STRUCTURAL REQUIREMENTS FOR PROTEIN TRAFFICKING

这项提案的总体目标是定义结构 细胞表面受体的细胞质尾部的特征是 对于内体途径中的蛋白质靶向和鉴定是必不可少的 辅助蛋白参与这一过程的辅助蛋白。转铁蛋白受体 包含各种目标信号的(TRS)将被用作 研究蛋白质转运和定位受体尾部 与网状蛋白包被坑的接合蛋白(AP)的相互作用。 靶向信号、辅助蛋白和一般的知识 蛋白质运输的特征对 了解细胞生长的调节，各种疾病状态，如 高胆固醇血症，以及靶向药物进入细胞。这个 具体目标是：1)描述tr细胞质尾部 与质膜包裹的PIT蛋白(AP-2复合体)的相互作用； 2)区分溶酶体膜的内化信号 靶向信号；3a)以确定胞质尾部序列是否 主要组织相容性复合体(MHC)不变链将以 一个混合不变链/TR直接从跨高尔基网络到 内体间隔，以及b)定义介导这一过程的信号 以及4)描述人口贩运之间的互动 不变链/tR杂交体与反式高尔基网络包被的PIT蛋白 (AP-1复合体)。 为了实现这些目标，突变的人类RR包含不同的区域 两种溶酶体膜蛋白的胞质尾部，溶酶体- 结合膜糖蛋白(LAMP-1、11残基尾巴)或溶酶体 酸性磷酸酶(LAP，19残基尾部)，或30个不同区域 残基不变链尾将被拼接到跨膜上并 并在细胞分析中检测以确定 溶酶体靶向或高尔基体到内体所需的信号 分别瞄准目标。突变的人类转录因子受体将在鸡中表达 使用逆转录病毒表达系统的胚胎成纤维细胞 细胞表面表达和内化活性。内吞体VS 溶酶体的贩运将通过TR半衰期测量进行监测 在脉冲追逐实验中，受体的分布分析 以及不含氯喹或布雷菲尔丁A，并进行了形态研究。 将使用CD对TR细胞质尾部的结构进行研究 光谱学。野生型或突变型tr尾巴包含 内化信号或假定的高尔基涂层凹坑信号将是 在细菌表达系统中表达并测试AP相互作用 采用体外结合试验。不变链/tr杂交体将是 用于未来的研究，旨在了解贩运人口是如何 该分子控制第二类MHC抗原的提呈。

The overall objective of this proposal is to define the structural features within the cytoplasmic tails of cell surface receptors that are essential for protein targeting within the endosomal pathway and identify the accessory proteins that mediate this process. Transferrin receptors (TRs) containing various targeting signals will be employed as a tool for studying both protein trafficking and mapping the receptor tail interactions with the adaptor proteins (APs) of clathrin-coated pits. A knowledge of targeting signals, the accessory proteins, and the general features of protein trafficking has important implications in understanding regulation of cell growth, various disease states such as hypercholesterolemia, and targeted drug delivery into cells. The specific aims are: 1) to characterize the TR cytoplasmic tail interactions with the plasma membrane coated pit proteins (AP-2 complex); 2) to differentiate internalization signals from lysosomal membrane targeting signals; 3a) to determine if the cytoplasmic tail sequence of the major histocompatibility complex (MHC) invariant chain will target a hybrid invariant chain/TR directly from the trans-Golgi network to the endosomal compartment, and b) to define the signal that mediates this trafficking; and 4) to characterize the interactions between the invariant chain/TR hybrid and the trans-Golgi network coated pit proteins (AP-1 complex). To accomplish these goals, mutant human TRs containing various regions of the cytoplasmic tails of two lysosomal membrane proteins, lysosome- associated membrane glycoprotein (LAMP-1, 11 residue tail) or lysosomal acid phosphatase (LAP, 19 residue tail), or various regions of the 30 residue invariant chain tail will be spliced to the transmembrane and extracellular domains of the TR and tested in cellular assays to identify the signals necessary for lysosomal targeting or Golgi to endosomal targeting, respectively. Mutant human TRs will be expressed in chicken embryo fibroblasts using a retroviral expression system and tested for cell surface expression and internalization activity. Endosomal versus lysosomal trafficking will be monitored with TR half-life measurements in pulse-chase experiments, receptor distribution assays in the presence and absence of chloroquine or brefeldin A, and morphological studies. Structural studies on the TR cytoplasmic tail will be performed using CD spectroscopy. Wild-type or mutant TR tails containing multiple internalization signals or putative Golgi coated pit signals will be expressed in a bacterial expression system and tested for AP interactions with in vitro binding assays. The invariant chain/TR hybrids will be used in future studies directed at understanding how the trafficking of this molecule controls class II MHC presentation of antigen.