Activation of mouse NBCe1-B by Xenopus laevis and mouse IRBITs: Role of the variable Nt appendage of IRBITs
Activation of mouse NBCe1-B by Xenopus laevis and mouse IRBITs: Role of the variable Nt appendage of IRBITs
复制标题
非洲爪蟾和小鼠 IRBIT 激活小鼠 NBCe1-B：IRBIT 可变 Nt 附属物的作用。
DOI：
10.1016/j.bbamem.2020.183240
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发表时间：
2020
影响因子：
3.4
通讯作者：
Chen Li-Ming
中科院分区：
文献类型：
--
作者：
Wang Meng;Wu Han;Liu Ying;Chen Li-Ming
1，4，5-三磷酸肌醇（inositol 1，4，5-trisphosphate，伊尔比特）释放的IP 3受体结合蛋白在调节细胞内Ca 2+信号和细胞内pH中起重要作用。IRBIT 1（由AHCYL 1编码）和IRBIT 2（由AHCYL 2编码）。爪蟾卵母细胞广泛用于离子通道和转运蛋白的生物物理研究。内源性伊尔比特是否在异种卵母细胞中表达尚不清楚。在这里，我们从青蛙卵母细胞中克隆了irbit2.L和irbit2.S，哺乳动物IRBIT 2的直系同源物。当过表达时，青蛙IRBIT与小鼠IRBIT 2-V2一样，能强烈刺激产电Na+/HCO 3-共转运蛋白NBCe 1-B。含有IRBIT结合结构域的NBCe 1-B的分离的Nt片段的表达大大降低了卵母细胞中的NBCe 1-B活性，表明NBCe 1-B的基础活性包含来自内源性青蛙伊尔比特刺激的大部分。蛙IRBIT与哺乳动物IRBIT在羧基端区域高度同源，但在氨基端（Nt）附件差异很大。有趣的是，截短研究表明，IRBIT 1的Nt附件和IRBIT 2-V2的长Nt附件适度增强，而IRBIT 2-V4的短Nt附件极大地抑制了伊尔比特和NBCe 1-B之间的功能相互作用。最后，Ala取代的Ser 68，一个关键的磷酸化位点的PEST结构域的伊尔比特，导致不同的功能后果，这取决于结构背景的Nt附件在不同的伊尔比特亚型。我们的结论是，Nt附件的IRBITs是不必要的，但发挥了重要的调节作用，在功能之间的相互作用伊尔比特和NBCe 1-B。
TheIP3receptorbinding protein released withinositol 1,4,5-trisphosphate (IRBIT) plays important roles in the regulation of intracellular Ca2+signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded byAHCYL1) and IRBIT2 (encoded byAHCYL2). The clawed frogXenopus laevisoocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed inXenopusoocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na+/HCO3–cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B.