Structural Analysis of the Mannose 6-Phosphate Receptors

甘露糖 6-磷酸受体的结构分析

• 批准号: 8661159
• 负责人: Nancy M. Dahms
• 金额: $ 45.57万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661159
• 关键词: Acids; Adaptor Signaling Protein; Affect; Affinity; Age; Angiostatins; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Biological Process; Birth; Carbohydrates; Cardiovascular system; Cations; Cell surface; Cells; Cellular Assay; Cessation of life; Chickenpox; Complement; Complex; Crystallization; Cytoplasmic Tail; Disease; Dissociation; Down-Regulation; Endocytosis; Endoplasmic Reticulum; Environment; Enzymes; Eukaryota; Extracellular Domain; Failure; Goals; Growth; Growth Factor; Herpes Simplex Infections; Herpes zoster disease; Human; Hydrolase; I-Cell Disease; IGF Type 2 Receptor; Individual; Insulin-Like Growth Factor II; Kringles; Laboratories; Length; Ligand Binding; Ligands; Lysosomes; Mammalian Cell; Mannose; Manuscripts; Mediating; Membrane; Modification; Molecular; Molecular Conformation; Mus; Mutation; N-terminal; Nutrient; Organism; Pathway interactions; Physiological Processes; Plasminogen; Play; Polysaccharides; Process; Property; Proteins; Receptor Gene; Regulation; Role; Signal Pathway; Site; Solutions; Spin Labels; Structure; Surface Plasmon Resonance; Techniques; Testing; Therapeutic; Transforming Growth Factors; Tretinoin; Tumor Suppressor Proteins; Urokinase Plasminogen Activator Receptor; Viral; Virus; Virus Diseases; antigen processing; base; cell growth; cell motility; design; enzyme replacement therapy; extracellular; human IGF2R protein; human disease; improved; inhibitor/antagonist; man; mannose 6 phosphate; novel; phosphodiester; plasminogen kringle 5; protein transport; receptor; receptor binding; three dimensional structure; trafficking; treatment strategy

DESCRIPTION (provided by applicant): The 300kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46kDa cation- dependent MPR (CD-MPR) play a key role in lysosome biogenesis by delivering ~60 different newly synthesized acid hydrolases to the lysosome by binding to mannose 6-phosphate (M6P) residues on their N-glycans. Disruption of this essential targeting pathway results in the most severe of the human lysosomal storage disorders, mucolipidosis II. In addition to lysosomal enzymes, the repertoire of identified extracellular ligands of the CI-MPR includes a diverse spectrum of M6P-containing proteins, such as growth factors (e.g., transforming growth factor-2) and pathogenic viruses (e.g., herpes simplex, varicella-zoster), and their interaction with the MPRs can result in the growth factor's activation or degradation, and facilitate viral entry into mammalian cells. The multifunctional CI-MPR also binds the non-M6P-containing ligands plasminogen, urokinase-type plasminogen activator receptor (uPAR), insulin-like growth factor-II (IGF-II), and retinoic acid. Together, these unique binding properties of the CI-MPR mediate its ability to regulate cell growth and motility, and to function as a tumor suppressor. However, the molecular basis governing the intracellular trafficking and ligand binding properties of these receptors has not been fully defined. Recent studies from our laboratory have provided the first, and to date the only, structural views of the CD-MPR's extracellular domain, and two out of the three carbohydrate binding sites of the CI-MPR. In the current proposal, we will use crystallographic and NMR approaches to determine the structure of the MPRs' carbohydrate (Aims 1 & 3) and plasminogen (Aim 2) binding sites under conditions which are physiologically relevant, including acidic conditions which are key for the ability of the receptors to release their cargo to undergo multiple rounds of lysosomal enzyme delivery. Phosphodiester-containing proteins will be identified using domain 5 of the CI-MPR as a novel affinity probe (Aim 1). Crystallographic and solution structures of the MPRs' cytoplasmic domain in the absence and presence of adaptor proteins will be determined (Aim 4). These studies will be complemented by EPR spectroscopic analyses of MPRs containing site-specific spin labels (Aim 4). The long term goal is to understand the molecular mechanisms by which these essential receptors carry out their diverse biological functions. These studies will also provide the structural basis for the design of improved therapeutics for the treatment of lysosomal storage disorders, and novel inhibitors of viral infection and growth factor activation.

描述（由申请人提供）：300kDa 阳离子非依赖性甘露糖 6-磷酸受体 (CI-MPR) 和 46kDa 阳离子依赖性 MPR (CD-MPR) 通过传递约 60 种不同的新合成的酸性水解酶，在溶酶体生物发生中发挥关键作用通过与 N-聚糖上的 6-磷酸甘露糖 (M6P) 残基结合而进入溶酶体。这一重要靶向途径的破坏会导致最严重的人类溶酶体贮积症，即粘脂沉积症 II。除了溶酶体酶外，CI-MPR 已鉴定的胞外配体库还包括多种含 M6P 的蛋白质，例如生长因子（例如转化生长因子-2）和致病病毒（例如单纯疱疹、水痘） -带状疱疹），它们与 MPR 的相互作用可以导致生长因子的激活或降解，并促进病毒进入哺乳动物细胞。多功能 CI-MPR 还结合不含 M6P 的配体纤溶酶原、尿激酶型纤溶酶原激活剂受体 (uPAR)、胰岛素样生长因子-II (IGF-II) 和视黄酸。总之，CI-MPR 的这些独特的结合特性介导了其调节细胞生长和运动以及充当肿瘤抑制因子的能力。然而，控制这些受体的细胞内运输和配体结合特性的分子基础尚未完全确定。我们实验室最近的研究提供了第一个也是迄今为止唯一的 CD-MPR 胞外结构域的结构视图，以及 CI-MPR 三个碳水化合物结合位点中的两个。在当前的提案中，我们将使用晶体学和核磁共振方法来确定 MPR 的碳水化合物（目标 1 和 3）和纤溶酶原（目标 2）结合位点的结构，这些条件是在生理相关的条件下进行的，包括酸性条件，这是关键的酸性条件。受体释放其货物以进行多轮溶酶体酶递送的能力。将使用 CI-MPR 的域 5 作为新型亲和探针来鉴定含磷酸二酯的蛋白质（目标 1）。将确定在不存在和存在衔接蛋白的情况下 MPR 胞质结构域的晶体结构和溶液结构（目标 4）。这些研究将得到对含有位点特异性自旋标签的 MPR 的 EPR 光谱分析的补充（目标 4）。长期目标是了解这些重要受体发挥其不同生物学功能的分子机制。这些研究还将为设计用于治疗溶酶体贮积症的改进疗法以及病毒感染和生长因子激活的新型抑制剂提供结构基础。