Z. Rahman, P. Sun, E. Meuillet, D. George, E.G. Bremer, A. AlQamari, A.S. Paller, Ganglioside modulates ligand binding to the EGFR, J. Invest. Dermatol. 116 (2001) 69–76. [12] Y. Liu, R. Li, S. Ladisch, Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization, J. Biol. Chem. 279 (2004) 36481–36489. [13] N. Hanai, T. Dohi, G.A. Nores, S. Hakomori, A novel ganglioside, de-Nacetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor kinase and as stimulator for cell growth, J. Biol. Chem. 263 (1988) 6296–6301.
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Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2 Simona Milani, Elena Sottocornola, Stefania Zava, Patrizia Berselli, Bruno Berra, Irma Colombo ⁎ Institute of General Physiology and Biological Chemistry, University of Milan, Via Trentacoste 2-20134, Milan, Italy Received 2 February 2007; received in revised form 10 April 2007; accepted 11 April 2007 Available online 20 April 2007
Abstract Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM3 in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM3 upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors coimmunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM3, but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM3 is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM3 on ErbB2 activation are mediated by EGFR. © 2007 Elsevier B.V. All rights reserved. Keywords: ErbB2; EGFR; GM3; Gangliosides; HC11 cell line
1. Introduction Gangliosides (sialic acid-containing glycosphingolipids, GSLs) are ubiquitous eukaryotic cell membrane components. They have been implicated in the modulation of cell differentiation and proliferation, and in oncogenesis, through their interaction with transmembrane signalling molecules such as growth factor receptor tyrosine-kinases [1–3]. In this view, ganglioside GM31 inhibits EGF-induced cell growth in the human epidermoid carcinoma cell line A431 through inhibition of EGFR auto-phosphorylation [5]. Similar behaviour has been observed for gangliosides GM3 and GM1 upon platelet-derived growth factor receptor (PDGFR) in Swiss 3T3 fibroblastic cells [6] and in neuroblastoma SH-SY5Y ⁎ Corresponding author. Tel.: +39 02 50315781; fax: +39 02 50315775. E-mail address: [email protected] (I. Colombo). 1 The gangliosides cited in the text are abbreviated according to Svennerholm's nomenclature (Ref. [4]). 1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2007.04.008
cells [7]. On the other hand, GM1, by tightly binding to nerve growth factor receptor (NGFR) with consequent dimer formation and its tyrosine auto-phosphorylation, enhances the activity of the receptor in NGF-responsive cells [8]. Other studies have also shown that gangliosides can preferentially alter the signalling of specific growth factors, as in the case of retinal glial cells, where GM3 alters the auto-phosphorylation of fibroblast growth factor receptor (b-FGFR) without affecting EGFR [9]. Altogether, these studies suggest that ganglioside modulation of growth factor receptor function may represent a general phenomenon and that specific gangliosides interact with specific receptors. Many data are available about the modulation of EGFR activation by gangliosides [10–13]; however, very little is known about the effects of gangliosides on other members of the ErbB receptor super-family. The ErbB receptor super-family belongs to the type I family of transmembrane receptor tyrosine-kinases and includes four members: EGFR (ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3) and ErbB4 (HER4) [14,15]. ErbB proteins are
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engaged in extensive network of homo- and hetero-associations resulting in signal diversification and multiple cellular responses [16]. The relative expression levels of the various receptors and the concentration of their respective ligands determine the composition of homo- and heterodimers [17,18]. Among the ErbB receptors, our attention focused on the ligand orphan ErbB2 tyrosine-kinase. ErbB2 plays an important role in growth and differentiation of the mammary gland; its amplification and over-expression is found in many kind of tumours and represents a main feature for a pour prognosis of breast cancer [19]. Furthermore, ErbB2 is the preferred heterodimerization partner of all the other ErbB proteins and enhances ligand binding affinity and signalling power due to its potent latent kinase activity [20]. To investigate the functional influence and molecular interactions between gangliosides and ErbB2, we used the HC11 mouse mammary epithelial cell line. These cells represent a suitable experimental model as they physiologically express ErbB2 in a manner dependent on the degree of cell confluence in culture and the stimulation with EGF: i.e., ErbB2 expression levels increase when cell confluence raises, whereas EGF stimulation produces the EGFR-mediated ErbB2 cross-phosphorylation and an appreciable increment of ErbB2 turn-over [21]. We first related ErbB2 expression levels and activation with the expression of gangliosides, particularly GM3, and hypothesized the important modulating role that GM3 may play [22]. Moreover, scanning confocal microscopy analyses and cell membrane fractionation experiments strongly pointed out the co-localization of ErbB2, EGFR and ganglioside GM3 within the glycosphingolipid enriched microdomains [23], important structural/functional platforms of the cell [24]. In the present study, to better understand the relationship between GM3 and ErbB2, we investigated their association at the molecular level, both before and after EGF stimulation of the cells, by co-immunoprecipitation experiments and subsequent characterization of the immunocomplexes by western blot, high performance-thin layer chromatography (HP-TLC) and TLCimmunostaining analyses. Our results show that ErbB2 is able to form heterodimers with EGFR, both before and after EGF stimulation; however the EGF is essential not only to trigger tyrosine-phosphorylation of the receptors but also to give stability to these high molecular complexes of receptors. Moreover, we demonstrate that ganglioside GM3 but not other gangliosides expressed by HC11 cells (GM2, GD2, GD1a) [20] is strictly and in a SDS-resistant manner associated to the activated ErbB2/EGFR heterodimers and, interestingly, to EGFR rather than to ErbB2 monomers. On the contrary, no ganglioside is associated to either ErbB2 or EGFR, in the absence of EGF stimulation. Our data give further and stronger boost in supporting the important role of GM3 as a regulatory factor in ErbB2 and EGFR activation events and suggest that the possible mechanism of these modulating effects upon ErbB2 may be mediated by a tight and specific molecular interaction with EGFR. 2. Materials and methods
(Institute for Pharmacological Research “Mario Negri”, Milan, Italy). HC11 cells were routinely maintained in RPMI-1640 medium containing 8% heatinactivated newborn calf serum (NCS), 2 mM L-glutamine, 50 μg/ml gentamycin and 5 μg/ml insulin from bovine pancreas (HC11 routine growth medium) (SIGMA, St. Louis, MO, USA). For EGF stimulation, HC11 cells were transferred in HC11 routine growth medium supplemented with 10 nM EGF (SIGMA, St. Louis, MO, USA) and incubated at 37 °C. After 15 min of stimulation, cells were washed twice with ice cold phosphate buffered saline (PBS) and lysed in culture dishes for further experiments.
2.2. Antibodies The C18 rabbit polyclonal anti-ErbB2 IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the Immobilized rProtein A™ (RepliGen Corporation, Cambridge, MA, USA) were employed in the immunoprecipitation experiments. The 1005 rabbit polyclonal anti-EGFR IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C18 anti-ErbB2 antibody were used in the subsequent western blot analyses. The PY20 mouse monoclonal anti-phosphotyrosine IgG2b antibody (Transduction Laboratories, Lexington, KY, USA) was utilized to investigate the tyrosine-phosphorylated form of both ErbB2 and EGFR receptors. Ganglioside GM3 was immunodetected both in TLC-immunostaining and in western blotting analyses by the GMR6 mouse antiGM3 IgM antibody (Seikegaku Corporation, Tokyo, Japan). Bound primary antibodies were visualized by the proper secondary horseradish peroxidase (HRP)linked antibodies (GE Healthcare, Little Chalfont, UK) and immunoreactivity assessed by chemiluminescence.
2.3. Cell lysis, immunoprecipitation and immunoblot analyses HC11 cells, stimulated or not with EGF, were washed twice with ice cold PBS and lysed in culture dishes in complete RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml PMSF, 50 KIU/ml aprotinin, 1 mM Na3VO4 in PBS) for 20 min at 4 °C on a rotary shaker. Collected crude lysates were allowed to stand for another hour on ice and then centrifuged 10 min at 10,000×g at 4 °C to remove nuclei and large cell debris. An aliquot of each sample supernatant (1 mg total proteins in 1 ml complete RIPA buffer) was immunoprecipitated with the anti-ErbB2 antibody. The immunocomplexes, collected by addition of Immobilized rProtein A™, were washed four times with complete RIPA buffer, loaded onto 7.5% SDS-PAGE, transferred onto nitrocellulose membrane (Bio-Rad, Richmond, CA, USA) with the Bio-Rad Transfer Blot Apparatus at 150 mA for 16 h at 4 °C in 25 mM Tris HCl, 190 mM glycine, 20% methanol and 0.05% SDS as transfer buffer and, finally, analysed by western blotting with anti-EGFR, anti-ErbB2, anti-phosphotyrosine, anti-GM3 antibodies. Briefly, nitrocellulose membranes were blocked for 1 h in washing buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20®, T-TBS) added with 5% blocking reagent (ECL System, GE Healthcare, Little Chalfont, UK) for anti-EGFR and anti-ErbB2 antibodies, or with 1% bovine serum albumin (BSA) for anti-GM3 and anti-phosphotyrosine antibodies and then incubated with the specific primary antibody for 1 h at room temperature (2 h for antiGM3) in the appropriate blocking buffer. After six 5-min washes in T-TBS, the blots were incubated for 1 h at room temperature with the proper horseradish peroxidase (HRP)-linked secondary antibodies in the suitable blocking buffer. After six 5-min washes with T-TBS, membranes were finally developed with the ECL western blotting detection reagents (GE Healthcare, Little Chalfont, UK) following manufacturer's instructions. Immunoreactive proteins were visualized by autoradiography on X-OMAT AR film (Kodak, Rochester, NY, USA). Manufacturer's instruction (30 min at 50 °C in 2% SDS, 62.5 mM Tris HCl pH 6.8, 100 mM mercaptoethanol; ECL manual, GE Healthcare, Little Chalfont, UK) were used to strip the nitrocellulose membranes and to re-probe the blots with other antibodies.
2.4. Extraction, purification and analysis of gangliosides from the immunocomplexes
2.1. Cell culture The HC11 mouse mammary epithelial cell line, clonally derived from the COMMA-D mouse mammary cell line [25], was a kind gift of E. Garattini
Gangliosides were extracted and purified from the lyophilised immunoprecipitates obtained with the anti-ErbB2 Ab from control and EGF-stimulated HC11 cell lysates, according to previously described methods [26].
S. Milani et al. / Biochimica et Biophysica Acta 1771 (2007) 873–878 In brief, lyophilised immunoprecipitates were extracted with three different chloroform/methanol mixtures 1:1, 1:2, 2:1 (vol:vol), partitioned with the theoretical upper phase (TUP, chloroform/methanol/water, 47:48:1, vol:vol:vol) and then with water and purified by dialysis against distilled water. Equal volumes of ganglioside fractions from each sample, together with pure reference ganglioside standards (Alexis Biochemicals, San Diego, CA, USA), were loaded onto a high-performance thin-layer chromatography (HP-TLC) plate (Merck GmbH, Darmstadt, Germany) and run in a mixture of chloroform/methanol/ 0.2% aqueous CaCl2 (60:40:9, vol:vol:vol), as solvent system. Gangliosides were colorimetrically visualized by spraying with the resorcinol reagent (200 mg resorcinol, 80 ml 37% HCl, 1 ml 25 mM CuSO4), under carefully controlled conditions (120 °C for 15 min). Each ganglioside was identified by comparing its HP-TLC mobility (Rf) with the Rf of the pure reference standards. For the steady identification of ganglioside GM3, one tenth of the volumes used for the HP-TLC assays were used to perform TLC-immunostaining with the anti-GM3 antibody. Briefly, a known amount of standard ganglioside GM3 (corresponding to 500 ng sialic acid) and equal volumes of the ganglioside fractions from control and EGF-stimulated cells were loaded onto an aluminium-baked TLC plate (Merck GmbH, Darmstadt, Germany) and run in the solvent system described above. The TLC plate was then dried and impermeabilized in 0.1% polyisobuthylmetacrylate in n-exane, sank for 2 h at room temperature in blocking buffer (1% BSA in PBS) and incubated with the antiGM3 Ab in blocking buffer for 2 h at room temperature. After three 5 min washes in blocking buffer, the TLC plate was incubated with the proper HRPlinked secondary antibody in the same buffer for 1 h at room temperature, washed as above described and subjected to the ECL detection reagents. Immunoreactive species were visualized by autoradiography on X-OMAT AR film.
3. Results 3.1. Dimerization and tyrosine-phosphorylation of ErbB2 and EGFR in EGF-stimulated HC11 cells To ensure that ErbB2 and EGFR are dimerization partners in the HC11 cell line, the receptors were co-immunoprecipitated from the whole cell lysates of control and EGF-stimulated cells with the specific anti-ErbB2 Ab and the immunocomplexes subsequently analysed by western blot with the anti-EGFR Ab and, as positive control, with the anti-ErbB2 Ab. Results are presented in Fig. 1, Panels A and B, respectively. As shown in Fig. 1, Panel A, both in control (lane 1) and in EGF-stimulated (lane 2) cells a protein with molecular mass of
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approximately 170 kDa, corresponding to EGFR, is detectable. Similarly, in Fig. 1, Panel B, an immunoreactive species with molecular mass of about 185 kDa, corresponding to ErbB2, is detectable both in control (lane 1) and in EGF-stimulated (lane 2) cells. The 185-kDa molecular species detected in EGF-stimulated cells (Fig. 1 Panel B, lane 2) shows a slower electrophoretic mobility if compared with the species identified in control cells (Fig. 1, Panel B, lane 1). As previously demonstrated [22], the slight decrease of electrophoretic mobility of ErbB2 is due to tyrosine-phosphorylation of the receptor. A species showing molecular mass much higher than those corresponding to EGFR and ErbB2 monomers, but specifically immunoreactive to anti-EGFR and anti-ErbB2 Abs, is evident in samples from EGF stimulated cells (lanes 2 of Fig. 1, Panels A and B, respectively). This molecular species may represent stable SDS-resistant ErbB2/EGFR heterodimers or, possibly, oligomers (see Discussion). To investigate the tyrosine-phosphorylation of ErbB2 and EGFR, western blot analyses were performed with the antiphosphotyrosine Ab using the same blots probed with the antiEGFR and anti-ErbB2 Abs, after careful stripping the nitrocellulose membrane. Results are shown in Fig. 1, Panel C. As expected, no tyrosine-phosphorylated band is evident in control cells (lane 1), whereas three molecular species with molecular masses consistent to those of EGFR, ErbB2 and ErbB2/EGFR high molecular complexes are clearly distinguishable after EGF stimulation (lane 2). Altogether these data indicate that ErbB2 and EGFR tightly, although transiently, interact even in the absence of the specific ligand and that EGF is indispensable to give stability to receptor aggregates and to trigger their tyrosine-phosphorylation. 3.2. Co-immunoprecipitation of ganglioside GM3 with ErbB2 and EGFR after EGF stimulation To assess the molecular association of ganglioside GM3 with ErbB2 and EGFR, the immunocomplexes from control and EGF-stimulated cells were lyophilized and subjected to lipid
Fig. 1. Western blot analyses of the ErbB2 immunocomplexes. Immunocomplexes were obtained by immunoprecipitation with anti-ErbB2 antibody from whole lysates of control and EGF-stimulated cells, separated by a 7.5% SDS-PAGE and analysed by western blotting, as described in Materials and methods. Panels A, B, C, D: western blot analyses with anti-EGFR, anti-ErbB2, anti-phosphotyrosine, anti-GM3 antibodies, respectively. In each Panel: lanes 1: immunocomplexes from control cells; lanes 2: immunocomplexes from EGF-stimulated cells. Molecular masses of EGFR (170 kDa) and ErbB2 (185 kDa) are reported. The asterisk positioned at the right side of Panel B indicates tyrosine-phosphorylated ErbB2. The arrows at the right side of each panel indicate high molecular mass species specifically immunoreactive to the used antibodies.
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extraction and partitioning; the resulting ganglioside fractions were analysed by HP-TLC. As shown in Fig. 2, Panel A, a unique ganglioside species, with retardation factor (Rf) comparable to the pure reference standard GM3 is clearly evident in EGF-stimulated cells (lane 2), whereas neither GM3 nor other ganglioside species are detectable in HC11 control cells (lane 1). To definitely identify the ganglioside detected in EGF-stimulated cells as GM3, one tenth of the same ganglioside fractions were further analysed by TLC-immunostaining with the specific anti-GM3 Ab. As shown in Fig. 2, Panel B, an immunoreactive species is evident in EGF-stimulated cells (lane 3), but not in control cells (lane 2).
No immunoreactive species is detectable in control cells (lane 1); on the contrary, two distinct anti-GM3 reactive species are clearly identifiable following EGF stimulation (lane 2). The slower molecular species displays an electrophoretic mobility almost similar to that of the ErbB2/EGFR aggregates previously detected by western blot with anti-EGFR, anti-ErbB2 and antiphosphotyrosine antibodies (see Fig. 1, lanes 2, Panels A, B and C, respectively). Interestingly, the molecular species having faster electrophoretic mobility seems to move almost like EGFR rather than ErbB2 (compare its electrophoretic mobility with that of tyrosine-phosphorylated EGFR of Fig. 1, Panel C, lane 2), indicating the preferential and SDS-resistant molecular association of GM3 with EGFR rather than ErbB2 monomers.
3.3. Specific association of ganglioside GM3 with EGFR and ErbB2/EGFR complexes
4. Discussion
Our data clearly demonstrate that GM3 co-immunoprecipitates with ErbB2 and EGFR exclusively after EGF stimulation; the specific association of GM3 with both or either the receptors remains to be clarified. To address this question, immunocomplexes from control and EGF-stimulated cells were loaded onto SDS-PAGE and sequentially analysed by western blotting with anti-phosphotyrosine antibody and, after accurate stripping of the membrane, with the specific anti-GM3 antibody. Results are shown in Fig. 1, Panel D.
Fig. 2. Analyses of ganglioside fractions of the ErbB2 immunocomplexes. Ganglioside fractions were extracted and purified from the immunoprecipitates obtained with the anti-ErbB2 antibody from the whole lysates of control and EGF-stimulated cells. Panel A: HP-TLC analysis. The ganglioside fractions of control and EGF-stimulated cells were loaded onto the HP-TLC plate. Ganglioside species were colorimetrically visualized by the resorcinol reagent. Lane 1: ganglioside fraction of control cells; lane 2: ganglioside fraction of EGFstimulated cells; lane 3: pure reference standard gangliosides (from top to bottom: GM3, GM2, GD1a, GD2). Panel B: TLC-immunostaining analysis. One tenth of the ganglioside fraction used in the HP-TLC experiment of Panel A was loaded onto aluminium-baked TLC plate and subjected to the immunostaining procedure with anti-GM3 antibody. Lane 1: pure reference standard GM3; lane 2: ganglioside fraction of control cells; lane 3: ganglioside fraction of EGFstimulated cells.
ErbB2, the ligandless member of the ErbB family of receptor tyrosine-kinases, represents an intriguing research topic. It is well documented that it plays a fundamental role in pathological and physiological processes in many tissues, including the mammary gland [27,28]. Many studies investigated the features of homo- or hetero-dimerization and tyrosine-phosphorylation of ErbB2, the subsequent signalling cascade and the factors capable to modulate these events [29–31]. In this context, we thought it is worth considering the functional relationship between ErbB2 and gangliosides; the latter are amphiphylic molecules that behave as important modulators of many growth factor receptors, such as FGFR, PDGFR, and, especially, EGFR [1,2]. In the HC11 epithelial cell line, we previously demonstrated the involvement of ganglioside GM3 in modulating the expression levels of tyrosine-phosphorylated ErbB2 [22]; the key role of this ganglioside in retaining and targeting the receptor into the so-called glycosphingolipid enriched microdomains was also demonstrated [23]. In the present study we investigated the molecular association of ErbB2 with EGFR (possible and frequent dimerization partner of ErbB2) and with GM3, depending on the activation state of the receptor. To this aim, samples from HC11 cells, stimulated or not with EGF, were subjected to experiments of co-immunoprecipitation by anti-ErbB2 antibody and, subsequently, analysed by western blot with anti-EGFR, anti-phosphotyrosine, and anti-ErbB2 antibodies. Furthermore, we analysed the ganglioside components associated to the receptors after lipid extraction and partitioning of the immunoprecipitates. Our results indicate that ErbB2 co-immunoprecipitates with EGFR, both in control and in EGF-stimulated cells, suggesting the existence of a close relationship between the receptors even in the absence of the proper activating stimuli; however EGF is indispensable to trigger tyrosine-phosphorylation of both ErbB2 and EGFR. Noteworthy, species with higher molecular mass, immunoreactive to anti-EGFR, anti-phosphotyrosine and antiErbB2 antibodies and displaying detergent and reducing agent resistance, are clearly visible in EGF-stimulated cells. These species may represent supra-molecular complexes of phosphorylated ErbB2/EGFR, either heterodimers or oligomers, indicating the strong stabilizing effect of the ligand on the activated receptors.
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Analogous effect, ascribed to the treatment with agonists, has been observed studying the dimerization and oligomerization of G-protein coupled receptors, like the D2 dopamine receptor and the 5-HT1B and 5-HT1D serotonin receptors [32,33]. Moreover, it has been well demonstrated, at least for EGFR, its ability to form not only homo-/hetero-dimers but also higher order association of receptors after stimulation by the ligand EGF [34,35]; time-resolved phosphorescence anisotropy decay experiments performed by Jovin and colleagues [36] established that the EGF-bound EGFR was aggregated in clusters containing 10–50 receptors on the surface of A431 cells. As in our experiments EGFR coimmunoprecipitated with ErbB2, we cannot exclude the possible organization of mixed clusters of receptor populations (i.e. ErbB2 and EGFR) at the HC11 cell surface. Although GM3 accounts for a low content of the various gangliosides expressed by HC11 cells, the HP-TLC analyses of the ganglioside fraction extracted from the immunoprecipitates displayed the presence of a unique ganglioside, with Rf analogous to the standard GM3, exclusively in EGF-stimulated cells. On the contrary, no ganglioside species was revealed in control cells. The identity of this species was assessed by TLC-immunostaining with anti-GM3 antibody. These data are consistent with and enforce our previous findings indicating a tight relationship among ErbB2, EGFR and GM3, from both a functional and a structural point of view [22,23]; moreover these results strongly support the hypothesis that GM3 could modulate ErbB2 and EGFR activation by specific interaction with either or both the receptor molecules. Literature data indicate that EGFR glycosylation is essential for GM3 binding and GM3-mediated suppression of EGFR activation [37]. More recently, the extracellular domain of the human recombinant EGFR was demonstrated to be able to directly bind GM3, in a site that seems different from the EGFbinding site; results indicate that GM3 has the highest affinity among many other ganglioside species, suggesting the importance of the characteristic extracellular saccharidic chain of GM3 molecule [38]. It has also been suggested that gangliosides, like GD1a and GM1, may act by altering the membrane topology and favouring a ligand-independent EGFR dimerization, termed pre-dimerization, that in turn enhances the efficiency of binding and signalling once stimulated by the growth factor [12]. To answer the question concerning the role of ErbB2 and its possible activation by a tight molecular interaction with ganglioside GM3, we analysed the immunocomplexes by western blotting with the specific anti-GM3 antibody; this approach is currently used to investigate the association of co-immunoprecipitated gangliosides and proteins [8,39–41]. Our results indicate that no immuno-reactive species was detectable in control cells, whereas the existence of two distinct immuno-reactive species was evident in EGF-stimulated cells. Comparison of their electrophoretic mobility with those of ErbB2, EGFR and ErbB2/EGFR higher molecular complexes indicated that GM3 joins with the tyrosine-phosphorylated ErbB2, in a stable manner, only within the ErbB2/EGFR complexes; moreover it is preferentially, or perhaps more stably, associated to the phos-
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phorylated EGFR monomer rather than to the phosphorylated ErbB2 monomer. These data are in agreement with our previous results [22] indicating that ganglioside GM3 plays a fundamental role in modulating tyrosine-phosphorylated ErbB2 levels when the receptor is cross-activated by EGF-stimulated EGFR; on the contrary no regulating effect is observed when ErbB2 undergoes constitutive auto-phosphorylation through an homodimerization mechanism. Collected data lead us to conclude that even if ganglioside GM3 plays a main role in modulating ErbB2 activation, its influence is not directly exerted on ErbB2, but it is mediated by molecular interaction with EGFR. Moreover, the formation of a ternary complex between ErbB2/EGFR/GM3 may produce effects that go beyond the modulation of receptor activation and could explain GM3 influence upon internalization of both the receptors, as previously hypothesized [22]. Acknowledgement We sincerely thank Dr. T. Topini for his kind help in drafting and preparation of the manuscript. References [1] A.A. Rampersaud, J.L. Oblinger, R.K. Ponnappan, R.W. Burry, A.J. Yates, Gangliosides and growth factor receptor regulation, Biochem. Soc. Trans. 27 (1999) 415–422. [2] A.J. Yates, A.A. Rampersaud, Sphingolipids as receptor modulators, an overview, Ann. N.Y. Acad. Sci. 845 (1998) 57–71. [3] S. Hakomori, Y. Igarashi, Functional role of glycosphingolipids in cell recognition and signalling, J. Biochem. 118 (1995) 1091–1103. [4] L. Svennerholm, Chromatographic separation of human brain gangliosides, J. Neurochem. 10 (1963) 613–623. [5] E.G. Bremer, J. Schlessinger, S.I. Hakomori, Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor, J. Biol. Chem. 261 (1986) 2434–2440. [6] E.G. Bremer, S.I. Hakomori, D.F. Bowen-Pope, E. Raines, R. Ross, Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation, J. Biol. Chem. 259 (1984) 6818–6825. [7] D.L. Hynds, M. Summers, J. van Brocklyn, M.S. O'Dorisio, A.J. Yates, Gangliosides inhibit platelet-derived growth factor-stimulated growth, receptor phosphorylation, and dimerization in neuroblastoma SH-SY5Y cells, J. Neurochem. 65 (1995) 2251–2258. [8] T. Mutoh, A. Tokuda, T. Miyadai, M. Hamaguchi, N. Fujuki, Ganglioside GM1 binds to the Trk protein and regulates receptor function, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5087–5091. [9] E. Meuillet, G. Cremel, H. Dreyfus, D. Hicks, Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Muller glia, Glia 17 (1996) 206–216. [10] A.R. Zurita, H.J. Maccioni, J.L. Daniotti, Modulation of EGFR phosphorylation by endogenously expressed gangliosides, Biochem. J. 335 (2001) 465–472. [11] X. Wang, Z. Rahman, P. Sun, E. Meuillet, D. George, E.G. Bremer, A. AlQamari, A.S. Paller, Ganglioside modulates ligand binding to the EGFR, J. Invest. Dermatol. 116 (2001) 69–76. [12] Y. Liu, R. Li, S. Ladisch, Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization, J. Biol. Chem. 279 (2004) 36481–36489. [13] N. Hanai, T. Dohi, G.A. Nores, S. Hakomori, A novel ganglioside, de-Nacetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor kinase and as stimulator for cell growth, J. Biol. Chem. 263 (1988) 6296–6301.
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