CELLULAR CONTROL OF FOLATE UPTAKE--CAVEOLAE V COATED PIT

描述：该提案的主要假设是叶酸受体 在胎盘和神经管上表达， 发育中的胎儿受光细胞增多的调节，这一过程被认为涉及 糖溶胶-磷脂酰肌醇（GPI）锚定蛋白和小窝。 在 此外，肾脏对肾小球滤液中叶酸的重吸收， 取决于同一个过程。 本研究的具体目的是：（1） 开发一系列具有不同荧光的叶酸受体， 锚定方案或靶向序列以将它们引导至小窝， 有涂层的凹坑 这些嵌合受体将用于精确地关联 途径特异性叶酸生物学与可能是无伪影的可视化 活细胞中的叶酸受体;和（2）使用条件致死突变体 细胞系与可诱导缺陷的包被坑内吞，以确定如何 特异性分子完成叶酸的区室化和调节 感受器在小窝而不是被纹孔中。 实验设计 使用cDNA构建体，当转染到CHO细胞中时， 含有锚定结构域或靶向序列的嵌合蛋白， 自体荧光报告蛋白和α-叶酸受体。 此外 在包被纹孔中有缺陷的温度敏感性突变细胞系 在非允许温度下的内吞作用将用于研究嵌合的 蛋白质分离和功能。 这种高度创新的方法， 叶酸受体生物学研究可能提供重要的新信息， 受体定位、再循环和功能机制的信息。

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.