Tyrosine Kinase Activity of Epidermal Growth Factor Receptor Is Regulated by GM3 Binding through Carbohydrate to Carbohydrate Interactions

Abstract

Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and β-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in α-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIB-knocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR. Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and β-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in α-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIB-knocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR. Epidermal growth factor receptor (EGFR) 4The abbreviations used are: EGFR, epidermal growth factor receptor; GM3, NeuAcα3Galβ4Glcβ1Cer; GM1, Galβ3GalNAcβ4(NeuAcα3)Galβ4Glcβ1Cer; ManIB, α-mannosidase IB; GlcNAc, N-acetylglucosamine; GS-II, G. simplicifolia II lectin; ConA, concanavalin A lectin; HRP, horseradish peroxidase; PA, pyridylamination; DMEM, Dulbecco’s modified Eagle’s medium; HRP, horseradish peroxidase; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; KD, knock-down; Os Fr. B, penta-antennary GlcNAc terminal N-glycan. is a 170-kDa transmembrane glycoprotein with an intracellular tyrosine kinase domain (1Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L.V. Whittle N. Waterfield M.D. Seeburg P.H. Nature. 1984; 309: 418-425Crossref PubMed Scopus (2096) Google Scholar). EGFR is expressed in epithelial cells, especially in basement membrane of stratified epithelium and squamous epithelium, and is overexpressed in solid cancers such as carcinoma cells. EGFR belongs to the erbB transmembrane receptor family, and plays a role in cell proliferation and differentiation of the epithelial surface. Ligand (EGF or α-celluline) binding leads to dimerization of EGFR and tyrosine phosphorylation, which then activates intracellular signal transducers such as mitogen-activated protein kinase (MAPK), signal transducers and activators of transcription (STAT), Akt, etc. (2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4918) Google Scholar, 3Oda K. Matsuoka Y. Funahashi A. Kitano H. Mol. Syst. Biol. 2005; 120050010Crossref Scopus (873) Google Scholar). The involvement of oligosaccharides on EGFR function has been widely studied. Twelve N-linked oligosaccharides (N-glycans) are attached to EGFR (1Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L.V. Whittle N. Waterfield M.D. Seeburg P.H. Nature. 1984; 309: 418-425Crossref PubMed Scopus (2096) Google Scholar), and loss of these N-glycans reduced EGFR activation (4Fernandes H. Cohen S. Bishayee S. J. Biol. Chem. 2001; 276: 5375-5383Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Moreover, Taniguchi’s group (5Sato Y. Takahashi M. Shibukawa Y. Jain S.K. Hamaoka R. Miyagawa J. Yaginuma Y. Honke K. Ishikawa M. Taniguchi N. J. Biol. Chem. 2001; 276: 11956-11962Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 6Gu J. Zhao Y. Isaji T. Shibukawa Y. Ihara H. Takahashi M. Ikeda Y. Miyoshi E. Honke K. Taniguchi N. Glycobiology. 2004; 14: 177-186Crossref PubMed Scopus (50) Google Scholar, 7Takahashi M. Tsuda T. Ikeda Y. Honke K. Taniguchi N. Glycoconj. J. 2004; 20: 207-212Crossref PubMed Scopus (62) Google Scholar, 8Wang X. Gu J. Ihara H. Miyoshi E. Honke K. Taniguchi N. J. Biol. Chem. 2006; 281: 2572-2577Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) also reported that EGFR function was changed in an N-glycanstructure-dependent manner. They demonstrated the biological significance of the core fucosylation of N-glycans on EGFR in intracellular signaling. Mice lacking fucosyltransferase 8 (Fut8–/– mice) have provided insight into the molecular mechanism of core fucosylation-regulated cell growth and cell differentiation. Studies using these mice demonstrated that a decrease in core fucose on EGFR caused a reduction in EGFR phosphorylation, which in turn reduced the affinity of EGF for EGFR (8Wang X. Gu J. Ihara H. Miyoshi E. Honke K. Taniguchi N. J. Biol. Chem. 2006; 281: 2572-2577Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). A bisecting N-acetylglucosamine (β-1,4-GlcNAc) modification of N-glycans also affects EGFR function (5Sato Y. Takahashi M. Shibukawa Y. Jain S.K. Hamaoka R. Miyagawa J. Yaginuma Y. Honke K. Ishikawa M. Taniguchi N. J. Biol. Chem. 2001; 276: 11956-11962Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The addition of bisecting GlcNAc influences the structure of N-glycans by inhibiting transfer by N-acetylglucosaminyltransferase IV and N-acetylglucosaminyltransferase V (9Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (498) Google Scholar). Bisecting GlcNAc-modified EGFR did not differ in its phosphorylation ratio, but caused an increase in extracellular signal-regulated kinase (ERK) phosphorylation due to the increase in EGFR internalization. EGFR regulation by glycolipids was also studied. Cell growth delay of the human epidermoid carcinoma cell line A431 is caused by GM3 (NeuAcα3Galβ4Glcβ1Cer)-dependent inhibition of EGFR tyrosine kinase activity (10Bremer E.G. Schlessinger J. Hakomori S. J. Biol. Chem. 1986; 261: 2434-2440Abstract Full Text PDF PubMed Google Scholar). GM3 is a ganglioside that binds to the extracellular domain of EGFR and inhibits its dimerization (11Miljan E.A. Meuillet E.J. Mania-Farnell B. George D. Yamamoto H. Simon H.G. Bremer E.G. J. Biol. Chem. 2002; 277: 10108-10113Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) without inhibiting ligand binding (10Bremer E.G. Schlessinger J. Hakomori S. J. Biol. Chem. 1986; 261: 2434-2440Abstract Full Text PDF PubMed Google Scholar). Wang et al. (12Wang X.Q. Sun P. Paller A.S. J. Biol. Chem. 2002; 277: 47028-47034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) reported that GM3 mediated binding of EGFR with caveolin-1. However, the GM3 binding site on the EGFR extracellular domain and the structurally bound form of EGFR are unknown. Hakomori (13Hakomori S. Glycoconj. J. 2004; 21: 125-137Crossref PubMed Scopus (140) Google Scholar, 14Hakomori S. An. Acad. Bras. Cienc. 2004; 76: 553-572Crossref PubMed Google Scholar, 15Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 225-232Crossref PubMed Scopus (480) Google Scholar) described the significance of carbohydrate to carbohydrate interactions on cell surface signaling and cell adhesion. It is commonly known that glycosphingolipids at the cell surface membrane are associated or complexed with signal transducers (small G-proteins, cSrc, Src family kinases), tetraspanins, growth factor receptors, and integrins. Microdomains containing glycosphingolipids complexed with growth factor receptors modulate and regulate cell growth through inhibition or activation of signal transduction. In addition to the above, interaction between GM3 and N-glycans with GlcNAc termini was recently reported (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar). These data may be consistent with the hypothesis that GM3 interacts directly with glycans on EGFR to modulate EGFR activity on the cell surface. Therefore, we designed experiments to elucidate the relationship between carbohydrate to carbohydrate interactions and EGFR function. The N-glycans on EGFR were remodeled by glycosidase digestion or by knockdown of the gene controlling N-glycan synthesis by using antisense oligonucleotides to study GM3-mediated inhibition of ligand-induced EGFR phosphorylation. In this study, we report that regulation of EGFR phosphorylation by GM3 was dependent on the structure of the N-glycans attached to EGFR. Cell Culture-Human ovarial epidermoid carcinoma A431 cells, which are known to overexpress EGFR in the membrane, were used in this study. A431 cells were cultured in DMEM (Sigma, number D5796) plus 10% heat-inactivated fetal bovine serum (Japan Bio Serum, number 82225), 100 μg/ml penicillin, 50 μg/ml streptomycin, and 110 μg/ml sodium pyruvate in 5% CO2 at 37 °C. Preparation of Membrane Fraction from A431 Cells-For preparation of membrane fractions, A431 cells were cultured, scraped, washed with lysis buffer (20 mm HEPES, 1 mm Na3VO4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1% aprotinin), and centrifuged at 5,800 × g for 5 min. Cell pellets were resuspended in lysis buffer and disrupted by a Dounce homogenizer (this step was repeated twice). The cell lysate was centrifuged at 1,750 × g for 5 min, and the supernatant was ultracentrifuged at 100,000 × g for 1 h at 4 °C. The resulting precipitate was collected as the membrane fraction. The membrane fraction was resuspended in an adequate amount of lysis buffer for determination of protein concentration or for use in further experiments. Glycosidase (Neuraminidase F and β-Galactosidase) Digestion-A431 cell membrane fraction (150 μg of protein) was centrifuged at 1,000,000 × g for 10 min at 4 °C, and the precipitate was washed and resuspended in 0.25 m HEPES buffer (pH 6.5). Glycosidase treatment was performed as follows: the membrane fraction suspended in 100 of HEPES buffer (pH was with 50 of 50 mm 100 of F number 100 in bovine serum and 100 of number 10 in bovine serum The was for 1 h at and centrifuged at 1,000,000 × g for 10 min at 4 °C. The was by the and fraction was resuspended in for 5 min at °C, by and a membrane transfer membrane, number with bovine serum a was performed with number to terminal GlcNAc, number to terminal and number to high mannose-type A was performed with number and number by was by enhanced using number In of GM3 on EGFR in vitro EGFR phosphorylation was performed as described by et al. Hakomori S. K. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and et al. S. Nakayama K. T. K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). of containing of GM3 number or μg of number in were was to these GM3 or that were was to the GM3 or by for 10 min. GlcNAc terminal penta-antennary Fr. B, or which was a N-linked from as described by et al. (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar), was to the GM3 and for h at 37 °C. The GM3, GM1, or a of GM3 and penta-antennary GlcNAc terminal N-glycan Os Fr. was to 10 of membrane fraction μg and for min at EGF of was to and for min at 5 of mm HEPES, 5 of 10 mm 5 of 100 and 5 of Na3VO4, the was on 5 of 50 were and for 10 min on buffer was and the was for min at °C. was to a concentration of and were for 5 min at °C. was by by using and The was by and by using In GM3-mediated of EGFR in EGFR phosphorylation was performed as described by et al. Hakomori S. K. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and et al. S. Nakayama K. T. K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). of containing or GM3, or in were was and the was DMEM was to the GM3 or and for 10 min. A of penta-antennary GlcNAc terminal N-glycan Os Fr. were to the GM3 and for h at 37 °C. A431 cells cultured in in DMEM containing 10% fetal bovine serum were in DMEM for h to were washed with 0.25 m HEPES buffer (pH and with F and as described for 1 h at 37 °C, cells were washed with DMEM and in DMEM containing GM3, GM1, or a of GM3 and penta-antennary GlcNAc terminal N-glycan Os Fr. for h at 37 °C. were washed with phosphorylation was by with of EGF and cells were for 1 h at 4 °C, and in of lysis buffer 5 mm 5 mm 1 mm for min at 4 °C. The cell lysate was to by using and of IB with for N-glycans in A431 cells, the α-mannosidase IB gene was down using the antisense A431 cells were with antisense and number in DMEM for 4 by addition of fetal bovine serum to a concentration of knockdown treatment was repeated EGFR was using and The A431 cell membrane fraction in 1% for 1 h at 4 was to the number with mm (pH at was at a the of mm (pH m NaCl, was to for min at a of 1 fraction was by and by and The that EGFR were in 1 of and further on a at a of EGFR were using a with and used for of N-glycan of of EGFR as described were using at 100 for oligosaccharides were on a and to using a were further using a oligosaccharides were by and was performed using × at a of with A 50 mm and 1 m a with the of was to for 110 min. by was performed using × at a of 1 with mm and mm Biochem. PubMed Scopus Google Scholar). The was to for 50 min. was performed using × at a of with (20 mm and F (20 mm 1% N. J. M. S. Y. Takahashi N. Biochem. PubMed Scopus Google Scholar). The was with F the of F was to for min. The oligosaccharides were by with of with were described by et al. S. Nakayama K. T. K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). A431 cell membrane μg of protein) were in 1% by for 1 h at 4 °C, and centrifuged at × g for min. The supernatant was then used for the GM3 was in 1 of × number were washed with and then washed with the GM3 was with the and with for 1 h at were washed with of with of for 1 h with at and centrifuged at × g for 5 min. with the were resuspended in of containing the membrane at 4 with and then washed with The and fraction was by in buffer and by with experiments using were as described GlcNAc on of EGFR by Glycosidase GlcNAc termini on N-glycans of EGFR, the A431 cell membrane fraction was with glycosidase (neuraminidase F and β-galactosidase) and by by with or by with simplicifolia II to GlcNAc termini or to EGFR from cells a whereas that from glycosidase-treated cells was not with that terminal were from the N-glycan on EGFR by the glycosidase the EGFR from cells not with glycosidase was with the whereas EGFR from glycosidase-treated cells was that GlcNAc termini were on EGFR from glycosidase-treated cells. of GM3-mediated of EGFR by Glycosidase of A431 Membrane Fraction in or A431 in glycosidase-treated or untreated A431 membrane GM3-mediated inhibition of EGFR phosphorylation in vitro was by with and in the or of GM3, or GlcNAc terminal penta-antennary N-glycan Fr. B, (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar). The of EGFR, to EGFR, is in the EGFR phosphorylation in the glycosidase-treated membrane fraction was reduced of by addition of of GM3 whereas EGFR phosphorylation in the untreated membrane fraction was of the the of EGF and of N-glycan with terminal GlcNAc and GM3 bind (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar), the increase in terminal GlcNAc on EGFR from glycosidase-treated membrane may have binding to GM3. effect of penta-antennary GlcNAc terminal N-glycan Os Fr. on inhibition of EGFR was observed in The inhibitory effect of GM3 on EGFR phosphorylation and was reduced by with GlcNAc terminal N-glycan Os Fr. for h 4 and and further reduced by of Os Fr. 5 and in the of the of EGF and GM3 These results indicate that GM3 binding to Os Fr. with GlcNAc termini reduced the inhibitory effect of GM3 on EGFR phosphorylation and that GM3 binds to GlcNAc termini in EGFR. A effect on inhibition of EGFR was observed in cultured A431 cells with (neuraminidase F and β-galactosidase) in in were with with GM3 or a of GM3 and penta-antennary GlcNAc terminal N-glycan Os Fr. B, EGFR phosphorylation was by addition of of and cell were to and GM3-mediated inhibition of EGFR phosphorylation in glycosidase-treated and untreated A431 cells were then EGFR phosphorylation was reduced of the by addition of GM3 in glycosidase-treated A431 cells whereas EGFR phosphorylation in untreated cells was of the the (20 of EGF and of effect of penta-antennary GlcNAc terminal N-glycan Os Fr. on inhibition of EGFR was also observed in The inhibitory effect of GM3 on EGF (20 EGFR phosphorylation was to of the by of GM3 and penta-antennary GlcNAc terminal N-glycan Os Fr. for h and of on of high mannose-type N-glycan on EGFR, α-mannosidase IB A. J. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was down by antisense The membrane fraction from cells was to a using A In this EGFR were observed for EGFR from cells to cells on of EGFR that EGFR from cells is for high mannose-type N-glycan. GM3-mediated of EGFR in the Membrane Fraction from EGFR phosphorylation and its regulation by GM3 were using the membrane fraction from A431 cells or A431 cells by using and EGFR phosphorylation in the of GM3 in the membrane fraction from A431 cells was of the of EGF but without However, EGFR phosphorylation in the membrane fraction from A431 cells was not by GM3 mannose-type N-glycan not bind to GM3 (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar), and inhibition of EGFR phosphorylation by GM3 was in the membrane fraction from A431 cells due to the high mannose-type N-glycan of the relationship between the of EGFR and GM3, N-glycan was EGFR from A431 cells, glycosidase-treated A431 cells, and A431 cells were using and as described EGFR by was and the N-glycans were with and by N-glycans were as in EGFR. The amount of N-glycans was reduced to in glycosidase-treated A431 cells and to in A431 cells, to in A431 cells oligosaccharides on EGFR from were The GlcNAc terminal N-glycan was to have in glycosidase-treated cells from to that N-glycans were by F and The high mannose-type N-glycan in cells from to These results are consistent with of the of N-glycans on EGFR from A431 cells, glycosidase-treated cells, and IB gene termini oligosaccharides in a N-linked on EGFR. oligosaccharides on EGFR from A431 cells, glycosidase-treated A431 cells, and cells were by as described N-linked oligosaccharides with GlcNAc termini were in glycosidase-treated EGFR, and high mannose-type oligosaccharides were in of EGFR with the interaction of GM3 with N-glycan on EGFR, were with GM3, with and by with S. Nakayama K. T. K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). were between the membrane fraction from and A431 cells. EGFR from the membrane fraction of glycosidase-treated A431 cells bound the with However, EGFR from the membrane fraction of A431 and A431 cells or binding to GM3, The of binding between GM3 and EGFR from the membrane fraction of was caused by of high mannose-type N-glycans. However, we the binding between GM3 and EGFR in glycosidase-treated cells to the of GlcNAc N-glycans. These results are consistent with GM3 inhibiting EGFR phosphorylation. In this we that regulation of EGFR kinase phosphorylation by GM3 is dependent on the binding of the GM3 carbohydrate to EGFR N-glycans. the study of et al. S. Nakayama K. T. K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar) in which GM3 regulation of EGFR was reported to be by addition of GlcNAc-terminated N-glycans. in that we the of the N-glycans on EGFR by treatment with and by down we remodeled the N-glycan to for EGFR with a high affinity or affinity for GM3. of EGFR from the glycosidase-treated cell membrane was by the addition of GM3 in vitro and in and In EGFR from the ManIB-knocked down cell membrane that for high mannose-type N-glycans was to have affinity for GM3 (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar), and regulation of EGFR phosphorylation was These results indicate that EGFR regulation by GM3 is by carbohydrate to carbohydrate interactions between GM3 and the N-glycan on EGFR. The results of the using Os Fr. with GlcNAc termini also GM3 directly binding N-glycans on EGFR. to carbohydrate interactions are with protein to carbohydrate GM3 bound to an EGFR we that a amount of penta-antennary GlcNAc terminal N-glycan Os Fr. be to binding of GM3 to EGFR Os Fr. and GM3 binding carbohydrate to carbohydrate However, or of Os Fr. were to with binding of GM3 to EGFR and and and These results also that GM3 binds to N-glycans on EGFR. structure that A431 EGFR a of The was the high and GlcNAc terminal were also GM3 bind to a of EGFR from A431 to regulate phosphorylation. In this study, we remodeled the N-glycan structure on EGFR by with or with an antisense for and then GM3-mediated inhibition of EGFR phosphorylation in N-glycan with GlcNAc termini on EGFR that of in glycosidase-treated cell membrane GM3-mediated inhibition of EGFR phosphorylation was enhanced and that N-glycans were by treatment with F and and the amount of GlcNAc termini enhanced GM3-mediated inhibition of EGFR phosphorylation. N-glycans with terminal GlcNAc were to bind to GM3, glycosidase-treated EGFR may also bind GM3. phosphorylation of glycosidase-treated EGFR was by GM3. In EGFR an amount of high mannose-type from to the high mannose-type has binding affinity for GM3, we that EGFR from the cells was not by GM3. evidence for GM3 and EGFR binding was observed in the using from and membrane were with and the binding of EGFR to was by EGFR from the membrane fraction of the cells was as a 170-kDa in the fraction binding of EGFR to the fraction was in the glycosidase-treated membrane fraction glycosidase but EGFR binding to was observed in the membrane fraction from A431 cells These results directly indicate that binding of EGFR and GM3 is dependent on carbohydrate to carbohydrate et al. Y. Hakomori S.I. K. PubMed Scopus Google Scholar) demonstrated affinity of EGFR for or for a using They reported that the effect of of N-glycans from EGFR not be observed by is consistent with a carbohydrate to carbohydrate interaction or a carbohydrate to protein interaction EGFR and GM3 results that GM3 binding to EGFR was dependent on the structure of the N-glycans on EGFR, binding GM3 to EGFR was dependent on a carbohydrate to carbohydrate However, results not the of a carbohydrate to protein of oligosaccharides in cells. The A431 cells that we used in this study are also cells that have N-glycans containing of EGFR by GM3 binding in cells is reduced GM3 binds to (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar). may be of the cells However, in cells, the is to cells. that cells GlcNAc-terminated N-glycans. These and N-glycans increase binding to GM3 (16Yoon S.J. Nakayama K.I. Takahashi N. Yagi H. Utkina N. Wang H.Y. Kato K. Sadilek M. Hakomori S.I. Glycoconj. J. 2006; 23: 639-649Crossref PubMed Scopus (35) Google Scholar). Therefore, EGFR in cells may be by GM3 binding to that this may be of the biological for the interactions between GM3 carbohydrate and EGFR N-glycan. In this study, we provide evidence that carbohydrate to carbohydrate interactions regulate EGFR signal transduction. Hakomori and of for for with N-glycan