ILEAL BILE ACID TRANSPORTER METABOLISM AND REGULATION

回肠末端钠/胆汁酸协同转运蛋白的鉴定和克隆 （ASBT）cDNA提供了关于其主要 结构、组织表达、调节、转运机制和在 人类疾病然而，很少有人知道的组装和结构的 功能性ASBT，其质膜表达和周转的调节， 或胆汁酸跨细胞转运和流出。拟议的研究将 重点研究肠上皮细胞摄取胆汁酸的分子机制。的 本研究的长期目标是了解 正常人肠道胆汁酸转运与饮食脂质代谢 和疾病状态。以下问题将得到解决：（1）什么是 一般结构和亚基化学计量的ASBT？有哪些步骤 ASBT生物起源与其一级结构相反， 功能转运蛋白复合物的化学计量和组装很差 明白初步研究表明，亚基内和可能的间 亚基二硫键是ASBT折叠的先决步骤， 转运蛋白复合物形成。在拟议的研究中， ASBT的化学计量将在未转染的MDCK细胞和正常的MDCK细胞中确定。 大鼠胆管细胞将确定相关亚单位的身份 使用化学交联、免疫共沉淀和酵母的组合， 双杂交筛选策略(2)细胞膜上的表达是怎样的？ ASBT监管？ASBT的表达受底物、激素如 糖皮质激素和促胰液素以及cAMP。而这一规定的一部分， ASBT蛋白或血浆的转录、翻译后调节 膜活性似乎是重要的组成部分。拟议 研究，ASBT的胞吐/胞吞和降解机制将是 测定(3)肠道Oatp 3在胆汁酸中的作用是什么 吸收？胆汁酸通过主动钠依赖性转运被吸收， 被动促进运输。一个候选的运输者， 转运蛋白Oatp 3已被分离。Oatp 3的细胞表达和作用 将测定肠胆汁酸转运。

The identification and cloning of the ileal apical Na/bile acid cotransporter (ASBT) cDNA has provided new information regarding its primary structure, tissue expression, regulation, transport mechanism, and role in human disease. However, little is known about the assembly and structure of the functional ASBT, the regulation of its plasma membrane expression and turnover, or bile acid transcellular transport and efflux. The proposed research will focus on the molecular mechanism for enterocyte bile acid uptake. The long-range goal of this work is to understand the mechanism and regulation of intestinal bile acid transport as it relates to diet lipid metabolism in normal and disease states. The following questions will be addressed: (1) What are the general structure and subunit stoichiometry of the ASBT? What are the steps in the ASBT biogenesis? In contrast to its primary structure, the subunit stoichiometry and assembly of the function transporter complex are poorly understood. Preliminary studies suggest that intra-subunit and possibly inter subunit disulfide bonding are prerequisite steps for ASBT folding and transporter complex formation. In proposed studies, the assembly and subunit stoichiometry of the ASBT will be determined in transfect MDCK cells and normal rat cholangiocytes. The identity of associated subunits will be determined using combination of chemical cross-linking, coimmunoprecipitation, and a yeast two-hybrid screening strategy. (2) How is the plasma membrane expression of the ASBT regulated? ASBT expression is regulated by substrate, hormones such as glucocorticoids and secretin, and by cAMP. Whereas part of this regulation is transcriptionally, post-translational regulation of ASBT protein or plasma membrane activity appears to be an important component. In the proposed studies, the mechanism for ASBT exocytosis/endocytosis and degradation will be determined. (3) What is the role of the intestinal Oatp3 in bile acid absorption? Bile acids are absorbed by active sodium dependent transport and passive facilitative transport. A candidate transporter for the facilitative transporter Oatp3, has been isolated. The Oatp3's cellular expression and role in intestinal bile acid transport will be determined.