Translocation of Activated Heterotrimeric G Protein Gαo to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum
2007; Elsevier BV; Volume: 282; Issue: 36 Linguagem: Inglês
10.1074/jbc.m705046200
ISSN1083-351X
AutoresKohei Yuyama, Naoko Sekino‐Suzuki, Yutaka Sanai, Kohji Kasahara,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the α-subunit of a heterotrimeric G protein, Go (Gαo). Using sucrose density gradient analysis of cerebellar membranes, Gαo, but not Gβγ, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5′-O-(thiotriphosphate) (GTPγS), which stabilizes Go in its active form. On the other hand, both Gαo and Gβγ were excluded from the DRM raft fractions in the presence of guanyl-5′-yl thiophosphate, which stabilizes Go in its inactive form. Only Gαo was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Gαo was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Gαo into the DRM rafts. Furthermore, Gαo was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1α, a physiological ligand for the G protein-coupled receptor, stimulated [35S]GTPγS binding to Gαo and caused Gαo translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-β-cyclodextrin. These results demonstrate the involvement of signal-dependent Gαo translocation to the DRM in the growth cone behavior of cerebellar granule neurons. The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the α-subunit of a heterotrimeric G protein, Go (Gαo). Using sucrose density gradient analysis of cerebellar membranes, Gαo, but not Gβγ, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5′-O-(thiotriphosphate) (GTPγS), which stabilizes Go in its active form. On the other hand, both Gαo and Gβγ were excluded from the DRM raft fractions in the presence of guanyl-5′-yl thiophosphate, which stabilizes Go in its inactive form. Only Gαo was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Gαo was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Gαo into the DRM rafts. Furthermore, Gαo was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1α, a physiological ligand for the G protein-coupled receptor, stimulated [35S]GTPγS binding to Gαo and caused Gαo translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-β-cyclodextrin. These results demonstrate the involvement of signal-dependent Gαo translocation to the DRM in the growth cone behavior of cerebellar granule neurons. Gangliosides, which are sialic acid-containing glycosphingolipids (GSLs), 3The abbreviations used are: GSLglycosphingolipidTxTriton X-100DRMdetergent-resistant membraneGAPgrowth-associated proteinCHOChinese hamster ovarySDFstromal cell-derived factorPTXpertussis toxinMβCDmethyl-β-cyclodextrinGTPγSguanosine 5′-O-(thiotriphosphate)GDPβSguanyl-5′-yl thiophosphateCSTCHO stable transfectants. are found in the outer leaflet of the plasma membrane of all vertebrate cells and are thought to play functional roles in cellular interactions and the control of cell proliferation (1Yamakawa T. Nagai Y. Trends Biochem. Sci. 1978; 3: 128-131Abstract Full Text PDF Scopus (260) Google Scholar, 2Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1480) Google Scholar). In the nervous system, where gangliosides are particularly abundant, the species and amounts of gangliosides undergo profound changes during development, suggesting that they play fundamental roles in this process. glycosphingolipid Triton X-100 detergent-resistant membrane growth-associated protein Chinese hamster ovary stromal cell-derived factor pertussis toxin methyl-β-cyclodextrin guanosine 5′-O-(thiotriphosphate) guanyl-5′-yl thiophosphate CHO stable transfectants. Exogenously administered gangliosides accelerate the regeneration of neurons in the central nervous system in vivo after lesioning (3Toffano G. Savoini G. Moroni F. Lombardi G. Calza L. Agnati L.F. Brain Res. 1983; 261: 163-166Crossref PubMed Scopus (191) Google Scholar). The addition of exogenous gangliosides to primary cultures of neurons and neuroblastoma cells in vitro stimulates cellular differentiation with concomitant neurite sprouting and extension (4Ledeen R.W. J. Neurosci. Res. 1984; 12: 147-159Crossref PubMed Scopus (288) Google Scholar). Glucosylceramide synthesis, the first glycosylation step in GSL synthesis, is required for embryonic development (5Yamashita T. Wada R. Sasaki T. Deng C. Bierfreund U. Sandhoff K. Proia R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9142-9147Crossref PubMed Scopus (407) Google Scholar). The transfection of the ganglioside GD3 synthase cDNA into neuroblastoma cells induces cholinergic differentiation and neurite sprouting (6Kojima N. Kurosawa N. Nishi T. Hanai N. Tsuji S. J. Biol. Chem. 1994; 269: 30451-30456Abstract Full Text PDF PubMed Google Scholar). GD3 synthase gene knock-out mice exhibit impairment in the regeneration of lesioned hypoglossal nerves (7Okada M. Itoh Mi M. Haraguchi M. Okajima T. Inoue M. Oishi H. Matsuda Y. Iwamoto T. Kawano T. Fukumoto S. Miyazaki H. Furukawa K. Aizawa S. J. Biol. Chem. 2002; 277: 1633-1636Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Finally, ganglioside-deficient mice exhibit central nervous system degeneration (8Yamashita T. Wu Y.P. Sandhoff R. Werth N. Mizukami H. Ellis J.M. Dupree J.L. Geyer R. Sandhoff K. Proia R.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2725-2730Crossref PubMed Scopus (197) Google Scholar). These findings show that gangliosides are involved in neural cell differentiation and brain development. However, the molecular mechanisms and signal transduction pathways underlying the ganglioside-dependent neural functions remain unclarified. GSLs exist in clusters and form microdomains containing cholesterol at the surface of the plasma membrane called rafts (9Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). Rafts are insoluble in cold nonionic detergents such as Triton X-100 (Tx) and can be isolated from the nonraft domains of the cell membrane (10Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar), and these rafts are called detergent-resistant membrane (DRM) rafts. GSL microdomains have been implicated in signal transduction because various signaling molecules, such as Src family kinases, are associated with them. However, the precise functions of GSL-enriched microdomains remain to be clarified. We have been investigating the association of gangliosides with specific proteins in the central nervous system. We previously demonstrated that an anti-ganglioside GD3 antibody (R24) co-immunoprecipitates phosphorylated proteins of 40, 53, 56, and 80 kDa and proteins of 135 and 16 kDa from rat cerebellar neurons. Of these proteins, the 53- and 56-kDa phosphoproteins were identified as the Src family kinase Lyn (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The 135-kDa protein was identified as the glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule TAG-1 (12Kasahara K. Watanabe K. Takeuchi K. Kaneko H. Oohira A. Yamamoto T. Sanai Y. J. Biol. Chem. 2000; 275: 34701-34709Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). We have demonstrated that TAG-1 transduces a signal via Lyn in the lipid rafts of primary cerebellar granule neurons and promotes neurite outgrowth (13Kasahara K. Watanabe K. Kozutsumi Y. Oohira A. Yamamoto T. Sanai Y. Neurochem. Res. 2002; 27: 823-829Crossref PubMed Scopus (48) Google Scholar). In this study, we identified the 40-kDa phosphoprotein as the α-subunit of the heterotrimeric G protein Go (Gαo) and demonstrated the activation-dependent translocation of Gαo to lipid rafts, leading to the growth cone collapse of cerebellar granule neurons. Materials—The anti-ganglioside GD3 monoclonal antibody (R24), anti-transferrin receptor monoclonal antibody, anti-Lyn monoclonal antibody (Lyn8), and anti-growth-associated protein (GAP)-43 monoclonal antibody were obtained from Signet Laboratories, Zymed Laboratories Inc., Wako Chemicals, and Chemicon International, respectively. The anti-Gαo polyclonal IgG (K-20), anti-Gαo monoclonal antibody (A2), and anti-Gβ polyclonal IgG (T-20) were purchased from Santa Cruz Biotechnology, except for the anti-Gαo (GC/2) polyclonal antibody (PerkinElmer Life Sciences) used for the in vitro kinase assay. The anti-Gαi-1 and anti-Gαi-2 polyclonal IgGs were obtained from Calbiochem. The anti-Lyn polyclonal IgG and anti-p44/42 mitogen-activated protein kinase polyclonal IgG were purchased from Cell Signaling Technology, Inc.; the anti-neurofilament IgG was purchased from Sigma. Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 568-conjugated phalloidin were obtained from Molecular Probes. An Enhanced Chemiluminescence kit was purchased from Amersham Biosciences. Complete Mini, a protease inhibitor mixture containing (p-amidinophenyl)methanesulfonyl fluoride, aprotinin, bestain, calpain inhibitor I, calpain inhibitor II, chymostatin, E-64, hirudin, leupeptin, α2-macroglobulin, Pefabloc SC, pepstatin, phenylmethylsulfonyl fluoride, 1-chloro-3-tosylamido-7-amino-2-heptanone-HCl, l-1-tosylamido-2-phenylethyl chloromethyl ketone, trypsin inhibitor, egg white, and soybean, was purchased from Roche Applied Science. Immunoprecipitation and in Vitro Kinase Assay—Membrane preparation from adult Wistar rat cerebella, immunoprecipitation with anti-ganglioside GD3 antibody, and an in vitro kinase assay were performed as described previously (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Briefly, the membrane fractions from the cerebella were solubilized in a lysis buffer (1% Tx, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl; 1 mm Na3VO4, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml pepstatin A) at 4 °C for 20 min. The supernatants were incubated with R24 and precipitated with protein G-Sepharose. Following immunoprecipitation, the in vitro kinase reaction was started by the addition of the 5 μCi of [γ-32P]ATP (3,000 Ci/mmol; PerkinElmer Life Sciences). Phosphorylation was stopped by the addition of Laemmli sample buffer, and the samples were subjected to SDS-PAGE followed by autoradiography. In a reimmunoprecipitation experiment, following the kinase reaction, the samples were boiled for 5 min in the lysis buffer with 1% SDS, diluted 10-fold with the lysis buffer, and then reimmunoprecipitated with anti-Gαo (GC/2). Expression of Gαo in Chinese Hamster Ovary (CHO) Cells—CHO cells or CST cells (CHO stable transfectants expressing ganglioside GD3 synthase) (14Ogura K. Nara K. Watanabe Y. Kohno K. Tai T. Sanai Y. Biochem. Biophys. Res. Commun. 1996; 225: 932-938Crossref PubMed Scopus (67) Google Scholar) were transfected transiently with the pcDNA-1 plasmid containing the rat Gαo1 gene (provided by Dr. T. Okamoto, RIKEN, Saitama, Japan) using Lipofectamine 2000 reagent (Invitrogen), according to the manufacture's instructions. Forty-eight hours after transfection, immunoprecipitation with R24 was performed as described previously (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Gαo was not endogenously expressed in the CHO cells. Treatment with GTPγS or GDPβS—The membrane fraction of the rat cerebellum was prepared as described previously (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Membrane aliquots (5 mg of protein) were suspended in 100 μl of the reaction buffer (20 mm Tris-HCl, 1 mm dithiothreitol, 250 mm (NH4)2SO4, and Complete Mini) containing 5 mm GTPγS and 5 mm MgCl2, or 5 mm GDPβS. After brief sonication and incubation for 30 min at 30 °C, the suspension was centrifuged at 11,500 × g for 10 min at 4 °C. The pellets as membrane fractions treated with GTPγS or GDPβS were solubilized in lysis buffer B (2% octyl glucoside, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm Na3VO4, 1 mm EGTA, and Complete Mini) at 4 °C. Immunoprecipitation manipulations were performed as described above using protein G-Sepharose and anti-Gαo antibody (A2), followed by the detection of Gαo-binding Gβ or GAP-43 by immunoblot analysis. Sucrose Density Gradient Analysis—For treatment with mastoparan (Wako Chemicals) or mouse recombinant chemokine stromal cell-derived factor (SDF)-1α (DAKO), rat cerebellar membranes (5 mg of protein) were incubated with 10 μm mastoparan or 100 μg/ml SDF-1α in 100 μl of reaction buffer B containing 100 μm GDP, 0.1 μm GTPγS, and 10 mm MgCl2 at 30 °C for 30 min. For comparison between a developing cerebellum (postnatal day 7) and an adult cerebellum, the membrane fractions of each cerebellum were prepared. In vitro pertussis toxin (PTX)-mediated ADP-ribosylation of Go proteins in the membrane fractions (5 mg of protein) isolated from a developing cerebellum was performed with nonradioactive NAD using a standard technique (15Carty D.J. Methods Enzymol. 1994; 237: 63-70Crossref PubMed Scopus (36) Google Scholar). Then sucrose gradient analysis with Tx was performed as described previously (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Briefly, the pellets as membrane fractions were homogenized using a Teflon glass homogenizer in 2 ml of TNE/Tx buffer (0.5% Tx, 25 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 1 mm EGTA). The sucrose content of the homogenate was then adjusted to 40% by adding 80% sucrose. A linear sucrose gradient (5–30%) in 6 ml of TNE without Tx was layered over the lysate. The gradients were centrifuged for 17 h at 200,000 × g at 4 °C using a Hitachi RPS40T rotor. Ten fractions were collected from the top of the gradient, followed by immunoblot analysis using various antibodies. The distribution of GD3 in the gradient fractions was observed by dot blotting with R24. [35S]GTPγS Binding Assay—Rat cerebellar membranes (postnatal day 7, 100 μg of protein) were resuspended in 50 μl of 50 mm Tris-HCl, pH 7.6, 2 mm EDTA, 100 mm NaCl, 5 mm MgCl2, 1 μm GDP, Complete Mini, and 50 nm [35S]GTPγS (2000 Ci/mmol) and incubated in the presence or absence of the indicated concentrations of mastoparan or SDF-1α at 30 °C. After 30 min, the reaction was terminated by adding 500 μl of immunoprecipitation buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 20 mm MgCl2, 0.5% Nonidet P-40, and Complete Mini). The extracts were incubated with 10 μl of protein G-Sepharose and centrifuged after 30 min to remove nonspecifically bound proteins. The extracts were incubated with anti-Gαo antibody (A2) for 1 h at 4 °C. The immune complex was then incubated with 10 μl of protein G-Sepharose, and the complexes were collected and washed three times in immunoprecipitation buffer. [35S]GTPγS binding in the immunoprecipitates was quantified by scintillation counting. Growth Cone Preparation—The neuronal growth cones of the rat cerebellum (postnatal day 7) were prepared according to the method of Pfenninger et al. (16Pfenninger K.H. Ellis L. Johnson M.P. Friedman L.B. Somlo S. Cell. 1983; 35: 573-584Abstract Full Text PDF PubMed Scopus (173) Google Scholar) with minor modifications. Briefly, rat cerebella were homogenized at 4 °C with six passes in a glass Teflon homogenizer in five volumes of 0.32 m sucrose, 1 mm Tris-HCl, pH 7.6, 1 mm MgCl2, and Complete Mini. The crude brain homogenate was centrifuged at 900 × g for 10 min. The supernatant was layered over a step gradient of sucrose at 0.75 and 1.0 m. The gradient was centrifuged at 250,000 × g for 1 h, and the 0.32/0.75 m interface was collected and diluted with 0.32 m sucrose. After centrifugation at 100,000 × g for 30 min, the pellets were used as growth cone samples. Primary Culture—Rat cerebellar granule neurons were cultured according to the method of Levi et al. (17Levi G. Aloisi F. Ciotti M.T. Thangnipon W. Kingsbury A. Balazs R. A Dissection and Tissue Culture Manual of Nervous System Alan R. Liss Inc., New York, NY. 1989; Google Scholar) with some modifications. Briefly, cerebella were dissected from 7-day-old rats, and cerebellar neurons were prepared using a dissociation solution (Sumitomo Bakelite Co., Ltd.). Then the dissociated cells were plated in a poly-l-lysine-coated chamber slide (8-well; Becton Dickinson Labware) at a density of 3.0 × 105 cells/well containing 0.5 ml of neurobasal medium (Invitrogen) with 25 mm KCl, 2 mm glutamine, and B27 supplement (Invitrogen). Growth Cone Collapse Assay—Cells cultured for 24 h were exposed to 10 μm mastoparan or 100 ng/ml SDF-1α and observed for their growth cone morphology by microscopy. For immunofluorescence analysis, after the treatment with mastoparan and SDF-1α for 5 min, the cells were fixed in 2% paraformaldehyde with 0.05% Tx for 20 min and incubated with an anti-neurofilament antibody for 1 h. Then the cells were incubated with the Alexa Fluor 488-labeled secondary antibody and Alexa Fluor 568-labeled phalloidin. Parts of the growth cones were stained red because of the high amount of actin filaments (18Arimura N. Inagaki N. Chihara K. Menager C. Nakamura N. Amano M. Iwamatsu A. Goshima Y. Kaibuchi K. J. Biol. Chem. 2000; 275: 23973-23980Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The images were captured at 400× using a Zeiss laser-scanning confocal imaging system (LSM510). The areas stained red were extracted with the image processing software Photoshop® (Adobe), and quantification was performed using the NIH Image program V1.62. The growth cone collapse percentage was calculated between the control and treated cells from three fields (0.2 mm2) selected randomly in three independent experiments. Anti-ganglioside GD3 Antibody (R24) Precipitates α-Subunit of Trimeric G Protein, Go (Gαo)—The immunoprecipitates obtained using R24 from the Tx extract of rat cerebellar membranes were analyzed for the presence of protein kinase activity. An in vitro kinase reaction resulted in the phosphorylation of several proteins of 40, 53, 56, and 80 kDa, as determined by SDS-PAGE (Fig. 1, lane 1). We previously identified p53/56 as two isoforms of the Src family kinase Lyn by sequential immunoprecipitation with R24 and the anti-Lyn antibody (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In this study, the same method was used in p40 identification. Briefly, the in vitro kinase assay was conducted, after which the immune complexes were disrupted by boiling in SDS-containing buffer and subjected to a second immunoprecipitation with the anti-Gαo antibody (Fig. 1, lane 3). As a result, the anti-Gαo antibody specifically precipitated p40 in the reimmunoprecipitation experiments. This result suggests that there is a specific association of Gαo with GD3 in the rat brain cell membrane. Gαo Is Associated with GD3 in cDNA Expression System—The association of Gαo with GD3 was confirmed using a cDNA expression system in CHO cells. GM3 is the only ganglioside synthesized in CHO cells and is an enzymatic substrate of GD3 synthase. We previously established a CHO cell line, namely, CST, which constitutively expresses GD3 synthase and demonstrated that R24 co-precipitated Lyn from CST cells expressing Lyn (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Both CHO and CST cells were transiently transfected with an expression plasmid carrying the Gαo gene. Gαo was co-immunoprecipitated by R24 (Fig. 2, lanes 2 and 5) from the CST cells but not from the CHO cells. This finding confirms the interaction of GD3 with Gαo. In addition, the specific binding of R24 to GD3 was verified. Gαo Is Abundant in DRMs in Developing Cerebellum but Not in Adult Cerebellum—GSLs form microdomains called "lipid rafts" or "rafts" in cellular membranes. Lyn is associated with lipid rafts in several types of cell including cerebellar neurons (11Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 19Zucchi I. Prinetti A. Scotti M. Valsecchi V. Valaperta R. Mento E. Reinbold R. Vezzoni P. Sonnino S. Albertini A. Dulbecco R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1880-1885Crossref PubMed Scopus (22) Google Scholar, 20Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). To investigate whether Gαo exists in lipid rafts, we isolated lipid rafts by treating cerebellar membranes with cold Tx and separating DRMs by sucrose gradient centrifugation. Go is a heterotrimeric GTP-binding protein composed of α, β, and γ subunits. The activation of G proteins was fundamentally initiated by the exchange of GDP by GTP bound to the α-subunits and the dissociation of the heterotrimer to an α-monomer and a βγ dimer. In this study, we examined the distribution of both monomeric and trimeric forms of Gαo on a sucrose gradient and compared the distribution between the membranes of a developing cerebellum (postnatal day 7) and an adult cerebellum. As shown in Fig. 3A, anti-Gαo antibody precipitated Gβ from the adult cerebellum (lane 6, the band is indicated by the upper arrow with the solid line) but not from the developing cerebellum (lane 4). This indicates that the level of the monomeric forms of Gαo was higher in the developing cerebellum than in the adult cerebellum. Sucrose density gradient analysis (Fig. 4B) showed that Gαo predominantly existed in the DRM fractions (lanes 3–5) in the postnatal developing cerebellum (62% of total in DRM fractions). In contrast, few Gαo molecules were present in the DRM fraction in the adult cerebellum (2% of total in DRM fractions). Most Gβ molecules existed in the nonraft fraction (lanes 7–10) in both the developing and adult cerebella (80 and 89% of total, in non-DRM fraction, respectively). The presence of Lyn in the DRM fractions and the exclusion of transferrin receptor, a nonraft marker protein, from the DRM fractions, confirmed the quality of the fractionation. The levels of Gαo and Gβ proteins as determined by immunoblotting were comparable between the developing and adult cerebellar membranes (data not shown). PTX ADP ribosylates several G proteins including Go and stabilizes the G proteins in their heterotrimeric forms (15Carty D.J. Methods Enzymol. 1994; 237: 63-70Crossref PubMed Scopus (36) Google Scholar). In the presence of PTX, most Gαo molecules were excluded from the DRM fractions in the developing cerebellum (92% of total in non-DRM fractions) (Fig. 3C). These observations suggest that Gαo undergoes translocation to the lipid rafts in the early stage of cerebellar development in an activation-dependent manner. The pretreatment of the cholesterol-depleting agent, methyl-β-cyclodextrin (MβCD) significantly reduced Gαo in the DRM fraction, suggesting that cholesterol depletion by MβCD disrupts the lipid rafts of the cerebellum (Fig. 3C).FIGURE 4Sucrose fractionation patterns of Gαo after treatment with GTPγS or GDPβS.A, immunoprecipitation of GTPγS- or GDPβS-treated cerebellar membranes with anti-Gαo monoclonal antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) using an anti-Gβ antibody. Precipitates obtained using an anti-Gαo antibody (lanes 3 and 5) or normal IgG (lanes 2 and 4) from GDPβS-treated (lanes 2 and 3) and GTPγS-treated (lanes 4 and 5) membranes. Lane 1 shows the lysate of the cerebellar membrane. The top arrow with the solid line indicates the bands of Gβ proteins, and the bottom arrow with the dotted line indicates the nonspecific bands. B, the cerebellar membrane fractions treated with GTPγS or GDPβS were fractionated by sucrose gradient centrifugation. Ten individual fractions were subjected to dot blotting (for GD3) and SDS-PAGE and Western blotting (for Gαo, Gβ, Gαi-1, Gαi-2, Lyn, and transferrin receptor (TfR)). Lanes 2–5 and 7–10 correspond to the DRM and non-DRM fractions, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gαo Is Localized to DRM after GTPγS Stimulation—The activation-dependent translocation of Gαo to the lipid rafts was confirmed using GTPγS, a nonhydrolyzable GTP analog, and GDPβS, an analog of GDP that cannot be converted to GTP (21Huang C. Duncan J.A. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 412-417Crossref PubMed Scopus (84) Google Scholar). After GTPγS treatment of the cerebellar membranes, Gαo was observed as a monomeric form dissociated from the βγ subunits (Fig. 4A, lane 5), and the majority of Gαo was localized in the DRM fractions as shown in the sucrose gradient analysis (Fig. 4B, 53% of total in DRM fraction, as estimated from blots by densitometry). In contrast, after GDPβS treatment, Gαo was precipitated with the β-subunit, which suggests that Gαo formed a heterotrimer with the βγ-subunits. Under this condition, almost all Gαo was excluded from the DRM fractions (Fig. 4B, 9% of total in DRM fractions). Most β-subunits were localized in the non-DRM fractions and did not show an obvious change upon GTPγS or GDPβS treatment (Fig. 4B, 98 or 99% of total in non-DRM fractions, respectively). Other PTX-sensitive G proteins that are expressed in the cerebellum, namely Gαi-1 and Gαi-2, did not show apparent changes in their localization patterns between these treatments. These results suggest the lateral translocation of Gαo on the membrane surface according to the state of activity of Gαo. Monomeric Gαo is solely localized to the lipid rafts, whereas trimeric Gαβγo may exist in adjacent regions of the membrane. Mastoparan and Chemokine SDF-1α Induce Recruitment of Gαo to Lipid Rafts—Mastoparan is a cationic amphiphilic tetradecapeptide isolated from wasp venom and has been reported to stimulate several G proteins including Go in a manner similar to that of the G protein-coupled receptor (22Higashijima T. Burnier J. Ross E.M. J. Biol. Chem. 1990; 265: 14176-14186Abstract Full Text PDF PubMed Google Scholar). SDF-1α, which induces leukocyte chemotaxis, triggers the chemoattraction of cerebellar granule cells (23Arakawa Y. Bito H. Furuyashiki T. Tsuji T. Takemoto-Kimura S. Kimura K. Nozaki K. Hashimoto N. Narumiya S. J. Cell Biol. 2003; 161: 381-391Crossref PubMed Scopus (167) Google Scholar). SDF-1α is the biological ligand for CXCR4, a G protein-coupled receptor. Both SDF-1α and CXCR4 are expressed in the developing cerebellum (24Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9448-9453Crossref PubMed Scopus (1434) Google Scholar, 25Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1484) Google Scholar). Mastoparan and SDF-1α significantly stimulated the Go-specific binding of GTPγS in the cerebellar membranes (Fig. 5A). After the exposure of the cerebellar membranes to mastoparan or SDF-1α, DRMs were isolated as described under "Experimental Procedures." As shown in Fig. 5B, mastoparan and SDF-1α induced the shift of Gαo into the DRM fractions (44 and 55% of total in DRM fractions, respectively; 9% of total in the DRM fractions for control samples). Almost all of the β-subunits were localized in the non-DRM fractions, and there were no obvious differences between the control and mastoparan- and SDF-1α-treated cerebella (86, 86, and 90% of total in non-DRM fractions, respectively). CXCR4 was mainly localized in the nonraft fractions (data not shown). These results indicate that Gαo translocates to the lipid rafts after activation by G protein-coupled receptor in the surrounding membrane region. Concentration of Gαo in Cerebellar Growth Cones—Growth cones are specialized neuronal compartments that are transiently generated at the tips of growing neurites. They play important roles in neurite outgrowth and the formation of neural circuits in a developing brain. Subcellular fractionation showed that the isolated growth cones (Fig. 6A, lane 1) contained higher
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