Translocation of Activated Heterotrimeric G Protein G alpha sub(o) to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum

• Published in: The Journal of biological chemistry 2007-09, Vol.282 (36), p.26392-26400
• Main Authors: Yuyama, Kohei, Sekino-Suzuki, Naoko, Sanai, Yutaka, Kasahara, Kohji
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X

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 sub(o) (G alpha sub(o)). Using sucrose density gradient analysis of cerebellar membranes, G alpha sub(o), but not G beta gamma , was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), which stabilizes G sub(o) in its active form. On the other hand, both G alpha sub(o) and G beta gamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G sub(o) in its inactive form. Only G alpha sub(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, G alpha sub(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of G alpha sub(o) into the DRM rafts. Furthermore, G alpha sub(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1 alpha , a physiological ligand for the G protein-coupled receptor, stimulated [ super(35)S]GTP gamma S binding to G alpha sub(o) and caused G alpha sub(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 G alpha sub(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.