STRUCTURE/FUNCTION OF ENDOCYTIC, RECYCLING RECEPTORS

该项目的长期目标是了解分子 配体加工和受体再循环的机制 在胞吞过程中受到调节。虽然肝脏去唾液酸糖蛋白 受体（ASGPR）是第一个内吞，回收受体鉴定 已经被很好地研究过了，关于这一点的重要信息 其它内吞受体的功能仍然未知。特别是， 内化配体和受体分离的机制 彼此之间，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 分离可以决定内吞作用的效率。如果该键 步骤是低效的，配体将重新结合到受体上， 被非生产性地回收到细胞表面，而不是被路由到 到溶酶体。在1986年，我们假设， ASGPR和其他内吞受体通过两种不同的途径发生， 细胞纹孔通路这两种途径假说现在已经被 其他人也证实了这一点，他们分离出的细胞系只在 这两个预测的途径，状态2途径。 第二相关 假设是内化状态2 ASGPR经历短暂的 失活和再活化（I/R）循环，因为它们穿过它们的 细胞内循环途径。I/R周期将确保 许多内吞受体的运作，因为失活的受体 在分离过程中不能重新结合配体。这两种假设都适用于 内吞受体。这个实验室现在已经重建了 ASGPR I/R循环，并发现其分子机制 基础;脂肪酸共价修饰。配体结合 状态2 ASGPR的活性受脂肪酸可逆调节 Cys残基的酰化/脱酰化。脂肪酰化的作用 ASGPR结构和功能中的两个人类亚基（H1和H2）将 现在可以阐明，我们的假设进一步完善。具体 目的是：1）确定Cys残基的脂肪酰化状态 在人ASGPR的突变Hl和H2亚基中。2)表征 表达Cys-突变体HI的稳定转染细胞系和 H2亚基介导ASGPR内吞功能。3)表征 细胞质和跨膜结构域中的修饰基团 野生型H1和H2。4)为了分析序列决定因素， H1中Cys 36和Cys 57的脂肪酰化或脱酰化， 人ASGPR的亚基，和5）鉴定特异性决定簇 在H1或H2中，其引导ASGPR进入状态1或状态2内吞作用 途径。这些研究的结果将确定，在分子水平上， 水平，调节正常ASGPR回收的机制， 从而导致用于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.