Translocation of activated heterotrimeric G protein Gαo to ganglioside-enriched detergent-resistant membrane rafts in developing cerebellum

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Abstract

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. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Yuyama, K., Sekino-Suzuki, N., Sanai, Y., & Kasahara, K. (2007). Translocation of activated heterotrimeric G protein Gαo to ganglioside-enriched detergent-resistant membrane rafts in developing cerebellum. Journal of Biological Chemistry, 282(36), 26392–26400. https://doi.org/10.1074/jbc.M705046200

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