CELLULAR CONTROL OF FOLATE UPTAKE--CAVEOLAE V COATED PIT

描述：这一建议的主要假设是叶酸受体 在胎盘和神经管上表达，叶酸摄取到 发育中的胎儿受到细胞增多的调节，这一过程被认为涉及 糖醇-磷脂酰肌醇(GPI)锚定蛋白和小窝。在……里面 此外，肾对肾小球滤液中的叶酸的重吸收是 依赖于相同的过程。本研究的具体目的是：(1) 开发一系列不同类型的自体荧光叶酸受体 锚定方案或靶向序列，以将它们定向到小窝和 有涂层的坑。这些嵌合受体将被用来精确地关联 途径特定的叶酸生物学，可能是无伪影的可视化 活细胞中的叶酸受体；以及(2)使用条件致死突变 有可诱导缺陷的细胞系在涂层小凹内吞作用中确定如何 特定分子完成叶酸的区隔和调节 小窝中的受体，而不是被包被的小窝。实验设计 采用了当导入CHO细胞时产生的 含有锚定结构域或靶向序列的嵌合蛋白 自身荧光报告蛋白和α-叶酸受体。此外 包被坑中缺陷的温度敏感突变细胞系 在不允许的温度下的内吞作用将用于研究嵌合体 蛋白质的分离和功能。这一高度创新的方法 叶酸受体生物学的研究可能提供重要的新的 受体定位、再循环和作用机制的信息。

DESCRIPTION: The major hypothesis of this proposal is that folate receptors are expressed on the placenta and neural tube and that folate uptake to the developing fetus is regulated by potocytosis, a process thought to involve glycosol-phosphatidylinositol (GPI)-anchored proteins and caveolae. In addition renal reabsorption of folate from the glomerular filtrate is dependent upon the same process. The specific aims of this study are: (1) to develop a series of autofluorescent folate receptors with differing anchoring schemes or targeting sequences to direct them to caveolae and coated pits. These chimeric receptors will be used to precisely correlate pathway-specific folate biology with what may be artifact-free visualization of folate receptors in live cells; and (2) to use conditional lethal mutant cell lines with inducible defects in coated pit endocytosis to determine how specific molecules accomplish compartmentation and regulation of folate receptors in caveolae rather than coated pits. The experimental design employs cDNA constructs that when transfected into CHO cells produce chimeric proteins containing anchoring domains or targeting sequences, an autofluorescent reporter protein and the alpha-folate receptor. In addition temperature-sensitive mutant cell lines that are defective in coated-pit endocytosis at non-permissive temperatures will be used to study chimeric protein segregation and function. This highly innovative approach to the study of folate receptor biology is likely to provide important new information on receptor localization, recycling and functional mechanism.