Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter β-subunit.
Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter β-subunit.
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有机溶质转运蛋白β亚基的细胞外、跨膜和细胞质域的整体功能。
DOI：
10.1042/bcj20161093
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Hinkle,PatriciaM
中科院分区：
文献类型：
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作者：
Christian,WhitneyV;Hinkle,PatriciaM
胆汁酸通过肠上皮细胞基底外侧膜的转运是由有机溶质转运蛋白（Ost）完成的，该转运蛋白由一个七跨膜结构域（TMD）亚基（Ostα）和一个辅助的单一TMD亚基（Ostβ）组成。尽管先前的研究已经证明了Ostβ的TMD对其活性的重要性，但进行了进一步的研究以评估Ostβ亚基的其他区域的贡献。当Ostβ被截短以仅含有TMD且每侧具有15个额外残基并与Ostα共表达时，转运活性得以保留，而较短片段无活性。为了探索Ostβ片段的更广泛功能，构建了嵌合蛋白，其中Ostβ的N-末端、TMD或C-末端区域与受体活性修饰蛋白（RAMP 1）的相应区域融合，RAMP 1是包括降钙素受体样受体（CGRP）在内的几种七TMD G蛋白偶联受体所需的单一TMD蛋白。Ostβ/RAMP 1嵌合体与Ostα和Ost β一起表达。正如预期的那样，替换Ostβ TMD会破坏转运活性;然而，用RAMP 1序列替换Ostβ的整个N-末端或整个C-末端结构域不会阻止质膜定位或支持[3 H]牛磺胆酸盐摄取的能力。免疫共沉淀实验表明，Ostβ的C-末端是一个以前未被识别的与Ostα相互作用的位点。所有含有N-末端RAMP 1片段的嵌合体允许共表达的cAMP对环AMP强烈增加的激动剂作出反应。这些结果提供了新的见解的结构和功能的异聚体Ost转运蛋白复合物。
Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostβ segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostβ were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostβ/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostβ TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostβ with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [3H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostβ is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex.