STRUCTURAL REQUIREMENTS FOR PROTEIN TRAFFICKING

蛋白质贩运的结构要求

• 批准号: 3465006
• 负责人: JAMES F. COLLAWN
• 金额: $ 9.17万
• 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/3465006
• 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.

本提案的总体目标是定义结构