Basis of Variability of Lung GPCR Signaling

描述（由申请人提供）：g蛋白偶联受体（gpcr）在整个肺中表达，介导稳态、适应性和致病性事件，是许多治疗剂的靶点。人气道平滑肌（HASM）表达数百种调节收缩、舒张、免疫反应和生长的gpcr，与哮喘和COPD直接相关。然而，在临床、离体和体外研究中，经常发现gpcr的功能、信号异质性和矛盾反应的个体间变异性。确定这种变异的分子基础一直是该资助的长期目标，对于理解疾病病理生物学和异质性、开发新药和药物基因组学至关重要。在过去的5年中，在参考人群中发现了单个HASM GPCR基因的多态性并进行了功能表征。这些研究确定了HASM信号变异性的一种机制，但也表明这种变异性不能完全用受体多态性来解释。发现了三种新的肺信号变异性机制：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受体组比以前认识到的要复杂得多。在Aim 1中，将确定HASM中表达的选择性剪接gpcr的结构和信号效应。我们最近发现，约50%的gpcr在HASM中表达多个受体“异构体”，这是由于mrna前剪接的选择性，这受到个体间差异的影响。这些同种异构体将在模型细胞系统中克隆、表达和表征，并在HASM中确认信号表型。在Aim 2中，将确定与阻塞性气道疾病相关的选定GPCR对形成异源二聚体并确定其功能。我们发现HASM gpcr可以形成异源二聚体，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检测和表征异源二聚体，以及研究转染细胞和HASM中与Gs、Gi和gq偶联受体相关的细胞内信号事件。在Aim 3中，将确定GRK5变异对与阻塞性气道疾病相关的选定gpcr的信号传导影响。GRKs调节大多数gpcr的激动剂促进功能。我们发现GRK5的多态性与WT GRK5相比，实质上改变了2AR功能，从而表明在个体受体之上的层次点上的变异性机制。选择的gpcr将在重组系统和HASM中进行研究，以确定GRK5在受体磷酸化、内化、脱敏和GRK/-阻滞蛋白信号传导方面的变异表型。总的来说，这些研究的结果将定义HASM gpcr的个体间变异性和异质性的机制，为阻塞性肺疾病的表型多样性和药物反应提供基础。公共卫生相关性：哮喘和慢性阻塞性肺病是主要的国家卫生问题。气道收缩的原因，以及某些药物的缓解，是由于气道肌肉受体的激活。但是，在收缩程度和缓解程度上，人与人之间存在很大差异，这一点尚不清楚。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气道收缩以及如何阻断或缓解它的能力。这笔拨款解决了这些差异的分子基础，以便我们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.