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Overexpression of Ganglioside GM1 Results in the Dispersion of Platelet-derived Growth Factor Receptor from Glycolipid-enriched Microdomains and in the Suppression of Cell Growth Signals

2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês

10.1074/jbc.m107756200

1083-351X

Teruhiko Mitsuda, Keiko Furukawa, Satoshi Fukumoto, Hiroshi Miyazaki, Takeshi Urano, Koichi Furukawa,

Erythrocyte Function and Pathophysiology

To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1+) lines. Compared with the vector control (GM1−) cell lines, GM1+ cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1+ cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1− cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1+ cells, while it was present in both GEM and non-GEM fractions in GM1− cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain. To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1+) lines. Compared with the vector control (GM1−) cell lines, GM1+ cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1+ cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1− cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1+ cells, while it was present in both GEM and non-GEM fractions in GM1− cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain. Gangliosides, sialic acid-containing glycosphingolipids are ubiquitously expressed in embryonal and adult tissues of mammals and birds (1.Wiegandt H. Glycolipids. Elsevier Science Publishing Co., Inc., New York1985: 199-260Google Scholar). In particular, they are enriched in nervous tissues, and the structures of the carbohydrate moiety are strictly regulated according to the developmental stages and tissue differentiation (1.Wiegandt H. Glycolipids. Elsevier Science Publishing Co., Inc., New York1985: 199-260Google Scholar, 2.Vanier M.T. Holm M. Ohman R. Svennerholm L. J. Neurochem. 1971; 18: 581-592Crossref PubMed Scopus (233) Google Scholar). Biological roles of gangliosides have been investigated in many studies, and various functions have been claimed such as receptors for bacterial toxins (3.King C.A. van Heyningen W.E. J. Infect. Dis. 1975; 131: 643-648Crossref PubMed Scopus (17) Google Scholar), receptors for some viruses (4.Markwell M.A. Svennerholm L. Paulson J.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5406-5410Crossref PubMed Scopus (210) Google Scholar), modulators for Ca2+ ions (5.Wu G.S. Vaswani K.K. Lu Z.H. Ledeen R.W. J. Neurochem. 1990; 55: 484-491Crossref PubMed Scopus (64) Google Scholar), those for adhesion molecules (6.Kleinman H.K. Martin G.R. Fishman P.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3367-3371Crossref PubMed Scopus (192) Google Scholar, 7.Cheresh D.A. Pierschbacher M.D. Herzig M.A. Mujoo K. J. Cell Biol. 1986; 102: 688-696Crossref PubMed Scopus (260) Google Scholar, 8.Blackburn C.C. Swank-Hill P. Schnaar R.L. J. Biol. Chem. 1986; 261: 2873-2881Abstract Full Text PDF PubMed Google Scholar), and for growth factor receptors (9.Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar). Some of them have also been assigned as a messenger of apotopsis signals (10.De Maria R. Rippo M.R. Schuchman E.H. Testi R. J. Exp. Med. 1998; 187: 897-902Crossref PubMed Scopus (137) Google Scholar). These functions can be classified into two major groups; recognition molecules for exogenous soluble molecules, and modulators of cis-acting receptor molecules for various growth/differentiation factors (9.Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar). However, the molecular mechanisms for ganglioside functions as described above have scarcely been clarified. A clear demonstration of the interaction between gangliosides and other receptor molecules has never been reported except for the binding of nerve growth factor receptor with GM1 (11.Mutoh T. Tokuda A. Miyadai T. Hamaguchi M. Fujiki N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5087-5091Crossref PubMed Scopus (398) Google Scholar). The platelet-derived growth factor (PDGF) 1The abbreviations used are: PDGFplatelet-derived growth factorPDGFRplatelet-derived growth factor receptorMAPKmitogen-activated protein kinase (meaning ERK1/2)GSL(s)glycosphingolipid(s)GEMglycolipid-enriched microdomainmAbmonoclonal antibodyβ14-GalNAc-T, β1,4-N-acetylgalactosaminyltransferase or GM2/GD2 synthaseβ13-Gal-T, β1,3-galactosyltransferase or GM1 synthaseFITCfluorescein isothiocyanateMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMES4-morpholineethanesulfonic acid. The nomenclature of gangliosides is based on that of Svennerholm (54.Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1307) Google Scholar) 1The abbreviations used are: PDGFplatelet-derived growth factorPDGFRplatelet-derived growth factor receptorMAPKmitogen-activated protein kinase (meaning ERK1/2)GSL(s)glycosphingolipid(s)GEMglycolipid-enriched microdomainmAbmonoclonal antibodyβ14-GalNAc-T, β1,4-N-acetylgalactosaminyltransferase or GM2/GD2 synthaseβ13-Gal-T, β1,3-galactosyltransferase or GM1 synthaseFITCfluorescein isothiocyanateMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMES4-morpholineethanesulfonic acid. The nomenclature of gangliosides is based on that of Svennerholm (54.Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1307) Google Scholar) receptor is a member of a family of tyrosine kinases that modulate multiple cellular processes in response to ligand binding. This receptor exerts roles through multiple phosphorylation cascades, each of which begins with the phosphorylation of the receptor itself. Recently, many of the participating molecules and substrates of PDGF/PDGFR effects have been identified, and their sites of interaction were mapped (12.DeMali K.A. Whiteford C.C. Ulug E.T. Kazlauskas A. J. Biol. Chem. 1997; 272: 9011-9018Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). These findings suggested the existence of a signaling module associated with PDGFR at the cell surface, consisting of components of the tyrosine kinase mitogen-activated protein kinase (MAPK) pathway. Anderson and co-workers (13.Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar) demonstrated that caveolae fractions from unstimulated fibroblasts contained PDGFR, Ras, Raf-1, MAP kinase kinase 1, and MAPK, and PDGF stimulation activated MAP kinase in the caveolae fraction, indicating that these components are functional in vivo. platelet-derived growth factor platelet-derived growth factor receptor mitogen-activated protein kinase (meaning ERK1/2) glycosphingolipid(s) glycolipid-enriched microdomain monoclonal antibody 4-GalNAc-T, β1,4-N-acetylgalactosaminyltransferase or GM2/GD2 synthase 3-Gal-T, β1,3-galactosyltransferase or GM1 synthase fluorescein isothiocyanate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 4-morpholineethanesulfonic acid. The nomenclature of gangliosides is based on that of Svennerholm (54.Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1307) Google Scholar) platelet-derived growth factor platelet-derived growth factor receptor mitogen-activated protein kinase (meaning ERK1/2) glycosphingolipid(s) glycolipid-enriched microdomain monoclonal antibody 4-GalNAc-T, β1,4-N-acetylgalactosaminyltransferase or GM2/GD2 synthase 3-Gal-T, β1,3-galactosyltransferase or GM1 synthase fluorescein isothiocyanate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 4-morpholineethanesulfonic acid. The nomenclature of gangliosides is based on that of Svennerholm (54.Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1307) Google Scholar) Caveolae have been thought to be specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules (14.Anderson R.G. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Crossref PubMed Scopus (658) Google Scholar). They are enriched in glycosphingolipids (GSLs), cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 °C. GSLs are enriched in this detergent-insoluble microdomain, that is almost equal to GEM (15.Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (717) Google Scholar, 16.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2549) Google Scholar). (GEM was used in this article for this microdomain.) In particular, GM1 has been suggested to be a useful marker of GEM as well as a main structural protein, caveolin. If gangliosides play roles in the modulation of various receptor molecules as described above, it may be possible that gangliosides such as GM1 are involved in the regulation of signaling as a functional component of GEM, being more than an indicator of the microdomain. In fact, Bremer et al. (17.Bremer E.G. Hakomori S. Bowen-Pope D.F. Raines E. Ross R. J. Biol. Chem. 1984; 259: 6818-6825Abstract Full Text PDF PubMed Google Scholar, 18.Bremer E.G. Schlessinger J. Hakomori S. J. Biol. Chem. 1986; 261: 2434-2440Abstract Full Text PDF PubMed Google Scholar) reported that GM3 and/or GM1 added to the culture medium of cells suppressed the cell growth and/or phosphorylation of epidermal growth factor receptor and PDGFR. Exogenous GM1, in turn, enhanced the phosphorylation of nerve growth factor receptor TrkA, resulting in neurite extension of rat pheochromocytoma PC12 cells (11.Mutoh T. Tokuda A. Miyadai T. Hamaguchi M. Fujiki N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5087-5091Crossref PubMed Scopus (398) Google Scholar). In this study, we have established transfectant cells of mouse fibroblast line Swiss 3T3 highly expressing GM1 using cloned cDNAs of GM2/GD2 synthase (19.Nagata Y. Yamashiro S. Yodoi J. Lloyd K.O. Shiku H. Furukawa K. J. Biol. Chem. 1992; 267: 12082-12089Abstract Full Text PDF PubMed Google Scholar) and GM1 synthase (20.Miyazaki H. Fukumoto S. Okada M. Hasegawa T. Furukawa K. J. Biol. Chem. 1997; 272: 24794-24799Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Then, we analyzed the effects of GM1 expression on the cell proliferation, phosphorylation of PDGFR/MAPK after PDGF stimulation, and also on the intracellular localization of PDGFR. The neo-expression of GM1 suppressed both cell proliferation and phosphorylation levels of PDGFR and MAPK in response to PDGF as reported in the experiments with exogenous GM1 (17.Bremer E.G. Hakomori S. Bowen-Pope D.F. Raines E. Ross R. J. Biol. Chem. 1984; 259: 6818-6825Abstract Full Text PDF PubMed Google Scholar). Surprisingly, the majority of PDGFR has moved from GEM to the non-GEM fraction, which appeared to be a major mechanism for the reduced PDGF/PDGFR signals in GM1+ cells. Moreover, it appeared that GM1 bound PDGFR in GEM, indicating that GM1 is not only a marker of the GEM microdomain, but an important functional component probably modulating both GEM structure and PDGFR activity. PDGF-BB was purchased from Genzyme (Cambridge, MA). Anti-PDGF receptor β (rabbit IgG, 958; goat IgG M-20), anti-caveolin-1 (rabbit IgG), anti-phosphotyrosine monoclonal antibody (PY20), donkey anti-goat IgG conjugated with horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPK rabbit IgG, anti-phospho MAPK rabbit IgG, anti-rabbit IgG conjugated with horseradish peroxidase were from New England Biolabs (Beverly, MA). Cholera toxin B subunit conjugated with biotin was from LIST Biological Laboratories (Campbell, CA). ECL detection reagent was from Invitrogen (Boston, MA). Protein A-Sepharose CL4B beads were from Amersham Bioscience Inc. (Buckinghamshire, UK). Polyvinylidene difluoride membrane was from Millipore (Bedford, MA). Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's essential medium supplemented with 7.5% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. For cell growth assay, cells were cultured in 48-well plates (Falcon, Lincoln Park, NJ), and serum-starved for 24 h before PDGF (50 ng/ml) treatment. After treatment for the indicated time, MTT assay was performed. Human β1,4-N-acetylgalactosaminyltransferase (β1,4-GalNAc-T, GM2/GD2 synthase) cDNA pM2T1–1 and β1,3-galactosyltransferase (β1,3-Gal-T, GM1 synthase) cDNA pM1T9 were digested by XhoI and the fragments containing the coding regions were inserted into the XhoI site of pMIKneo to obtain the pMIKneo/β1,4-GalNAc-T and pMIKneo/β1,3-Gal-T, respectively. pMIKneo is a mammalian expression vector with SRα promoter and was provided by Dr. Maruyama of Tokyo Medical and Dental University. Swiss 3T3 cells used for cDNA transfection were plated in a 60-mm plastic tissue culture plate (Falcon). Two kinds of cDNAs were transfected into cells with LipofectAMINETM (Invitrogen, Rockville, MD) according to the manufacturer's instructions. Stable transfectants were selected in the presence of 250 μg/ml G418 (Sigma). The cell surface expression of gangliosides was analyzed by flow cytometry (Beckton Dickinson, Mountain View, CA), using the anti-ganglioside monoclonal antibodies (mAb); M2590 (anti-GM3), mAb10-11 (anti-GM2), mAb92-22 (anti-GD1a), mAbR24 (anti-GD3), mAb3F8 (anti-GD2), mAb370 (anti-GD1b), and mAb549 (anti-GT1b). The cells were incubated with mAbs for 45 min on ice and then stained with FITC-conjugated goat anti-mouse IgM or IgG (Cappel, Durham, NC). To analyze GM1, cells were incubated with the cholera toxin B subunit-biotin conjugates for 45 min on ice, and then stained with FITC-conjugated avidin (EY Laboratories, San Mateo, CA). Control cells were prepared using the second antibody alone. Two × 104 cells were seeded in 48-well plates. After serum deprivation for 24 h, they were cultured in the presence of PDGF (50 ng/ml) and 1% fetal calf serum. At day 0, 1, and 2 of culture, MTT assay was performed. Growth of cells was quantitated by assessing the reduction of MTT to formazan, measured as the absorbance at 590 nm using an enzyme-linkd immunosorbent assay reader (System Instruments, Japan). Cells were plated at a density of 2 × 106 cells/12 ml in two 10-cm plates, and serum-starved for 6 h before PDGF treatment. After treatment, cells were washed three times with phosphate-buffered saline, and lysed in lysis buffer (25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mmNa3VO4, 1.5 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 20 units/ml aprotinin). Lysed cells were centrifuged at 8,000 rpm for 10 min. For MAPK phosphorylation assay, lysates were subjected to 12% SDS-PAGE. Proteins were transferred electrophoretically onto a polyvinylidene difluoride membrane and immunoblotted with antibodies reactive with MAPK (ERK1/2) or phospho-MAPK. For PDGF receptor phosphorylation assay, lysates were immunoprecipitated with anti-PDGFR antibody (958) before immunoblotting. Briefly, precleared samples were incubated with antibody for 1 h, and then incubated with protein A-Sepharose CL4B beads overnight. The beads were washed 3 times with lysis buffer. The precipitates were subjected to 8% SDS-PAGE, electroblotted, and then immunoblotted with anti-PDGFR antibody or PY20. The bands were detected by peroxidase-conjugated anti-rabbit IgG and the ECL detection system (PerkinElmer Life Science) in both assays. Five × 107 cells were plated in five 15-cm dishes, and cultured up to 90% confluency. Then the cells were serum-starved for 6 h, and treated with PDGF (50 ng/ml) for 10 min. After treatment, cells were washed with phosphate-buffered saline containing 1 mmNa3VO4, collected, suspended in 1 ml of MNE buffer (25 mm MES, pH 6.5, 150 mm NaCl, 5 mm EDTA) containing 1% Triton X-100, then Dounce homogenized 20 times, and mixed with an equal volume of 80% sucrose (w/v) in MNE buffer. Then, samples were placed on the bottom of Ultra-Clear Centrifuge Tubes (Beckman Instruments). Two ml of 30% sucrose in MNE buffer was overlaid, and 1 ml of 5% sucrose in MNE buffer was layered on the top. These samples were centrifuged for 16 h at 20,000 × g. The entire procedure was performed at 4 °C. Five hundred ml each was fractionated from the top. An opaque band located immediately above the 5% interface (fraction 3) was collected and designated the GEM fraction. A sample from the bottom fraction (fraction 10) was collected and designated the non-GEM fraction. Sample fractions isolated as described above were 500 μl each. Fifty μl of each sample fraction was precipitated with trichloroacetic acid, washed with acetone 3 times, and subjected to SDS-PAGE and immunoblotted. The remaining samples were dialyzed twice for 4 h against the dialysis buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 50 mm NaF, 10 mmNa pyrophosphate, 0.2% Nonidet P-40, 1 mmNa3VO4, 1 mm EDTA) before immunoprecipitation with anti-PDGFR antibody. After immunoprecipitation, beads were washed 4 times with washing buffer (50 mm Tris-HCl, pH 7.5, 0.3 m NaCl, 0.5% (w/v) sodium deoxycholate, 0.5% (v/v) Nonidet P-40/0.1% SDS), and subjected to SDS-PAGE and immunoblotting. Cells were plated at a density of 1 × 107/15 ml in five 15-cm plates, and serum starved for 6 h before PDGF treatment. Then, cells were washed three times with phosphate-buffered saline containing 1 mmNa2VO4, and cross-linked at room temperature using 8.7 mm bis(sulfosuccinimidyl)suberate (BS3; Pierce, Rockford, IL) in 25 mm HEPES (pH 8.5), 120 mmNaCl, 6 mm KCl, 1 mm MgCl2, and 10 mm EGTA. The reaction was terminated after 30 min. Cells were then subjected to GEM isolation. Lipid fractions were extracted from about 200 μl of packed cells using chloroform/methanol (2:1, 1:1, 1:2) sequentially. After desalting, gangliosides were isolated by anion exchange column chromatography with DEAE-Sephadex A-50 (Amersham Biosciences Inc.). Thin layer chromatography (TLC) was performed with high performance TLC plates (Merck, Darmstadt, Germany) using a solvent system of chloroform, methanol, 0.2% CaCl2 (55:45:10). GEM fraction was isolated from V1 cells and served for immunoprecipitation with anti-PDGFR antibody. The precipitates were mixed with 20 μl of tyrosine kinase assay buffer (50 mm HEPES, pH 7.4, 20 mmMnCl2, 5 mm MgCl2, 1 mmdithiothreitol, 100 μm Na3VO4). Then, 5 μCi of [γ-32P]ATP and GM1 were added before incubation at 30 °C for 10 min. Then, samples were subjected to 8% SDS-PAGE, and relative kinase activity was measured with autoradiography. Intracelular localization of GM1 and PDGFR was analyzed by antibody staining. Cells were cultured on a cover glass and fixed with cold acetone containing 10% phosphate-buffered saline at −20 °C for 10 min. PDGFR was stained with anti-PDGFR antibody (958) and FITC anti-rabbit antibody (BIOSOURCE Int., Camarillo, CA). GM1 was stained with rhodamine-conjugated choleratoxin B (LIST Biological Laboratories Inc.). Staining pattern was analyzed with confocal microscopy (μRadianceTM, Bio-Rad Microscience Lab, Tokyo). After the transfection of Swiss 3T3 cells with β1,4-GalNAc-T expression vector (pMIKneo/β1,4-GalNAc-T) and β1,3-Gal-T expression vector (pMIKneo/β1,3-Gal-T), or pMIKneo alone, two transfectants (M1 and M3) and two vector control lines (V1 and V2) were established. Expression profiles of gangliosides were examined by flow cytometry (Fig. 1). Among the gangliosides examined, expression levels of GM1 markedly increased in the transfectants (M3 and M6). Expression levels of other gangliosides showed no change compared with those in the vector control lines (V1 and V2). The proliferation of the GM1 transfectants (M3 and M6) and the vector control lines (V1 and V2) were compared by MTT assay. As shown in Fig. 2, the GM1 transfectants showed a reduced growth rate in the presence of PDGF. The absorbance (590 nm) of transfectants (M3 and M6) in MTT assay was about 30–40% of that of vector control lines (V1 and V2). In the absence of PDGF, the growth of the transfectants was similar to that of vector control cells (data not shown). Consequently, we found endogenously expressed GM1 suppressed PDGF-dependent growth of Swiss 3T3 cells. To analyze the alteration of PDGF receptor (PDGFR) and its downstream signaling molecules, the activation of MAPK was examined by Western immunoblotting (Fig. 3A). After 6 h of serum deprivation, 50 ng/ml PDGF was added and incubated for 5, 10, 30, 60, or 120 min. Then cells were lysed, resolved by SDS-PAGE, and immunoblotted with anti-MAPK antibody. After that, the membrane was reblotted with anti-phosphorylated MAPK antibody. The intensity of MAPK bands was almost equivalent between transfectants (M3 and M6) and control lines (V1 and V2) and no apparent change in the intensity was observed during the incubation. For 5–10 min after PDGF addition, the phosphorylation level of MAPK was equivalent between the two groups. However, after 30–120 min, the transfectants showed earlier reduction in the activation levels of MAPK than those of vector control lines (Fig. 3B). After analysis of the phosphorylation level of MAPK, the activation of PDGFR was examined by immunoprecipitation and immunoblotting (Fig. 4). After 6 h of serum deprivation, 50 ng/ml PDGF was added and incubated for 5 or 60 min. Then, lysates were immunoprecipitated with anti-PDGFR antibody. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (PY20), (Fig. 4A, upper). Subsequently, the membrane was reblotted with anti-PDGFR antibody (Fig. 4A, lower). Phosphorylation levels of PDGFR in the transfectants were generally lower than those in vector control lines (Fig. 4B). It is well known that GM1 localizes in GEM and has been used as a marker of GEM. PDGFR was found in caveolae by Anderson and co-workers (21.Liu P. Ying Y. Ko Y.G. Anderson R.G. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Therefore, we isolated GEM to compare the phosphorylation levels of PDGFR in GEM and non-GEM. To confirm the technique for the isolation of GEM, we isolated GEM of transfectant cells (M3) and immunoblotting was performed with anti-caveolin-1 antibody and cholera toxin B subunit to identify GM1. As shown in Fig. 5, both caveolin-1 and GM1 were detected mainly in fraction 3. Both were present only in the low density fractions, and not in high density fractions such as fractions 9 and 10 in which soluble proteins were present. Therefore, we used fraction 3 as the GEM, and fraction 10 as the non-GEM fraction in the following experiments. We isolated the GEM fraction and the non-GEM fraction from each cell line. In all cell lines, the isolated GEM fraction contained 0.05 mg of total protein, and the non-GEM fraction contained 0.5 mg of total protein. They were subjected to immunoprecipitation with anti-PDGFR antibody. Immunoprecipitates were then immunoblotted with anti-PDGFR antibody (Fig. 6A, top). Surprisingly, PDGFR in GEM of the transfectants (M3 and M6) was clearly and significantly reduced compared with that in non-GEM, although the vector control lines (V1 and V2) showed an almost equal distribution of PDGFR between GEM and non-GEM (Fig. 6, A and B, and Table I). Then, the membrane was reblotted with anti-phosphotyrosine antibody (PY20) to determine the phosphorylation of PDGFR (Fig. 6A). The phosphorylation of PDGFR was detected only in PDGF-treated samples in both the transfectants and vector controls, and the intensity of the phosphorylated bands was almost proportional to that of PDGFR bands stained with anti-PDGFR antibody (Fig. 6, A and C, and Table I). A part of GEM and the non-GEM fractions was trichloroacetic acid precipitated, separated on SDS-PAGE, and immunoblotted with anti-caveolin-1 antibody or the cholera toxin B subunit (Fig. 6A, bottom), confirming that caveolin-1 was present in GEM in both transfectants and vector controls, and that GM1 was enriched in the GEM fraction of the transfectants.Table IPDGFR was dispersed from GEM/DIM in GM1+transfectant cells−PDGFR+PDGFRM3M6M3M6V10.006ap values obtained with paired t test.0.0070.0130.009V20.0230.0130.0090.030GEMGEM + non-GEMM3M6M3M6V10.008ap values obtained with paired t test.0.0220.0120.010V20.0060.0080.0150.011A. Significant differences in the ratio of PDGFR as GEM/non-GEM shown in Fig. 6BB. Significant differences in the relative intensity of phospho-PDGFR bands shown in Fig. 6Ca p values obtained with paired t test. Open table in a new tab A. Significant differences in the ratio of PDGFR as GEM/non-GEM shown in Fig. 6B B. Significant differences in the relative intensity of phospho-PDGFR bands shown in Fig. 6C Cross-linking of PDGFR revealed that dimer bands could be detected in GEM fractions of control cells after PDGF treatment, but not in GEM fractions of the transfectant cells. Only a faint dimer band appeared in non-GEM of M6, but not in that of M3 (Fig. 7). To analyze the complex formation of PDGFR with GM1, the GEM fraction and non-GEM fraction were subjected to immunoprecipitation with anti-PDGFR antibody. Then, immunoprecipitates were extracted with chloroform/methanol (1:1), and the extracted samples were subjected to TLC immunostaining. The polyvinylidene difluoride membrane was blotted with cholera toxin B subunit, resulting in the detection of definite GM1 bands in the GEM fraction and faint bands in the non-GEM fraction (Fig. 8A). There was no significant difference in the intensity of GM1 bands between transfectants and vector controls (Fig. 8B), suggesting that the majority of the increased GM1 in the transfectants existed in GEM, but it was difficult for them to bind to PDGFR. The ratio of immunoprecipitated GM1 to PDGFR in GEM was markedly and significantly high in GM2+ cells (Fig. 8C). The immunoprecipitates were also subjected to immunoblotting with anti-caveolin-1 antibody. Caveolin-1 could not be detected (data not shown). To analyze the effects of GM1 on the PDGFR kinase activity, immunoprecipitated PDGFR served for kinase assay in the presence of various amounts of GM1. As shown in Fig. 9, a low concentration of GM1 rather enhanced the kinase activity, while relatively high concentration of GM1 (higher than 100 μm) suppressed the kinase in a dose-dependent manner. To confirm the dispersion of PDGFR from GEM in the transfectants, cytostaining of GM1 and PDGFR was performed. GM1 was stained mainly on the cell membrane in both vector controls and transfectants, whereas the intensity was much stronger in the transfectant cells as expected (Fig. 10, left column). As for PDGFR, it was stained in membrane and cytoplasm as a granular pattern (Fig. 10, middle column). Merge of these two staining patterns revealed contrasting results between GM1− cells and GM1+ cells. The majority of GM1 was overlapped with PDGFR showing yellow color mainly around the cell membrane in the control cells (Fig. 10, right column). The faint green color seemed to represent PDGFR in the non-GEM fraction. In contrast, the yellow color indicating co-localizing GM1 and PDGFR was scarcely detected in the transfectants, and either the single red color (GM1/GEM) or single green color (PDGFR/non-GEM) was prominently detectable. These results as shown with higher magnification at the bottom were in good accordance with the results of the biochemical fractionation experiments as summarized in Fig. 11.Figure 11A scheme to present the change in PDGFR localization by the overexpression of GM1 in GEM. Overexpressed GM1 might alter the GEM organization resulting in the dispersion of PDGFR to the non-GEM fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cellular events regulated with signals via PDGF/PDGFR include cytoskeletal rearrangement and migration (22.Seppa H. Grotendorst G. Seppa S. Schiffmann E. Martin G.R. J. Cell Biol. 1982; 92: 584-588Crossref PubMed Scopus (560) Google Scholar, 23.Mellstrom K. Heldin C.H. Westermark B. Exp. Cell Res. 1988; 177: 347-359Crossref PubMed Scopus (130) Google Scholar), mitogenesis (24.Cochran B.H. Reffel A.C. Stiles C.D. Cell. 1983; 33: 939-947Abstract Full Text PDF PubMed Scopus (614) Google Scholar), differentiation (25.Noble M. Murray K. Stroobant P. Waterfield M.D. Riddle P. Nature. 1988; 333: 560-562Crossref PubMed Scopus (686) Google Scholar), calcium mobilization (26.Moolenaar W.H. Tertool