Basis of Variability of Lung GPCR Signaling

G蛋白偶联受体(GPCRs)在整个肺组织中都有表达，介导内环境平衡，适应性， 和致病事件，是许多治疗剂的靶标。人呼吸道平滑肌(HASM) 表达数百种调节收缩、放松、免疫反应和生长的GPCR，直接 与哮喘和COPD的相关性。然而，功能上的巨大个体间差异性， GPCRs的信号转导和矛盾反应常见于临床、体外和体外研究。 确定这种变异性的分子基础一直是这笔赠款的广泛长期目标，而且是 对于了解疾病的病理生物学和异质性、新药的开发以及 药物基因组学。在过去的5年中，单个HASM GPCR基因的多态性是 在参考种群中鉴定，并在功能上表征。这些研究确定了一种机制 HASM中信号的可变性，但也表明这种可变性不能完全用受体来解释 多态现象。发现了肺信号可变性的三种新机制：选择性剪接， 异二聚体的形成和G蛋白偶联受体激酶(GRKs)的遗传变异。因此，HASM 受体组比以前认识的要复杂得多。 在目标1中，在HASM中表达的选择性剪接GPCRs的结构和信号效应将是 下定决心。我们最近发现，大约50%的GPCRs在HASM中表达多种受体亚型 到另一种前mRNA剪接，这是受个体间差异的影响。这些异构体将被克隆， 在模型细胞系统中表达和鉴定，并在HASM中确认信号表型。在AIM 2、来自与阻塞性呼吸道疾病相关的选定GPCR对的异源二聚体的形成 确定并确定它们的功能。我们发现HASM GPCRs可以形成异二聚体，通常 在非常不同的受体之间，它们充当不同的信号转导单位，改变呼吸道的反应性。 共振能量转移技术将被用于检测和表征异二聚体，如 以及与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GRK5变体对选定的GPCR的信号影响 与阻塞性呼吸道疾病有关。对于大多数GPCRs，GRKs调节激动剂促进的功能。我们有 发现与WT GRK5相比，GRK5的多态显著改变了2AR的功能，因此表明 个体感受器上方某一等级点上的可变性机制。选定的GPCR将在#年进行研究 重组系统和HASM用于确定GRK5受体变异表型 磷酸化、内化、脱敏和GRK/-arrestin信号转导。总体而言，这些结果 研究将定义HASM GPCRs个体间变异性和异质性的机制，提供 阻塞性肺病的表型多样性和药物反应的基础。

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.