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Cancer Research 67, 1935, March 1, 2007. doi: 10.1158/0008-5472.CAN-06-3023
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

Shunichi Yokoe1, Motoko Takahashi3, Michio Asahi1, Seung Ho Lee1, Wei Li1, Daisuke Osumi1, Eiji Miyoshi1 and Naoyuki Taniguchi1,2

1 Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka; 2 Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Center for Advanced Science & Innovation, 2-1 Yamadaoka, Suita, Osaka, Japan; and 3 Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga, Japan

Requests for reprints: Naoyuki Taniguchi, Department of Disease Glycomics, Research Institute for Microbial Diseases, 4th Floor, Center for Advanced Science & Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-4137; Fax: 81-6-6879-4137; E-mail: tani52{at}wd5.so-net.ne.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ErbB2 and ErbB3, two members of the ErbB family, form a high-affinity heregulin coreceptor that elicits potent mitogenic and transforming signals, and clinical studies indicate that these receptors play an important role in tumor incidence and progression. To determine whether N-glycosylation is involved in the function of ErbB3, a series of human ErbB3 molecules devoid of N-glycans were prepared and transfected to Flp-In-CHO cells for stable expression. A cross-linking study showed that the Asn418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin. The wild-type or N418Q mutant of ErbB3 was next coexpressed with ErbB2 in Flp-In-CHO cells, and the effect of N-glycan on heterodimerization was examined. The N418Q mutant of ErbB3 was autodimerized with ErbB2 without ligand stimulation, and receptor tyrosine phosphorylation and subsequent extracellular signal-regulated kinase (ERK) and Akt phosphorylation were promoted in the absence of heregulin. A cell proliferation assay and a soft agar colony formation assay showed that the N418Q mutant of ErbB3 coexpressed with ErbB2 promoted cell proliferation and colony formation in soft agar in an ERK- and Akt-dependent manner. The mutation also promoted the growth of tumors in athymic mice when injected s.c. These findings suggest that the Asn418-linked N-glycan in ErbB3 plays an essential role in regulating receptor heterodimerization with ErbB2 and might have an effect on transforming activity. [Cancer Res 2007;67(5):1935–42]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ErbB family of receptor tyrosine kinases, consisting of the epidermal growth factor receptor (EGFR or ErbB1), ErbB2, ErbB3, and ErbB4, are expressed in numerous types of epithelial tissues. They play an essential role in cell proliferation, differentiation, survival, migration, and adhesion, and their aberrant signaling has been implicated in the development of many types of human cancers (1, 2). By binding to their ligands, they form a wide array of homo- and heterodimers, each with distinct signaling abilities. Each dimer has a different tyrosine autophosphorylation site, which serves as a docking site for specific Src homology 2–containing proteins and recruit different signaling molecules, including the members of extracellular signal-regulated kinase (ERK), phospholipase C-{gamma}, and phosphoinositide-3-kinase (PI3K) pathways.

ErbB3 is a member of ErbB family, a 185-kDa type I transmembrane glycoprotein consisting of 1,342 amino acid residues (3). Although its cytoplasmic domain is devoid of kinase activity, ErbB3 mediates cellular responses of isoforms of heregulin by forming a heterodimer with other ErbB family members. It is well known that the ErbB3 and ErbB2 heterodimer is the most prevalent and potent complex among the heterodimers formed within the ErbB family to activate the intrinsic kinase domain, resulting in the activation of mitogenic signaling pathways such as the Ras/ERK and PI3K pathways (4, 5).

It has been reported that glycosylation regulates the function of glycoproteins by inducing conformational changes or by affecting intramolecular interactions (6–10). Many membrane-bound proteins, such as receptors, are glycosylated, and it has been reported that glycosylation status is crucial for their function (11–15). All the members of ErbB family are glycoproteins. For example, EGFR contains 11 potential N-glycosylation sites in its extracellular domain, and glycosylation is essential for its function (16–21). An initial N-glycosylation is required for the proper sorting of EGFR to the membrane as well as for ligand binding (16–18). We previously reported that the N-glycan on Asn420 played an important role in the suppression of ligand-independent spontaneous oligomerization (19). To determine the role of N-glycans of ErbB3, we prepared mutants of ErbB3 that lack each of the 10 N-glycosylation sites and examined their in vitro and in vivo effects. The results show that the N-glycan on Asn418, which corresponds to the glycan on Asn420 in EGFR, plays a crucial role in regulating both homodimerization and heterodimerization and subsequent signal transduction, which affects tumor formation in vivo.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. All chemicals and reagents were purchased from Nacalai Tesque (Kyoto, Japan) or Wako Pure Chemicals (Osaka, Japan), unless otherwise noted. Human recombinant heregulin was purchased from Oncogene (San Diego, CA). An anti-ErbB3 antibody was from Neomarkers (Westinghouse, CA). An anti-ErbB2 antibody for immunoprecipitation (SV2-61{gamma}) was from Nichirei (Tokyo, Japan), and an anti-ErbB2 antibody for Western blotting (NCL-CB11) was from Novocastra (Newcastle, United Kingdom). An anti-phosphotyrosine antibody (PY20) was from Transduction Laboratories (Lexington, KY). An anti–phospho-p44/42 mitogen-activated protein (MAP) kinase and anti–total-p44/42 MAP kinase antibodies, anti–phospho-Akt (Ser473), and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA). The Flp-In-CHO cell line was obtained from Invitrogen (Carlsbad, CA). The MAP/ERK kinase (MEK) inhibitor, PD98059, and PI3K inhibitor, wortmannin, were purchased from Calbiochem (San Diego, CA).

Expression vector for ErbB3 mutants. Human ErbB3 cDNA was subcloned in pcDNA5/FRT for a stable high-expression Flp-In System (Invitrogen). Mutations were prepared via PCR amplification using a mutant sequence oligonucleotide (summarized in Supplementary Table S1). All mutations were verified by DNA sequencing.

Establishment of mutant ErbB transfectants. Flp-In-CHO cells were grown in Ham's F12 medium, supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Wild-type or mutant ErbB3 constructs were transfected into Flp-In-CHO cells using LipofectAMINE 2000 (Invitrogen). After 24 h, cells were selected in the medium with 600 µg/mL hygromycin B (Calbiochem) for 3 to 4 weeks. To establish ErbB2 and ErbB3 stable coexpressing cells, wild-type ErbB2 cDNA (kindly provided by Dr. Tadashi Yamamoto, University of Tokyo, Japan) in pcDNA3.1 Hygro were transfected into the cells, which stably expressed wild-type or mutants of ErbB3, using LipofectAMINE 2000.

Western blot analysis. Cells were harvested in lysis buffer containing 20 mmol/L Tris-HCl (pH, 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% (w/v) Nonidet P-40, 10% (w/v) glycerol, 5 mmol/L sodium PPi, 10 mmol/L NaF, 1 mmol/L sodium orthovanadate, 10 mmol/L ß-glycerophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 5 µg/mL leupeptin, and 1 mmol/L DTT. Cell lysates were centrifuged at 15,000 rpm for 10 min at 4°C, and the protein concentration of the supernatant was determined using a Protein Assay CBB kit (Nacalai Tesque). Cell lysates containing equal amounts of protein were subjected to 8% SDS-PAGE, transferred to nitrocellulose membrane, and after blocking with 5% skim milk in TBST [20 mmol/L Tris-HCl (pH, 7.5), 150 mmol/L NaCl, 0.05% Tween 20] for 1 h at room temperature, the membrane was incubated with antibodies against ErbB3 (1:2,500) or ErbB2 (1:2,500) as first antibodies at 4°C overnight. An anti-phosphotyrosine antibody (PY20, 1:2,500), an anti–phospho-p44/42 MAP kinase (ERK) antibody (1:2,500), or an anti–phospho-Akt (Ser473) antibody (1:2,500) were used for the detection of tyrosine phosphorylation, activation of ERK, or Akt, respectively. After washing with TBST, the membranes were treated with second antibodies for 1 h at room temperature. The membranes were washed with TBST, and the bands were detected using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

Cell surface biotinylation and immunoprecipitation of ErbB3. Wild-type or mutants of ErbB3 were expressed in Flp-In-CHO cells grown in 100-mm dishes. After washing the cells twice with ice-cold PBS containing 0.1 mmol/L CaCl2 and 1 mmol/L MgCl2, biotinylation was initiated by adding 0.2 mg/mL of sulfo-N-hydroxysuccinimidobiotin (EZ-Link Sulfo-NHS-Biotin; Pierce, Rockford, IL) and incubated for 30 min at 4°C. Reactions were stopped by adding 50 mmol/L of Tris-HCl (pH, 7.4). After washing twice with ice-cold PBS, cells were harvested with a lysis buffer centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants, containing equal amounts of protein, were immunoprecipitated with an ErbB3 antibody and 15 µL of protein G sepharose at 4°C with rotation. After washing with lysis buffer, the precipitates were boiled, subjected to 8% SDS-PAGE gel, and transferred to nitrocellulose membranes. The biotinylated wild-type or mutants of ErbB3 were detected by Vectastain ABC kit (Vector Laboratories, Burlingame, CA).

Cross-linking experiment. After treating the cells with FCS-free medium containing 0.1% bovine serum albumin in a 100-mm dish for 2 h, the culture medium was removed, followed by stimulation with or without heregulin for 5 min. Cross-linking was initiated by adding bis-(sulfosuccinimidyl)-suberate (BS3; Pierce) to a final concentration of 1 mmol/L, followed by incubation for 2 h at room temperature. The reactions were stopped by adding 50 mmol/L of Tris-HCl (pH, 7.4), and cross-linking was detected by a Western blot analysis as described above.

Cell proliferation assay. About 100 µL (2.5 x 103 cells) of Flp-In-CHO cells, which stably coexpressed wild-type or N418Q mutant ErbB3 with wild-type ErbB2, were plated in each well of 96-well microtiter plates in the presence or absence of wortmannin or PD98059. After culturing the cells for various durations (0, 24, 48, 72, and 96 h), a cell proliferation assay was done using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) to estimate anchorage-dependent cell growth according to the manufacturer's instructions.

Soft agar colony formation assay. To assess anchorage-independent cell growth, a soft agar colony formation assay was done as described previously (22, 23). Briefly, 2 x 102 cells in culture medium were mixed with a volume of 0.5% top agar twice higher and seeded in 60-mm plates onto a base layer of complete medium containing 0.5% agar in the presence or absence of wortmannin or PD98059. The cells were incubated at 37°C in 5% CO2 in air. After 2 to 3 weeks, the number of colonies was counted and statistically analyzed.

Tumor formation assay in athymic mice. To evaluate tumor formation in the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells, we injected 1 x 106 cells s.c. into athymic nude mice (5-week-old female BALB/c mice; Charles River Laboratories Japan, Inc., Kanagawa, Japan). Briefly, the cells were plated on a 100-mm dish in complete medium. After treatment with PBS containing 1 mmol/L EDTA, the cells were suspended to a single-cell level with Hank's buffer. A total of 1 x 106 cells were s.c. injected into athymic mice. At 1 month after the injection, the size of the tumor was measured. These mice were maintained in temperature-controlled rooms at the Institute of Experimental Animal Science, Osaka University Graduate School of Medicine, and were treated according to NIH guidelines.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of transfectants with mutated ErbB3s that lack N-glycan consensus sites. ErbB3 contains 10 potential N-glycosylation sites in its extracellular domain (Supplementary Fig. S1). To determine which N-glycan of ErbB3 is responsible for receptor function, we prepared the mutants for each of the 10 glycosylation sites of ErbB3. When the wild-type or mutant ErbB3s were stably expressed in Flp-In CHO cells, all the single-site mutants were expressed at levels comparable to the wild type (Fig. 1A ). The mutant in which all five N-glycosylation sites of domain III were changed (N334Q/N389Q/N395Q/N418Q/N450Q, shown as 5N III 5Q in figures) was also expressed in Flp-In CHO cell (Fig. 1B). Moreover, mutants in which the amino acid adjacent of Asn418 was changed (L417Q) or the mutant in which the amino acids adjacent to all N-glycosylation sites of domain III were changed (V333Q/H388Q/S394Q/L417Q/L449Q, shown as V, H, S, L, L III 5Q in figures) were also expressed in Flp-In CHO cells (Fig. 1B).


Figure 1
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  		Figure 1. Expression levels of ErbB3 mutants in Flp-In-CHO cells. A and B, wild-type or mutants of ErbB3 were expressed in Flp-In CHO cells, and their expression was examined by a Western blot analysis using an anti-ErbB3 antibody. 5N III 5Q: N334Q/N389/N395Q/N418Q/N450Q, V.H.S.L.L III 5Q: V333Q/H388Q/S394Q/L417Q/L449Q. C and D, the wild type or mutants of ErbB3 on cell surface were biotinylated and immunoprecipitated with the anti-ErbB3 antibody, detected using an ABC kit. Details are described in Materials and Methods.

 
Cell surface expression of each mutant of ErbB3s. To assess the contributions of N-glycans of ErbB3 for sorting to the cell surface, we investigated the cell surface expression level of ErbB3 mutants that lack one or more N-glycosylation sites. As a result, the expression level on the cell surface of each of the ErbB3 mutants (N107Q, N231Q, N334Q, N389Q, N395Q, N418Q, N450Q, N503Q, N547Q, N597Q) were the same as that of the wild-type ErbB3 (Fig. 1C). The cell surface expression levels of mutants of L417Q or the mutants in which the amino acids adjacent to the N-glycosylation sites of domain III were changed to glutamine (V333Q/H388Q/S394Q/L417Q/L449Q) were also not changed (Fig. 1D). On the other hand, when all of the N-glycosylation sites in domain III were changed (N334Q/N389Q/N395Q/N418Q/N450Q), cell surface expression level was significantly reduced. These results clearly suggest that a lack of any of the 10 N-glycans of ErbB3 has no effect on cell surface expression; however, a lack of all 5 N-glycans in domain III impairs the trafficking of the receptor to the cell surface.

N418Q mutant of ErbB3 was autodimerized in the absence of heregulin. We compared the dimerization level of the mutants with that of wild type by cross-linking using BS3. As shown in Fig. 2A , the N418Q mutant autodimerized in the absence of its ligand, heregulin, indicating that the loss of the N-glycan linked to Asn418 leads to spontaneous dimerization. However, the other single-site mutants of ErbB3 as well as wild-type ErbB3 did not undergo autodimerization (Fig. 2A). The mutant in which all of the N-glycosylation sites in domain III of ErbB3 were changed (N334Q/N389Q/N395Q/N418Q/N450Q), the L417Q mutant, or the mutant in which all of the amino acids adjacent to the N-glycosylation sites of domain III were substituted (V333Q/H388Q/S394Q/L417Q/L449Q) were also examined; however, no dimerization was observed (Fig. 2B). The extent of dimerization of the N418Q mutant was enhanced in the presence of heregulin (Fig. 2A).


Figure 2
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  		Figure 2. Autodimerization and heregulin-induced dimerization of ErbB3 mutants. A and B, after serum starvation for 2 h, Flp-In-CHO cells stably expressing wild type or mutants of ErbB3 were treated with or without 5 nmol/L of heregulin for 5 min. Cross-linking was done by adding BS3 to a final concentration of 1 mmol/L, followed by incubation for 2 h at room temperature. The reactions were stopped by adding 50 mmol/L of Tris-HCl (pH, 7.4) and then solubilized in lysis buffer as described in Materials and Methods. Whole cell lysates were subjected to 8% SDS-PAGE and detected with anti-ErbB3 antibody. HRG, heregulin; BS3, bis-(sulfosuccinimidyl)-suberate; 5N III 5Q, N334Q/N389/N395Q/N418Q/N450Q; V.H.S.L.L III 5Q, V333Q/H388Q/S394Q/L417Q/L449Q.

 
N418Q mutant of ErbB3 dimerized with ErbB2 and elicited ERK and PI3K/Akt signaling in the absence of heregulin. To determine the effect of N-glycans on heterodimerization with ErbB2, a cross-linking study was also done using ErbB2/ErbB3 coexpressing cells. The result showed that the N418Q mutant of ErbB3 underwent heterodimerization with wild-type ErbB2 in the absence of heregulin (Fig. 3 ). The formation of a heterodimer of the N418Q mutant of ErbB3 with wild-type ErbB2 promoted receptor tyrosine phosphorylation (Fig. 3) and activated the ERK and PI3K/Akt pathways (Fig. 4 ) in the absence of heregulin. However, we observed no significant morphologic changes between those transfectants.


Figure 3
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  		Figure 3. Coexpression of the N418Q mutant of ErbB3 and wild-type ErbB2 induces heterodimer formation and tyrosine phosphorylation in the absence of a ligand. After serum starvation for 2 h, wild type or the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing Flp-In-CHO cells were treated with or without 5 nmol/L of heregulin for 5 min. Cross-linking was initiated by adding BS3 to a final concentration of 1 mmol/L and incubated for 2 h at room temperature. The reactions were stopped by adding 50 mmol/L of Tris-HCl (pH, 7.4), and cross-linked products were separated by 8% SDS-PAGE and probed with an anti-ErbB3 antibody or an anti-ErbB2 antibody. Cross-linked products were also immunoprecipitated with anti-ErbB2 antibody, subjected to 8% SDS-PAGE and probed with anti-phosphotyrosine antibody (PY20).

 

Figure 4
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  		Figure 4. Up-regulation of phosphorylated ERK and Akt in N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells. After serum starvation 2 h, wild-type or the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing Flp-In-CHO cells were pretreated with or without 10 mol/L of PD98059 (A) or 1 mol/L of wortmannin (B) for 30 min. The cells were then treated with or without 5 nmol/L of heregulin for 5 min and solubilized in lysis buffer, and whole cell lysates were subjected to 12.5% SDS-PAGE. The blots were probed with anti–phospho-ERK and anti-ERK, anti–phospho-Akt and anti-Akt. PD98059, MEK inhibitor; wortmannin, PI3K inhibitor.

 
Cell proliferation and anchorage-independent cell growth are up-regulated by the N418Q mutant of ErbB3. To assess the receptor function of the N418Q mutant of ErbB3, we investigated cell proliferation using a WST-8 assay. The N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells proliferated faster than wild-type ErbB3 and ErbB2 coexpressing cells (Fig. 5 ). The difference was significant after day 4. Cell proliferation was inhibited by PD98059 or wortmannin. Furthermore, to assess the anchorage dependency of cell growth, soft agar assays were done, which is considered to be related to malignant transformation. As shown in Fig. 6A and B , the number of colonies was increased in the plate containing the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells, compared with the plate containing wild-type ErbB3 and wild-type ErbB2 coexpressing cells. The enhanced growth was inhibited by both PD98059 and wortmannin.


Figure 5
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  		Figure 5. Proliferation was promoted in N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells. Cell proliferation was detected using a cell counting kit as described in Materials and Methods. N418Q mutant or wild-type of ErbB3 and wild-type ErbB2 coexpressing cells were plated at a density of 2.5 x 103 cells per well in 96-well plates in the presence or absence of PD98059 or wortmannin and cultured in Ham's F12 medium supplemented with 10% FCS. At the indicated time points (0, 24, 48, 72, and 96 h), the absorbance of each well was read at OD450 nm after the addition of the WST-8 reagents according to manufacturer's instructions. Graph indicates the number of cells expressing N418Q mutant (solid line) or wild-type (broken line) of ErbB3 and wild-type ErbB2. Points, means; bars, SD.

 

Figure 6
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  		Figure 6. Anchorage-independent cell growth and tumor formation were promoted in N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells. To assess anchorage-independent cell growth and to evaluate tumor formation in the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells, a soft agar colony formation assay and a tumor formation assay in athymic mice were done as described in Materials and Methods. A and B, 2 x 102 of cells, expressing N418Q mutant or wild-type ErbB3 and wild-type ErbB2, were seeded in soft agar plate in the presence or absence of PD98059 or wortmannin. After 2 to 3 wks, the number of colonies was counted. Columns, means; bars, SD. C, 1 x 106 of N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells or wild-type ErbB3 and wild-type ErbB2 coexpressing cells were s.c. injected into 5-wk-old female athymic mice (BALB/c mice; n = 9 in each group). At 1 mo after the injection, tumor formation was monitored. D, for quantitative analysis, tumor volume (mm3) was measured (P < 0.02). Columns, means; bars, SD.

 
Tumor formation was promoted for N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells in athymic mice. To determine the effects of the increased activity of the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells on tumor growth, cells were injected into 5-week-old female athymic mice, and tumor growth was monitored. As shown in Fig. 6C, the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells permitted the vigorous formation of tumor, 1 month after the injection. The mean volumes of tumors of mice injected with the N418Q mutant of ErbB3 and wild-type ErbB2 coexpressing cells were about thrice as large as that of mice injected wild-type ErbB3 and wild-type ErbB2 coexpressing cells (Fig. 6D). These results suggest that the N418Q mutation enhances malignant transformation both in vitro and in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ErbB family is ubiquitously expressed in various cells and plays an essential role in cell proliferation, differentiation, survival, migration, and adhesion. In this study, we focused on the role of N-glycans on the function of ErbB3. Mutants of ErbB3 that lack each N-glycosylation site were prepared and characterized. It has been suggested that the Asn418-linked N-glycan plays a role in regulating both the homodimerization and heterodimerization of ErbB3, and the N-glycan seems to be involved in cell proliferation, survival, and malignant transformation both in vitro and in vivo.

It has been reported that wild-type ErbB3 does not homodimerize upon neuregulin binding, whereas it forms a heterodimer with ErbB2 in the presence of neuregulin (24). In this study, we showed that only the N418Q mutant underwent autodimerization in the absence of neuregulin among all the mutants of ErbB3 that lack each N-glycosylation site. To verify that substituted glutamine has no effects on homodimerization, we prepared the mutant in which the amino acid adjacent to Asn418 was changed (L417Q) or mutants in which the amino acids adjacent to all of the N-glycosylation sites of domain III were changed (V333Q/H388Q/S394Q/L417Q/L449Q) and observed that those mutants were not autodimerized in the absence of heregulin (Fig. 3C), thus confirming that deleting N-glycan affects the dimerization status.

It has been proposed that domains I and III of EGFR or ErbB3 form a ligand-binding domain (25–28), and in the case of EGFR, it has been suggested that ligand binding alters the relative orientation of domains I and III, followed by a change in the conformation of the dimerization loop projecting from each domain II to enable receptor dimerization. In contrast, in the conformation that is interpreted to be the inactive form, the dimerization loop interacts with a disulfide-bounded loop in subdomain IV termed the tether loop, preventing exposure of the dimerization loop and changing the relative orientation of domains I and III, preventing the ligand from binding (27, 29, 30). Asn418 in ErbB3 corresponds to Asn420 in EGFR, and both residues are located in domain III. Because the attachment of N-glycans seems to be involved in receptor dimerization, it is possible that these N-glycans are crucial for maintaining the conformation of the inactive form. The issue of whether the conformations of spontaneous dimerization of N420Q mutant of EGFR (19) or N418Q mutant of ErbB3 are similar to that of ligand-induced dimerization is not known at present; however, the oligosaccharide on Asn420 of EGFR or Asn418 of ErbB3 might be involved in tethering, and thus, deleting those oligosaccharides would result in spontaneous dimerization (31). It has recently been suggested that the glycosylation at Asn579 of EGFR plays a role in ligand binding and dimerization (32); the dimerization level of N579Q-EGFR is elevated compared with wild-type EGFR, and it has been suggested that the N-glycan on N579, situated at the tip of the subdomain IV autoinhibitory tether loop, serves to strengthen the tether. The addition of an N-glycan may alter the asparagine side-chain torsion angle distribution and reduces its flexibility and may be involved in stabilizing the protein folding (33). It is possible that specific N-glycans on ErbB receptors serve to regulate conformational changes that are required for dimerization. Although the basic mechanisms of oligomerization may differ, the involvement of N-glycans in oligomerization is suggested for other receptors as well; a mutation in the {alpha}2A/ß1-adrenergic receptor at the asparagine in the N-glycan consensus sequence enhances heterodimerization (34). A mutation in the N-glycan consensus sequence of Musk, a muscle-specific receptor tyrosine kinase, results in an increase in ligand-independent activation (35). Thus, it is assumed that some N-glycans might act to prevent unnecessary protein-protein interactions.

In the case of the N420Q mutant of EGFR, downstream signaling such as ERK signaling is not activated;4 however, in the N418Q mutant of ErbB3/wild-type ErbB2 coexpressing cells, the up-regulation of downstream signaling, such as the ERK and PI3K pathways, were observed. We assume that, in the latter case, the dimerization is sufficient for downstream signaling because ErbB2 is intact. It is known that Ras/ERK pathway and PI3K/Akt pathway are involved in the ErbB-related carcinogenesis (2, 36–39). In the present study, cell proliferation and anchorage-independent cell growth were increased by the N418Q mutation of ErbB3 through both the ERK and PI3K/Akt pathways (Figs. 5 and 6A and B). The N418Q mutant of ErbB3 also enhanced the growth rate of tumors that were s.c. injected into athymic mice (Fig. 6C and D). These findings suggest that signaling through the N418Q-mutated ErbB3/ErbB2 heterodimer can contribute to tumor growth both in vitro and in vivo. The Asn418-linked N-glycan in ErbB3 may modify the transforming activity of ErbB3, and it is a possible additional example of the glycoconjugate regulation of cancer malignancy (40).

It has been reported that N-glycosylation affects the trafficking of many glycoproteins such as EGFR, TrkA, or insulin receptor (41–44). We previously reported that a bisecting GlcNAc modification of N-glycans results in the enhancement of endocytosis of EGFR (20). It was also reported that an N-glycan with a GlcNAcß1,6 branch regulates the expression levels of EGFR by inhibiting its endocytosis (13). In the case of the insulin receptor, N-glycans may regulate the transport of the receptor to the cell surface and also take part in autophosphorylation (45). The mutant in which all of the N-glycosylation sites in domain III of ErbB3 were changed to glutamine (N334Q/N389Q/N395Q/N418Q/N450Q) was not expressed on the cell surface (Fig. 2B), suggesting that at least some combination of N-glycans in domain III of ErbB3 might be crucial for receptor trafficking to the cell surface.

It is not known whether N-glycosylation on Asn418 in domain III of ErbB3 is implicated in human cancer. We have to examine the possibility of the involvement of N-glycans of the ErbB family in oncogenesis and determine the mechanisms by which N-glycans regulate ErbB oligomerization in a future study.


   Acknowledgments
 
Grant support: Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan; the 21st Century Center of Excellence Program from Japan Society for the Promotion of Science; Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency; and Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (S. Yokoe).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

4 Unpublished observation. Back

Received 8/15/06. Revised 10/20/06. Accepted 12/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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