Basis of Variability of Lung GPCR Signaling

描述(申请人提供)：G蛋白偶联受体(GPCRs)在整个肺中表达，介导稳态、适应性和致病事件，是许多治疗药物的靶标。人的呼吸道平滑肌(HASM)表达数百种GPCRs，调节收缩、松弛、免疫反应和生长，与哮喘和COPD直接相关。然而，在临床、体外和体外研究中，GPCRs的功能、信号的异质性和矛盾的反应经常被发现。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，对于了解疾病病理生物学和异质性、新药开发和药物基因组学至关重要。在过去的5年中，在一个参考人群中发现了单个HASM GPCR基因的多态性，并对其进行了功能鉴定。这些研究发现了HASM中信号可变性的一种机制，但也表明这种可变性不能完全用受体的多态来解释。发现了肺信号变异的三种新机制：选择性剪接、异源二聚体形成和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，将确定在HASM中表达的选择性剪接GPCRs的结构和信号效应。我们最近发现，大约50%的GPCRs在HASM中表达多种受体“异构体”，这是由于前mRNA的选择性剪接，这是受个体间差异的影响。这些异构体将在模型细胞系统中克隆、表达和鉴定，并在HASM中确认信号表型。在目标2中，将确定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并确定其功能。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，作为不同的信号转导单位，改变呼吸道反应性。共振能量转移技术将用于异源二聚体的检测和表征，以及与转基因细胞和HASM中Gs、GI和GQ偶联受体相关的细胞内信号事件的研究。在目标3中，将确定GRK5变体对选定的与阻塞性呼吸道疾病相关的GPCRs的信号影响。对于大多数GPCRs，GRKs调节激动剂促进的功能。我们发现与WT GRK5相比，GRK5的一个多态性显著改变了2AR的功能，从而表明在个体受体之上的一个层级点上存在一种可变性机制。选定的GPCRs将在重组系统和HASM中进行研究，以确定GRK5变异在受体磷酸化、内化、脱敏和GRK/-arrestin信号转导方面的表型。总之，这些研究的结果将确定HASM GPCRs的个体间变异性和异质性的机制，为阻塞性肺疾病的表型和药物反应的多样性提供基础。公共卫生相关性：哮喘和慢性阻塞性肺疾病是主要的国家健康问题。呼吸道收缩的原因，并通过某些药物缓解，是由于激活了呼吸道肌肉上的受体。但是，在紧缩的程度和它的缓解方面，每个人都有很大的不同，这一点还不被理解。这种人与人之间的差异限制了我们更多地了解这些疾病中的呼吸道狭窄以及如何阻断或缓解它的能力。这笔赠款解决了这些差异的分子基础，以便我们将更多地了解这些疾病以及如何治疗它们。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Asthma and chronic obstructive pulmonary disease represent major national health problems. The cause of the airway constriction, and its relief by certain medications, is due to activation of receptors on the airway muscle. But, there are large differences in the extent of constriction, and its relief, from person to person that is not understood. This between-person variability limits our ability to understand more about airway constriction in these diseases and how to block or relieve it. This grant addresses the molecular basis for these differences so that we will understand more about these diseases and how to treat them.