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TSH RECEPTOR MULTIMERIZATION

TSH 受体多聚化

基本信息

批准号：
7931718
负责人：
TERRY Francis DAVIES
金额：
--
依托单位：
JAMES J PETERS VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7931718
关键词：
1,2-diacylglycerol 1-Phosphatidylinositol 3-Kinase AKT Signaling Pathway ARRB2 Acetates Actins Adenylate Cyclase Affect Affinity Age Agonist Alanine Alexa594 Antibodies Antibody Activation Antigen-Antibody Complex Arrestin Beta 1 Arrestins Aspirate substance Attention Autoantibodies Autoantigens Autoimmune Diseases Autoimmune Process Bathing Behavior Binding Binding Proteins Biochemical Bioinformatics Biological Assay Biology Buffers Calibration Canis familiaris Carbon Dioxide Caring Cattle Caveolae Cell Culture Techniques Cell Line Cell Survival Cell membrane Cell physiology Cell surface Cells Centrifugation Characteristics Chills Chinese Hamster Ovary Cell Chloroform Cholera Toxin Protomer B Cholesterol Clathrin Cleaved cell Cluster Analysis Co-Immunoprecipitations Color Complementary DNA Complex Computer software Core Facility Coupled Coupling Cybernetics Cyclic AMP Cysteine Cytochalasin D Cytolysis Data Databases Defect Degradation Pathway Deposition Detection Detergents Diffusion Digitonin Diglycerides Dimerization Discrimination Disease Dissociation Dithiothreitol Docking Down-Regulation Dryness Dyes Endocytosis Energy Transfer Environment Epithelial Cells Epitopes Equus caballus Ethers Exposure to Fab Immunoglobulins Face Felis catus Fluorescein Fluorescein-5-isothiocyanate Fluorescence Fluorescence Resonance Energy Transfer Fostering Fractionation G Protein-Coupled Receptor Genes GTP-Binding Proteins Ganglioside GM1 Gangliosides Gel Glass Glycols Glycoproteins Glycosphingolipids Grant Gray unit of radiation dose Growth Heating Hemorrhage High Pressure Liquid Chromatography Hormones Horseradish Peroxidase Housing Human Hydrocortisone Hyperthyroidism Ice Image Image Analysis Imagery Immune Immunoassay Immunoblotting Immunoglobulin G Immunoprecipitation In Vitro Incubated Individual Initiator Codon Inositol Inositol Phosphates Instruction Insulin Insulin-Dependent Diabetes Mellitus International Interphase Isoleucine Isopropanol Isothiocyanates Knock-out LH Receptors Label Laboratories Lateral Lead Learning Legal patent Length Life Ligands Link Lipids Lysine Lysosomes Magnesium Chloride Manufacturer Name Measurable Measurement Measures Mediating Membrane Membrane Microdomains Methanol Methods Microscope Microscopy Milk Mission Modeling Molecular Molecular Models Molecular Weight Monoclonal Antibodies Movement Multiple Sclerosis Mus Mutate Mutation Mutation Analysis Nature Needles Nuclear Oils Oligonucleotides Organic solvent product Oryctolagus cuniculus PDPK1 gene Pain Pathway interactions Patients Penicillins Phenanthrolines Phenylalanine Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphorylation Phosphotransferases Physiological Physiology Pigments Play Plug-in Polyvinyls Population Positioning Attribute Preparation Prevalence Problem Solving Procedures Process Proline Protease Inhibitor Proteins Proto-Oncogene Proteins c-akt Protocols documentation Publications Publishing RNA Rattus Reading Reagent Receptor Signaling Recycling Regulation Relative (related person)Reporting Research Resistance Resort Rheumatoid Arthritis Role Running Saline Sampling Scaffolding Protein Secondary to Sepharose Series Serine Serum Shapes Shoes Signal Pathway Signal Transduction Signaling Molecule Site Slide Small Interfering RNA Solubility Somatostatin Specificity Speed Sphingolipids Spottings Staging Staining and Labeling Staining method Stains Streptavidin Streptomycin Stroke Structural Models Structure Structure-Activity Relationship Subfamily lentivirinae Sucrose Surface System T-Lymphocyte Tablets Tail Techniques Temperature Testing Threonine Thyroid Diseases Thyroid Gland Thyrotropin Receptor Time Tissues Transfection Transferrin Translating Transmembrane Domain Tromethamine Tryptophan Tube Tweens Universities Valine Vesicle Virginia Water Western Blotting Woman Work antibody conjugate autoimmune thyroid disease base beta-arrestin cell growth cell motility cell type cellular imaging charge coupled device camera chemical reaction coated pit crosslink dalton dichlorodifluoromethane dimer disulfide bond experience fetal bovine serum flotillin fluorescence microscope fluorophore gene therapy human disease improved in vivo instrument interest knock-down men milliliter molecular modeling monomer mutant novel phospholipase C beta prototype ratiometric receptor receptor binding receptor expression receptor function receptor internalization reconstitution reconstruction research study thyroid associated ophthalmopathies time use trafficking

项目摘要

DESCRIPTION (provided by applicant): The thyrotropin receptor (TSHR), expressed on the plasma membrane of thyroid epithelial cells as well as a variety of extra-thyroidal sites, is central to the regulation of thyroid growth and function. The TSHR is also the major autoantigen in the autoimmune hyperthyroidism known as Graves' disease where T cells and autoantibodies are directed at the TSHR. Our laboratory first demonstrated the existence of multimeric TSHR forms in native thyroid tissue and more recently we modeled such forms in transfected cells by using functionally tagged TSHRs and fluorescence resonance energy transfer (FRET). This competitive renewal application continues these studies with four specific aims: Specific AIM 1 - To determine the transmembrane contact sites of TSHR multimers. In previous studies we have determined that Y110 in the TSHR ectodomain is an important contact site for ectodomain dimerization and are currently pursuing complex mutational approaches, based on detailed molecular modeling, to determine the sites within the transmembrane domain which are responsible for the major multimeric forms of the full length TSHR. Specific AIM 2 - To determine the role of TSH ligand in TSH receptor multimerization.. Our preliminary data have suggested that TSH reduces TSHR multimers in FRET and Co-IP experiments but the mechanism for this effect is unclear. We will, therefore, further evaluate the influence of TSH induced regulation of the TSHR using bioinformatic and biochemical approaches. Specific AIM 3 - To illustrate that TSHRs reside within lipid rafts, are TSH ligand regulated, and are primary centers of signal initiation. Our data indicate that TSHR forms can be found in lipid raft compartments. Since lipid rafts are centers for signal transduction, we hypothesize that TSHRs within lipid rafts are major signal initiators and will examine this concept in detail. Specific AIM 4 - To test the hypothesis that TSHRs in lipid rafts are resistant to internalization. Internalization leads to the termination of signal transduction and would normally be expected to control excessive hyperstimulation as seen in Graves' disease. Internalization of TSHRs is regulated by the binding of ¿-arrestin to phosphorylated receptors. Lipid rafts are centers of arrestin binding and in other receptor systems have been found to reduce the speed of receptor internalization. This may prolong receptor signaling which can have profound consequences in thyroid disease. We will therefore perform a detailed analysis including time-lapse imaging, to determine the role of arrestin and the signaling consequences. Significance Of the study: The overall aim of this grant is to understand the structure-function relationship of TSH receptor multimers and their role within and without lipid rafts. Understanding the biology of TSHRs may lead to better strategies to control excessive TSHR activation seen in human disease. Relevance: Thyroid disease is common amongst our VA patients and affects some 10% of the population with increasing prevalence with age. Autoimmune thyroid disease, while most common in women, often affects men to a worse degree. This is particularly true for Graves' eye disease. Hence, studies to investigate the cause of autoimmune thyroid disease are highly relevant to the mission of the VA and in addition serve as a model for all other autoimmune diseases including rheumatoid arthritis, multiple sclerosis and Type 1 Diabetes.

描述（由申请人提供）：促甲状腺激素受体（TSHR）表达于甲状腺上皮细胞的质膜以及各种甲状腺外部位，是调节甲状腺生长和功能的中心。TSHR也是自身免疫性甲状腺功能亢进症（称为Graves病）中的主要自身抗原，其中T细胞和自身抗体针对TSHR。我们的实验室首先证明了天然甲状腺组织中存在多聚体TSHR形式，最近我们通过使用功能标记的TSHR和荧光共振能量转移（FRET）在转染细胞中模拟了这种形式。这一竞争性续期申请继续这些研究，有四个具体目标：特异性AIM 1 -确定TSHR多聚体的跨膜接触位点。在先前的研究中，我们已经确定TSHR胞外域中的Y110是胞外域二聚化的重要接触位点，并且目前正在基于详细的分子建模来追求复杂的突变方法，以确定跨膜结构域内负责全长TSHR的主要多聚体形式的位点。特异性目的2 -确定TSH配体在TSH受体多聚化中的作用。我们的初步数据表明，TSH减少TSHR多聚体在FRET和Co-IP实验，但这种影响的机制尚不清楚。因此，我们将使用生物信息学和生物化学方法进一步评估TSH诱导的TSHR调节的影响。特异性目的3 -说明TSHR位于脂筏内，受TSH配体调节，是信号起始的主要中心我们的数据表明，TSHR形式可以发现在脂筏车厢。由于脂筏中心的信号转导，我们假设脂筏内的TSHR是主要的信号启动器，并将详细研究这一概念。特定目的4 -检验脂筏中TSHR对内化具有抵抗性的假设。内化导致信号转导的终止，并且通常预期控制如在格雷夫斯病中所见的过度过度刺激。TSHR的内化受抑制蛋白与磷酸化受体的结合调节。脂筏是arrestin结合的中心，并且在其他受体系统中已发现其降低受体内化的速度。这可能会延长受体信号传导，这可能对甲状腺疾病产生深远的影响。因此，我们将进行详细的分析，包括延时成像，以确定arrestin的作用和信号的后果。研究的意义：这项研究的总体目标是了解结构-功能 TSH受体多聚体的关系及其在脂筏内外的作用。了解TSHR的生物学可能会导致更好的策略来控制人类疾病中出现的过度TSHR激活。相关性：甲状腺疾病在我们的VA患者中很常见，影响约10%的人群，患病率随着年龄的增长而增加。自身免疫性甲状腺疾病虽然在女性中最常见，但对男性的影响往往更严重。这对于格雷夫斯眼病尤其如此。因此，研究调查自身免疫性甲状腺疾病的原因与VA的使命高度相关，此外，作为所有其他自身免疫性疾病，包括类风湿性关节炎，多发性硬化症和1型糖尿病的模型。