STRUCTURE/FUNCTION OF ENDOCYTIC, RECYCLING RECEPTORS

内吞、再循环受体的结构/功能

基本信息

批准号：
6179760
负责人：
PAUL H WEIGEL
金额：
$ 23.99万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 2002-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6179760
关键词：
3T3 cells carbohydrate receptor cysteine fatty acylation glycoproteins high performance liquid chromatography laboratory rat liver cells membrane proteins mutant protein sequence protein structure function receptor mediated endocytosis site directed mutagenesis transfection

项目摘要

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.

该项目的长期目标是了解分子配体加工和受体回收的机制内吞作用期间受到调节。虽然肝脏脱唾液酸糖蛋白受体（ASGPR）是第一个被识别的内吞、再循环受体并已得到充分研究，关于如何做到这一点的重要信息其他内吞受体的功能尚不清楚。尤其，内化配体和受体分离的机制彼此之间是未知的。这对于疾病可能很重要患有 ASGPR 缺陷，例如糖尿病和酗酒，因为分离可以决定内吞作用的效率。如果这个键步骤效率低下，配体将重新与受体结合并被非生产性地回收到细胞表面，而不是被路由到溶酶体。 1986年，我们假设内吞作用是由 ASGPR 以及其他内吞受体通过两种不同的方式发生细胞涂层凹坑通路。这两条路径假说现已被经其他人验证，他们分离出仅存在一种缺陷的细胞系这两条预测的路径，即状态 2 路径。第二个相关假设是内化状态 2 ASGPR 经历了短暂的当它们穿过它们的过程时，失活和再激活（I/R）循环细胞内循环途径。 I/R 周期将确保高效许多内吞受体的运作，因为失活的受体在分离过程中无法重新结合配体。两个假设都适用于一般内吞受体。该实验室现已重建通透细胞中的 ASGPR I/R 循环并发现了其分子基础;脂肪酸的共价修饰。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（HI 和 H2）将现在得到了阐明，我们的假设也得到了进一步完善。具体的目标是： l) 确定 Cys 残基的脂肪酰化状态存在于人类 ASGPR 的突变 HI 和 H2 亚基中。 2）表征表达 Cys 突变体 HI 的稳定转染细胞系的能力 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) 分析必要的序列决定因素 H1 中 Cys36 和 Cys57 的脂肪酰化或脱酰化，是主要的人类 ASGPR 的亚基，以及 5) 确定特定的决定因素在 HI 或 H2 中，引导 ASGPR 进入状态 l 或状态 2 内吞作用途径。这些研究的结果将在分子水平上定义水平，调节正常 ASGPR 回收的机制，并且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。