Regulated expression and functional modulation of norepinephrine transporter by isoform interaction

通过异构体相互作用调节去甲肾上腺素转运蛋白的表达和功能调节

• 批准号: 13680844
• 负责人: KITAYAMA Shigeo
• 金额: $ 2.18万
• 依托单位: OKAYAMA UNIVERSITY (2002)Hiroshima University (2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680844/
• 关键词: norepinephrine; transporter; neurotransmitter; alternative splicing; antidipressant

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.

为了探索通过选择性剪接产生的去甲肾上腺素转运蛋白（NET）亚型之间可能的相互作用以实现其功能表达，我们首先鉴定了一系列来自人类和牛的 NET 剪接变体。通过比较编码COOH末端区域的两个NET基因的3'-侧翼区域，证明人和牛NET基因的组织结构相似，均由来自外显子13〜16的C0、C1、C2和C3盒组成。通过选择外显子 15 或 16 以及包含/排除外显子 14，在该区域发生选择性剪接，产生类型 1a (C0-C1-C2)、1b (C0-C2)、2a (C0-C1-C3)、2b (C0-C3)。人类 NET 显示 1a 和 2a，但不显示 1b 或 2b，而牛 NET 显示所有亚型。在人类 NET 中，存在一种额外的亚型，称为 2c 型，它是由上游 12nt 外显子 16 编码的替代盒 C3' 产生的，导致另一个由 3 个氨基酸组成的 COOH 末端。 1a 型和 2a 型均显示出功能性表达，而 1b、2b 和 2c 型在 COS-7 细胞中瞬时表达时缺乏转运活性。结合我们之前对通过选择性剪接产生的大鼠 NET 同工型的发现，得出的结论是，这些同工型揭示了不同的功能特性和受调节的表达，并且同工型之间的相互作用可能在不同的步骤（包括膜运输、寡聚化和功能偶联）产生对其功能表达的调节。

项目成果

Toshihiro Dohi et al.: "Inhibition of monoamine neurotransmitter transporter and central nervous system stimulation induced by synthetic local anesthetics and cocaine : a"Current Medicinal Chemistry-Central Nervous System Agents : Mini-Review. 2. 295-315

Toshihiro Dohi 等人：“合成局部麻醉剂和可卡因诱导的单胺神经递质转运蛋白和中枢神经系统刺激的抑制：a”当前药物化学-中枢神经系统药物：小型评论。

Shigeo Kitayama et al.: "Functional characterization of the splicing variants of human norepinephrine transporter"Neurosci.Lett.. 312. 108-112 (2001)

Shigeo Kitayama 等：“人去甲肾上腺素转运蛋白剪接变体的功能表征”Neurosci.Lett.. 312. 108-112 (2001)

Shigeo Kitayama, Kei Kumagai, Katsuya Morita and Toshihiro Dohi: "Identification and functional characterization of the novel isoforms of bovine norepinephrine transporter produced by alternative splicing"Brain.Res.. 934. 152-156 (2002)

Shigeo Kitayama、Kei Kumagai、Katsuya Morita 和 Toshihiro Dohi：“通过选择性剪接产生的牛去甲肾上腺素转运蛋白的新型亚型的鉴定和功能表征”Brain.Res.. 934. 152-156 (2002)

Mitsutaka Sugimura, Shigeo Kitayama, Katsuya Morita, Masahiro Irifune, Tohru Takarada, Michio Kawahara and Toshihiro Dohi: "Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously

Mitsutaka Sugimura、Shigeo Kitayama、Katsuya Morita、Masahiro Irifune、Tohru Takarada、Michio Kawahara 和 Toshihiro Dohi：“挥发性麻醉剂和静脉麻醉剂对其转运蛋白异源摄取 GABA、谷氨酸和多巴胺的影响