RECEPTOR MEDIATED ENDOCYTOSIS OF COBALAMIN (VITAMIN B12)

描述：本申请的总体目标是定义 受体介导的内吞和转运的分子和细胞基础 维生素B12/钴胺素（Cbl）。 膳食Cbl被回肠吸收 肠上皮细胞通过特异性内在因子-Cbl受体（IFCR）。 Cbl是 穿过这些细胞，进入循环并被转运到 外周组织与转钴胺素II（TCII）结合， 通过TCII受体（TCIR）摄取。 Cbl缺乏可能出现在 许多不同的位点，如TCII合成减少或有缺陷， 胃粘膜中的IFCR、TCII-R或IF或由于缺陷 细胞内分选 申请人实验室最近的研究旨在 表征Cbl摄取和细胞内 运输 初步数据表明，Cbl可以进入流通，而不是 不仅通过众所周知的IFCR途径，而且通过顶端衍生的TCIIR。 本提案的具体目标将在这些意见的基础上加以扩展， 其它初步数据显示分离了IFCR的推定cDNA克隆， TC IIR通过：1）研究细胞内Cbl分选途径， IF介导的顶侧至基底侧转胞吞; 2）表征 内化后Cbl和TCII的细胞内分选途径 从顶侧和基底侧域; 3）研究的途径和动力学， 将IFCR和TC II受体递送至其各自的质膜; 研究Cbl的发育、区域和激素调节 Cbl受体及其构效关系的初步研究 通过分离和表达编码IF-Cbl和TC-II的cDNA克隆， 受体。 人膀胱来源的Caco-2细胞系将用作 目标1-3和监管框架中提出的大多数研究的模式 目标4中提出的研究。 负鼠肾（OK）细胞， 恒定和相对高水平的放射性标记钴胺素-IFCR将被 用于确定排序机制是否类似于 肠和肾来源的细胞系。 大鼠肠道的体内研究 125 I标记的TC II-Cbl和差异标记的IF-Cbl的摄取， TCII-Cbl模型将被用于试图确认Cbl确实可以被 在通过GI转运期间，与TC II结合转运而不降解 小肠的远端。 选择Caco-2细胞是因为 尽管ICFR结合较低并且随传代变化很大， 钴胺素-TCII-R结合和抗体交叉反应性，124 kDa Westerns上的膜蛋白似乎比 而不是正常细胞。 为实现本提案的目标而计划的实验方法 从标准的生物化学应用，例如放射性标记的钴胺素 结合测定，蛋白质分离和纯化，抗体产生， 免疫沉淀，密度梯度分离，Western和北方 更复杂和技术要求更高的自由流动分析 电泳、受体克隆、体外转录和翻译 和爪蟾表达方案。 修订后的具体目标4现在也 包括用于鉴定配体结合区的体外诱变 和TC II-R的细胞质基底外侧靶向结构域。 的影响 将通过克隆检查TC II-R基底外侧靶向突变 导入表达载体pSFFV-neo，并转染Caco-2细胞。

DESCRIPTION: The overall objectives of this application are to define the molecular and cellular basis of receptor-mediated endocytosis and transport of vitamin B12/cobalamin (Cbl). Dietary Cbl is absorbed by ileal enterocytes via a specific intrinsic factor-Cbl receptor (IFCR). Cbl is trancytosed across these cells, enters the circulation and is transported to peripheral tissues bound to transcobalamin II (TCII) whereupon the complex is taken up via TCII receptors (TCIIR). Cbl deficiency may arise at a number of different sites such as reduced or defective synthesis of TCII, IFCR, TCII-R or IF in the gastric mucosa or as the result of defective intracellular sorting. Recent studies in the applicant's laboratory have been aimed at characterizing the intestinal mechanisms of Cbl uptake and intracellular transport. Preliminary data suggests that Cbl can enter the circulation not only via the well-known IFCR pathway but also via apically derived TCIIR. The specific aims of this proposal will expand on these observations and other preliminary data showing isolation of putative cDNA clones of IFCR and TC IIR by: 1) studying the intracellular pathways of Cbl sorting during IF-mediated apical to basolateral transcytosis; 2) characterizing the intracellular sorting pathways for Cbl and TCII following internalization from apical and basolateral domains; 3) studying the route and kinetics of delivery of IFCR and TC II-receptors to their respective plasma membranes; and 4) studying developmental, regional and hormonal regulation of Cbl receptors and initiating studies of structure-function relationships of Cbl receptors by isolating and expressing cDNA clones encoding IF-Cbl and TC-II receptors. The human intestinal-derived Caco-2 cell line will be used as a model for the majority of studies proposed in aims 1-3 and in regulatory studies proposed in aim 4. Opossum kidney (OK) cells, which appear to bind constant and relatively high levels of radiolabeled cobalamin-IFCR will be used to determine whether or not sorting mechanisms are similar in intestinal and kidney-derived cell lines. In vivo studies of rat intestinal uptake of 125I-labeled TC II-Cbl and of differentially labeled IF-Cbl and TCII-Cbl models will be used in an attempt to confirm that Cbl can indeed be transported bound to TC II without degradation during transit through the GI tract to the distal small intestine. Caco-2 cells were chosen because although ICFR binding is lower and does vary greatly with passage, cobalamin-TCII-R binding and antibody cross-reactivity with a 124 kDa membrane protein on Westerns appears to be approximately 10 times greater than in OK cells. Planned experimental approaches to address the aims of this proposal range from standard biochemical applications such as radiolabeled cobalamin binding assays, protein isolation and purification, antibody generation, immunoprecipitation, density gradient separations, Western and Northern analyses to more sophisticated and technically demanding free flow electrophoresis, receptor cloning, in vitro transcription and translation and Xenopus expression protocols. The revised specific aim 4 also now includes in vitro mutagenesis for identification of ligand binding regions and the cytoplasmic basolateral targeting domain(s) of TC II-R. Effects of mutations on basolateral targeting of TC II-R will be examined by cloning into the expression vector, pSFFV-neo, and transfection into Caco-2 cells.