权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Basis of Variability of Lung GPCR Signaling

肺 GPCR 信号传导变异的基础

基本信息

批准号：
8516557
负责人：
Stephen B Liggett
金额：
$ 35.23万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8516557
关键词：
Address Agonist Alternative Splicing Amino Acid Substitution Arrestins Asthma Blood Vessels Cell model Cell physiology Cells Characteristics Chronic Obstructive Airway Disease Clinical Co-Immunoprecipitations Complex Detection Development Dinoprostone Disease Drug Targeting Electrolytes Energy Transfer Epithelial Event G Protein-Coupled Receptor Genes G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G protein-coupled receptor 50 G-Protein-Coupled Receptors GRK5 gene Genes Genetic Genetic Polymorphism Genetic Variation Genome Goals Grant Growth Health Heterogeneity Homeostasis Human Immune response In Vitro Individual Life Ligands Liquid substance Lung Mediating Membrane Methods Molecular Mucous body substance Muscle Muscle Tonus Muscle function Muscle relaxation phase Obstructive Lung Diseases Pathologic Persons Pharmaceutical Preparations Pharmacogenomics Phenotype Phosphorylation Physiological Population Process Property Prostaglandins Protein Isoforms Proteins RNA Splicing Receptor Activation Receptor Signaling Recombinants Relaxation Signal Transduction Smooth Muscle Myocytes Structure System Techniques Testing Therapeutic Therapeutic Agents Translating Variant base cell type constriction desensitization gene cloning genetic variant immune clearance insight mRNA Precursor novel receptor receptor coupling respiratory smooth muscle response trafficking

项目摘要

G-protein coupled receptors (GPCRs) are expressed throughout the lung, mediating homeostatic, adaptive, and pathogenic events, and are targets for many therapeutic agents. Human airway smooth muscle (HASM) express hundreds of GPCRs regulating contraction, relaxation, immune response, and growth, with direct relevance to asthma and COPD. However, substantial inter-individual variability in function, heterogeneity of signaling, and paradoxical responses of GPCRs are frequently found in clinical, ex vivo, and in vitro studies. Defining the molecular basis of this variability has been the broad long-term goal of this grant, and is critical for understanding disease pathobiology and heterogeneity, development of new drugs, and pharmacogenomics. During the past 5 years, polymorphisms of individual HASM GPCR genes were identified in a reference population and functionally characterized. These studies identified one mechanism of signaling variability in HASM, but also showed that the variability cannot be entirely explained by receptor polymorphisms. Three novel mechanisms of lung signaling variability were uncovered: alternative splicing, heterodimer formation, and genetic variation of G-protein coupled receptor kinases (GRKs). Thus the HASM receptorome is much more complex than previously recognized. In Aim 1, the structural and signaling effects of alternatively spliced GPCRs expressed in HASM will be determined. We have recently found that ~50% of GPCRs express multiple receptor "isoforms" in HASM due to alternative pre-mRNA splicing, which is subject to inter-individual variation. These isoforms will be cloned, expressed, and characterized in model-cell systems, and the signaling phenotypes confirmed in HASM. In Aim 2, heterodimer formation from selected GPCR pairs relevant to obstructive airway disease will be ascertained and their function determined. We find that HASM GPCRs can form heterodimers, often between very different receptors, that act as distinct signal-transduction units and alter airway responsiveness. Resonance energy transfer techniques will be utilized for detection and characterization of heterodimers, as well as studies of intracellular signaling events related to Gs, Gi, and Gq-coupled receptors in transfected cells and HASM. In Aim 3, the signaling impact of a GRK5 variant will be determined for selected GPCRs relevant to obstructive airway disease. GRKs regulate agonist-promoted function for most GPCRs. We have found a polymorphism of GRK5 that substantially alters 2AR function compared to WT GRK5, thus indicating a mechanism of variability at a hierarchic point above an individual receptor. Selected GPCRs will be studied in recombinant systems and HASM to ascertain the GRK5 variant phenotype in regards to receptor phosphorylation, internalization, desensitization, and GRK/-arrestin signaling. Collectively, results from these studies will define mechanisms of inter-individual variability and heterogeneity of HASM GPCRs, providing a basis for the diversity of phenotypes and drug-responses in obstructive lung disease.

G蛋白偶联受体（GPCR）在整个肺中表达，介导体内平衡，适应性，和致病事件，并且是许多治疗剂的靶点。人气道平滑肌（HASM）表达数百种GPCR，调节收缩、舒张、免疫应答和生长，与哮喘和COPD的关系。然而，功能的个体间变异性、在临床、离体和体外研究中经常发现GPCR的信号传导和矛盾反应。确定这种变异性的分子基础一直是这项资助的广泛的长期目标，对于理解疾病的病理生物学和异质性，新药的开发，药物基因组学在过去的5年中，个体HASM GPCR基因的多态性是在参考群体中鉴定并进行功能表征。这些研究确定了一种机制， HASM中的信号变异性，但也表明这种变异性不能完全由受体多态性发现了三种新的肺信号可变性机制：选择性剪接，异二聚体形成和G蛋白偶联受体激酶（GRKs）的遗传变异。因此，受体组比以前认识到的要复杂得多。在目标1中，将研究HASM中表达的选择性剪接GPCR的结构和信号作用。测定我们最近发现，由于HASM中约50%的GPCR表达多种受体“亚型”，前体mRNA的可变剪接，这是受个体间变异。这些同种型将被克隆，在模型细胞系统中表达和表征，并在HASM中确认信号表型。在Aim中 2，将从与阻塞性气道疾病相关的所选GPCR对形成异二聚体。确定其功能和作用。我们发现HASM GPCR可以形成异二聚体，通常在非常不同的受体之间，作为不同的信号转导单位并改变气道反应性。共振能量转移技术将用于异二聚体的检测和表征，以及转染细胞中与Gs、Gi和Gq偶联受体相关的细胞内信号事件的研究和HASM。在目标3中，将确定GRK 5变体对所选GPCR的信号传导影响与阻塞性气道疾病相关。GRKs调节大多数GPCR的激动剂促进的功能。我们有发现GRK 5的多态性与WT GRK 5相比显著改变了2AR功能，因此表明在个体受体之上的层次点上的可变性机制。选定的GPCR将在重组系统和HASM，以确定GRK 5变体表型，磷酸化、内化、脱敏和GRK/-抑制蛋白信号传导。总的来说，这些结果研究将定义HASM GPCR的个体间变异性和异质性的机制，阻塞性肺疾病表型和药物反应多样性的基础。