Regulated expression and functional modulation of norepinephrine transporter by isoform interaction

为了探索由选择性剪接产生的去甲肾上腺素转运体（NET）亚型之间可能的相互作用，我们首先从人类和奶牛中鉴定了一系列NET剪接变体。通过比较两种NET基因编码COOH末端区域的3'-侧翼区域，发现人和牛的NET基因组织结构相似，均由13 ~ 16外显子的C0、C1、C2和C3磁带组成。通过选择15或16外显子和包含/排除14外显子，在该区域发生选择性剪接，产生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，存在另一种异构体，称为2c型，它是由上游12nt处16外显子编码的备选盒C3'产生的，导致另一个由3个氨基酸组成的COOH末端。1a和2a型均显示功能性表达，而1b、2b和2c型在COS-7细胞中短暂表达时缺乏转运活性。结合我们之前对选择性剪接产生的大鼠NET异构体的研究结果，我们得出结论，这些异构体具有不同的功能特性和调控表达，异构体之间的相互作用可能在包括膜运输、寡聚化和功能偶联在内的各个步骤中对其功能表达进行调控。

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.