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 alpha-subunit of a heterotrimeric G protein, G(o) (Galpha(o)). Using sucrose density gradient analysis of cerebellar membranes, Galpha(o), but not Gbetagamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTPgammaS), which stabilizes G(o) in its active form. On the other hand, both Galpha(o) and Gbetagamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G(o) in its inactive form. Only Galpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Galpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Galpha(o) into the DRM rafts. Furthermore, Galpha(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1alpha, a physiological ligand for the G protein-coupled receptor, stimulated [(35)S]GTPgammaS binding to Galpha(o) and caused Galpha(o) 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-beta-cyclodextrin. These results demonstrate the involvement of signal-dependent Galpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.
Highlights
Administered gangliosides accelerate the regeneration of neurons in the central nervous system in vivo after lesioning [3]
Anti-ganglioside GD3 Antibody (R24) Precipitates ␣-Subunit of Trimeric G Protein, Go (G␣o)—The immunoprecipitates obtained using R24 from the Triton X-100 (Tx) extract of rat cerebellar membranes were analyzed for the presence of protein kinase activity
We examined the distribution of fractions in the postnatal developing cerebellum both monomeric and trimeric forms of G␣o on a sucrose gradi- (62% of total in detergentresistant membrane (DRM) fractions)
Summary
Administered gangliosides accelerate the regeneration of neurons in the central nervous system in vivo after lesioning [3]. We identified the 40-kDa phosphoprotein as the ␣-subunit of the heterotrimeric G protein Go (G␣o) and demonstrated the activation-dependent translocation of G␣o to lipid rafts, leading to the growth cone collapse of cerebellar granule neurons. Anti-ganglioside GD3 Antibody (R24) Precipitates ␣-Subunit of Trimeric G Protein, Go (G␣o)—The immunoprecipitates obtained using R24 from the Tx extract of rat cerebellar membranes were analyzed for the presence of protein kinase activity.
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