MORITA Katsuya Hiroshima University, Graduate School of Biomedical Sciences, associate professor, 大学院・医歯薬学総合研究科, 助教授 (10116684)
DOHI Toshihiro Hiroshima University, Graduate School of Biomedical Sciences, professor, 大学院・医歯薬学総合研究科, 教授 (00034182)
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¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
To explore the possible interaction between isoforms of norepinephrine transporter (NET) produced by alternative splicing for their functional expression, we first identified a series of NET splice variants from human and cow. By comparing 3'-flanking region of both NET gene encoding COOH terminal region, it was demonstrated that organization of human and bovine NET gene displays similar structure, which consists of cassettes C0, C1, C2 and C3 from exon 13〜16. Alternative splicing occurs at this region by selection of exon 15 or 16 and inclusion/exclusion of exon 14, resulting in types 1a (C0-C1-C2), 1b (C0-C2), 2a (C0-C1-C3), 2b (C0-C3). Human NET reveals 1a and 2a, but not 1b or 2b, whereas bovine NET shows all isoforms. In human NET, one additional isoform, designated type 2c, exists, which results from alternative cassette C3' encoded by exon 16 at 12nt upstream resulting in another COOH terminal composed of 3 amino acids. Both type 1a and 2a revealed functional expression, while type 1b, 2b and 2c lacked transport activity when expressed transiently in COS-7 cells. Taken together with our previous findings of rat NET isoforms produced by alternative splicing, it is concluded that these isoforms reveal different functional properties and regulated expression, and that interaction between isofoms produces regulation of their functional expression probably at various steps including membrane trafficking, oligomerization and functional coupling.