Molecular properties of B-adrenergic receptors in Asthma

 性状（由申请方提供）：作用于人气道平滑肌（HASM）上β2-肾上腺素能受体（β2AR）的β-激动剂可松弛肌肉，从而扩张气道，用于哮喘的急性（抢救）和慢性（维持）治疗。由于许多哮喘患者不能得到控制，因此需要了解在慢性治疗期间观察到的急性β受体激动剂的疗效丧失和耐受性或快速耐受的发展。这些问题部分是由于缺乏对HASM中β2AR信号传导的基本方面的理解。该提案有三个目标来缩小这一差距，其广泛的长期目标是开发哮喘的最佳β受体激动剂治疗以降低发病率。在目标1中，我们将筛选4000万个化合物库，以发现稳定有利于哮喘的特定β2AR构象的β-激动剂。这种“偏向”将朝向Gs/cAMP偶联（改善支气管扩张）并且远离β-抑制蛋白募集（因此使耐受性最小化）。这将通过连续筛选方法来实现，该方法测量cAMP，然后测量人气道中的β-arrestin募集，然后测量生理功能。筛选出的化合物将成为哮喘临床前试验的候选药物。此外，它们的结构将告诉我们工程激动剂从受体发挥特定功能的分子基础。目的2：研究β 2 AR与另一种支气管扩张受体苦味受体（TAS 2 R）之间的相互作用。TAS 2 R的激动剂正在开发中用于治疗哮喘，并且设想TAS 2 R和β2AR激动剂将伴随施用。然而，这两种受体似乎在细胞表面以异二聚体的形式交织在一起，其中一种受体的激活改变了另一种受体的功能。异源二聚体及其功能将使用双分子荧光互补，免疫共沉淀，生物素化，共聚焦显微镜进行研究。将通过测量每个单体组分的第二信使（TAS 2 R为Ca 2 +; β2AR为cAMP）来确定功能。在目标3中，miRNAs对β2AR基因的翻译抑制机制将定义β2AR蛋白的静息水平如何在HASM上建立。该水平也是对β-激动剂的急性反应的决定因素，并且miRNA本身似乎受到激动剂的调节。这些事件将通过模型细胞和HASM中的过表达、敲低以及基因和蛋白质表达测量，用let 7、miR 15和miR 30 miRNA家族的选定成员进行研究。总的来说，这些研究将确定在β-激动剂途径中指向新疗法的机制，以改善哮喘的治疗。

 DESCRIPTION (provided by applicant): β-agonists acting at the β2-adrenergic receptor (β2AR) on human airway smooth muscle (HASM) relax the muscle and thus dilate the airways, and are used for acute (rescue) and chronic (maintenance) therapy for asthma. With many asthmatics not achieving control, there is a need to understand the loss of efficacy observed with acute ß-agonists and the development of tolerance, or tachyphylaxis, during chronic therapy. These issues are due in part to a lack of understanding of fundamental aspects of β2AR signaling in HASM. This proposal has three aims to close this gap, with the broad, long-term objective of developing optimal ß-agonist treatment for asthma to reduce morbidity. In Aim 1, we will screen a 40 million compound library to discover ß-agonists that stabilize a specific β2AR conformation that is favorable for asthma. This "biasing" would be towards Gs/cAMP coupling (improves bronchodilation) and away from ß-arrestin recruitment (thus minimizing tolerance). This will be accomplished through a sequential screening approach that measures cAMP, then ß-arrestin recruitment, and then physiologic function in human airways. The compounds that come from this screening will be candidates for preclinical trials in asthma. Moreover, their structures will teach us the molecular basis for engineering agonists to exert specific functions from receptors. In Aim 2, the interactions between β2AR and another airway receptor that bronchodilates, the bitter taste receptor (TAS2R), will be ascertained. Agonists for TAS2Rs are in development for treating asthma, and it is envisioned that TAS2R and β2AR agonists will be administered concomitantly. Yet, the two receptors appear to be intertwined at the cell surface in heterodimers, where activation of one receptor alters function of the other. Heterodimers and their function will be studied using bimolecular fluorescent complementation, co-immunoprecipitation, biotinylation, and confocal microscopy. Function will be ascertained by measuring the second messengers from each monomeric component (Ca2+ for TAS2R; cAMP for β2AR). In Aim 3, the mechanism of translational repression of the β2AR gene by miRNAs will define how the resting level of β2AR protein is established on HASM. This level is also a determinant of the acute response to ß-agonist, and miRNAs themselves appear to be modulated by agonists. These events will be studied with selected members of the let7, miR15, and miR30 miRNA families by overexpression, knock-downs, and gene and protein expression measurements in model cells and HASM. Collectively, these studies will define mechanisms that point to new therapeutics within the ß-agonist pathway for improved therapy of asthma.