STRUCTURAL REQUIREMENTS FOR PROTEIN TRAFFICKING

蛋白质贩运的结构要求

• 批准号: 2146855
• 负责人: JAMES F. COLLAWN
• 金额: $ 9.08万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146855
• 关键词: Golgi apparatus; acid phosphatase; antigen presentation; binding proteins; biological signal transduction; cell growth regulation; chick embryo; circular dichroism; clathrin; cytoplasm; endocytosis; glycoproteins; histocompatibility antigens; immunofluorescence technique; intracellular transport; lysosomes; membrane transport proteins; mutant; protein transport; receptor mediated endocytosis; transferrin receptor

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.

本建议的总体目标是确定结构 细胞表面受体的细胞质尾内的特征， 对于内体途径内的蛋白质靶向至关重要并识别 介导这一过程的辅助蛋白。 转铁蛋白受体 (TRs)包含各种靶向信号将被用作工具， 研究蛋白质运输和绘制受体尾部 与网格蛋白包被的凹坑的衔接蛋白（AP）的相互作用。 了解靶向信号、辅助蛋白和一般蛋白质， 蛋白质运输的特征对 了解细胞生长的调节，各种疾病状态， 高胆固醇血症和靶向药物递送到细胞中。 的 具体目标是：1）表征TR胞质尾区 与质膜包被的小窝蛋白（AP-2复合物）的相互作用; 2)区分来自溶酶体膜的内化信号 靶向信号; 3a）确定细胞质尾序列是否 主要组织相容性复合体（MHC）不变链将靶向 一个混合不变链/TR直接从trans-Golgi网络到 内体区室，和B）定义介导该信号的信号 （4）确定人口贩运与人口贩运之间的相互作用 不变链/TR杂合体和trans-Golgi网络包被的凹坑蛋白 (AP-1复合物）。 为了实现这些目标，含有不同区域的突变人类TR 两种溶酶体膜蛋白质的胞质尾部，溶酶体- 相关膜糖蛋白（LAMP-1，11残基尾）或溶酶体 酸性磷酸酶（19个残基尾），或30个残基尾的各个区域 残基不变链尾将被剪接到跨膜上， TR的胞外结构域，并在细胞测定中进行测试以鉴定 溶酶体靶向或高尔基体到内体所必需的信号 分别瞄准。 突变的人TR将在鸡中表达 胚胎成纤维细胞使用逆转录病毒表达系统，并测试 细胞表面表达和内化活性。 内体与 将通过TR半衰期测量监测溶酶体运输 在脉冲追踪实验中， 和不存在氯喹或布雷菲德菌素A，以及形态学研究。 将使用CD对TR胞质尾区进行结构研究 谱 含有多个的野生型或突变型TR尾 内化信号或假定的高尔基体包被的凹坑信号将被 在细菌表达系统中表达并测试AP相互作用 用体外结合试验。 不变链/TR混合体将是 在今后的研究中使用，以了解贩运人口 该分子控制抗原的II类MHC呈递。