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¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
RGS (regulators of G protein signaling) proteins comprise a large family of more than 30 members, which modulate heterotrimeric G protein signalling. We initially identified RGS8 as a brain specific RGS, then RGS8 was found to be specifically expressed in cerebellar Purkinje cells. Since the apparent site of action of RGS8 is considered to be adjacent to G proteins at the plasma membrane, we examined the subcellular distribution of the RGS8 protein by expression of RGS8 in non-neural DDT1MF2 cells. We found that RGS8 was concentrated in the nuclei, and that co-expression of constitutively active Gao (GaoQL) resulted in the translocation of RGS8 to the plasma membrane. Concerning detail distribution and the mechanism regulating the subcellualr distribution of RGS8 protein, here, we investigated the following four points.
1. Various Gα subtypes were co-expressed with RGS8. Inactive and active forms were expressed for each Gα subunit, and their effects on the distribution of RGS8 protein w
ere investigated. Only when the Gαi family was expressed, did the membrane translocation of RGS8 occur. Expression of the inactive or active form of the Gαi family showed similar effects. By generating and using a point mutant RGS8(L153F) that does not bind to Gαo, we determined whether the membrane recruitment of RGS8 might be the direct result of physical association with the Gαi family. This RGS8(L153F) was co-expressed with an inactive or active form of the Gαi family. Only active Gαo caused the membrane shift of RGS8(L153F). These results demonstrated that activation of Go can specifically induce membrane-translocation of RGS8 without their direct interaction.
2. The cellular distribution of the RGS8 protein in cerebellar Pukinje cells was studied in detail using cultured Purkinje cells and frozen sections of the cerebellum. It was shown that the protein is excluded from the nuclei and distributed in the cell body and dendrites except the axons of Purkinje cells.
3. We examined the possibility that distribution of RGS8 protein is differently regulated in neuron. The subcellular distribution of RGS8 protein in neuronally differentiated P19 cells was studied. We observed nuclear distribution and Gα dependent membrane translocation of RGS8, both of which were quite similar to observation in nonneural cells.
4. We identified a new short isoform of RGS8, RGS8S, that arises by alternative splicing. RGS8S cDNA encodes a N- terminus of 7 amino acids instead of aa 1-9 of RGS8, and 10-180 of RGS8. We examined the effects of RGS8 and RGS8S on Gq-mediated signaling. RGS8 decreased the amplitude of the response upon activation of ml muscarinic or substance P receptors, but did not remarkably inhibit signaling from m3 muscarinic receptors. In contrast, RGS8S showed much less inhibition of the response of either of these Gq-coupled receptors. Thus, we found that 9 amino acids in the N-terminus of RGS8 have contribution to the function to inhibit Gq-coupled signaling in a receptor type-specific manner, suggesting that a certain type of Gq-coupled receptors may recruit RGS8 to the plasma membrane by direct interaction. Less