STRUCTURE AND FUNCTION OF ENDOCYTIC RECYCLING RECEPTORS

肝去唾液酸糖蛋白受体（ASGP-R）是第一个内吞， 回收受体进行鉴定和表征。 目前， 这种类型的至少9个受体，其功能是去除各种 循环配体 尽管这些受体已经被研究过， 广泛地说，关于它们如何运作的许多细节仍然是未知的。 为 例如，内吞的配体和受体被 彼此分离的情况尚未阐明。 我们的长期目标 是了解分离和其他步骤的分子基础 沿着ASGP和其它受体的受体再循环途径。 我们已经发现，内化的ASGP-R经历了一个 失活/再活化（I/R）循环，因为它穿过其细胞内 回收行程 我们的中心假设是， 失活是使分离步骤（从而使整个分离步骤） 内吞过程）如此有效。 由于解离的配体不能重新结合 对于失活的ASGP-R，它将被递送到溶酶体，而不是被 再循环到细胞表面。 我们现在成功地重建了 ASGP-R失活和再活化（I/R循环）在体外 渗透性细胞系统 ASGP-R失活仅需要外源性ATP， 而ASGP-R的再活化需要酰基辅酶A。 最后一个令人兴奋的发现 是这个项目的基础 我们的具体目标是：1。到 表征ASGP-Rs在可渗透性细胞中的失活和再活化， 肝细胞 动力学、对辅因子的需求和特异性 将检查脂肪酸链长度。 2.以确定是否，并在 ASGP-R的哪些位点、亚基被脂肪酸酰化。 3 h-或 14 C-棕榈酸盐将用于评估ASGP-R的共价修饰 在I/R周期中。 将使用HPLC、GLC、 蛋白水解酶的质谱和氨基酸序列分析 片段 3.为了确定受体I/R周期是否发生在其他细胞中， 内吞再循环受体系统。 8个类似受体 将使用各种方法评估系统是否发生I/R循环 细胞类型和合适的配体。 4.为了确定损失是否 ASGP-R活性与糖尿病或慢性酒精相关 在大鼠中的消耗是由于正常I/R周期的扰动。 如果我们证实了我们的假设，即ASGP-R I/R周期改变， 这些疾病，那么其他疾病可能与其他疾病有关。 循环受体系统也出现故障。 这些研究可能 揭示了一类新的病理学， 内吞受体以去除和降解它们各自的配体。

The hepatic asialoglycoprotein receptor (ASGP-R) was the first endocytic, recycling receptor to be identified and characterized. There are now at least 9 receptors of this type, whose function is to remove a variety of circulating ligands. although these receptors have been studied extensively, many details about how they function are still unknown. For example, the mechanism by which endocytosed ligand and receptor are segregated from each other has not been elucidated. Our long-term goal is to understand the molecular basis for segregation and other steps along the receptor recycling pathway for the ASGP and other receptors. We have discovered that the internalized ASGP-R undergoes an inactivation/reactivation (I/R) cycle as it traverses its intracellular recycling itinerary. Our central hypothesis is that transient ASGP-R inactivation is what makes the segregation step (and thereby the whole endocytic process) so efficient. Since dissociated ligand cannot rebind to inactive ASGP-R, it will be delivered to lysosomes rather than being recycled to the cell surface. We have now succeeded in reconstituting both ASGP-R inactivation and reactivation (the I/R cycle) in an in vitro permeable cell system. ASGP-R inactivation requires only exogenous ATP, while ASGP-R reactivation requires acyl-CoA. The latter exciting finding is the basis for much of this project. Our Specific Aims are: 1. To characterize the inactivation and reactivation of ASGP-Rs in permeable hepatocytes. The kinetics, requirement for cofactors and specificity for fatty acid chain length will be examined. 2. To determine if, and at what site(s), subunits of the ASGP-R are fatty acid acylated. 3H- or 14C-Palmitate will be used to assess covalent modification of the ASGP-R during the I/R cycle. Acylated sites will be defined using HPLC, GLC, mass spectrometry and amino acid sequence analyses of proteolytic fragments. 3. To determine if receptor I/R cycles occur in other endocytic, recycling receptor systems. Eight other similar receptor systems will be assessed for the occurrence of an I/R cycle using various cell types and the appropriate ligands. 4. To determine whether the loss of ASGP-R activity associated with diabetes or chronic alcohol consumption in rats is due to a perturbation of the normal I/R cycle. If we confirm our hypothesis that the ASGP-R I/R cycle is altered in these diseases, then other diseases may be associated with other malfunctioning recycling receptor systems as well. These studies may uncover a new class of pathologies related to the inability of different endocytic receptors to remove and degrade their respective ligands.