STRUCTURE/FUNCTION OF ENDOCYTIC, RECYCLING RECEPTORS

这个项目的长期目标是了解分子 配体加工和受体循环的机制 在内吞作用过程中受到调节。尽管肝脏去唾液酸糖蛋白 受体(ASGPR)是第一个被鉴定的内吞、循环受体 并得到了很好的研究，这是关于这一点和 其他内吞受体的功能尚不清楚。特别是， 内化配体和受体分离的机制 彼此之间的距离是未知的。这在疾病中可能很重要。 ASGPR缺陷，如糖尿病和酗酒，因为 分离能决定内吞作用的效率。如果此密钥 STEP效率低下，配体将重新与受体结合并被 非生产性回收到细胞表面，而不是被路由 到溶酶体。1986年，我们假设内吞作用是由 ASGPR以及其他内源性受体通过两种不同的方式发生 细胞包被的坑道。这两条路径假说现在已经被 只在一种情况下分离缺陷细胞系的其他人的验证 这两条预测的途径是状态2途径。第二个相关 假说是内在化状态2 ASGPR经历了短暂的 停用和重新激活(I/R)循环 细胞内循环路线。I/R周期将确保高效 许多内胞受体的运作，因为失活的受体 在分离过程中不能重新结合配体。这两个假设都适用于 一般情况下，内吞受体。这个实验室现在已经重组了 通透性细胞内ASGPR I/R循环及其分子的发现 基础；通过脂肪酸进行共价修饰。配基结合 状态2 ASGPR活性受脂肪酸可逆调节 半胱氨酸残基的酰化/脱酰化。脂肪酰化作用的研究进展 ASGPR结构和功能中的两个人类亚基(HI和H2)将 现在被澄清了，我们的假设进一步完善了。具体的 目的：L)测定半胱氨酸残基的脂肪酰化状态 在突变的人ASGPR的HI和H2亚基中。2)描述 稳定表达Cys-突变体HI和Hi的细胞系的能力 H2亚基介导ASGPR内吞功能。3)刻画 胞质和跨膜区的修饰基团 通配型H1和H2。4)分析所需的序列行列式 Cys36和Cys57在H1中的脂肪酰化或去酰化 人ASGPR的亚单位，以及5)识别特定决定因素 在HI或H2中引导ASGPR进入L状态或状态2内吞作用 小路。这些研究的结果将在分子水平上定义 层面，规范正常的ASGPR回收的机制，应该 导致识别和治疗异常内吞的策略 人类疾病中的受体调节。

The long term goal of this project is to understand the molecular mechanisms by which ligand processing and receptor recycling are regulated during endocytosis. Although the hepatic asialoglycoprotein receptor (ASGPR) was the first endocytic, recycling receptor identified and has been well studied, important information about how this and other endocytic receptors function is still not known. In particular, the mechanism by which internalized ligand and receptor are segregated from each other is unknown. This is likely to be important in diseases with ASGPR defects, such as diabetes and alcoholism, because segregation can determine the efficiency of endocytosis. If this key step is inefficient, ligands will rebind to receptors and be nonproductively recycled to the cell surface, rather than being routed to lysosomes. In 1986 we hypothesized that endocytosis mediated by ASGPRs, and also other endocytic receptors occurs via two different cellular coated-pit pathways. This Two Pathway hypothesis has now been validated by others who isolated cell lines defective in only one of these two predicted pathways, the State 2 pathway. A second related hypothesis is that internalized State 2 ASGPRs undergo a transient inactivation and reactivation (I/R) cycle as they traverse their intracellular recycling route. I/R cycles would ensure the efficient operation of many endocytic receptors, since inactivated receptors could not rebind ligand during segregation. Both hypotheses apply to endocytic receptors in general. This laboratory has now reconstituted the ASGPR I/R cycle in permeable cells and discovered its molecular basis; covalent modification by fatty acids. The ligand-binding activity of State 2 ASGPRs is reversibly regulated by fatty acid acylation/deacylation of Cys residues. The role of fatty acylation of the two human subunits (HI & H2) in ASGPR structure and function will now be elucidated, and our hypotheses further refined. The specific aims are: l) To determine the fatty acylation status of Cys residues in mutant HI and H2 subunits of the human ASGPR. 2) To characterize the ability of stably transfected cell lines expressing Cys-mutant HI and H2 subunits to mediate ASGPR endocytic functions. 3) To characterize the modifying groups in the cytoplasmic and transmembrane domains of wildtype H1 and H2. 4) To analyze sequence determinants necessary for fatty acylation or deacylation of Cys36 and Cys57 in H1, the major subunit of the human ASGPR, and 5) To identify specific determinants in HI or H2 that direct ASGPRs into the State l or State 2 endocytosis pathways. Results from these studies will define, at the molecular level, the mechanisms that regulate normal ASGPR recycling and should lead to strategies for identifying and treating abnormal endocytic receptor regulation in human diseases.