Molecular properties of B-adrenergic receptors in Asthma

 描述(申请人提供)：作用于人呼吸道平滑肌上的β2肾上腺素能受体(β2AR)的β激动剂松弛肌肉，从而扩张呼吸道，用于哮喘的急性(救援)和慢性(维持)治疗。由于许多哮喘患者没有得到控制，因此有必要了解在慢性治疗过程中，急性β-激动剂的疗效丧失以及耐受性或快速反应的发展。这些问题的部分原因是缺乏对β2AR信号的基本方面的了解。这项提案有三个目标来缩小这一差距，一个广泛的、长期的目标是开发治疗哮喘的最佳？？激动剂疗法，以减少发病率。在目标1中，我们将筛选4,000万个化合物文库，以发现稳定有利于哮喘的特定β2AR构象的？激动剂。这种“偏向”将倾向于Gs/cAMP偶联(改善支气管扩张)，而远离？arrestin募集(从而将耐受性降至最低)。这将通过一种连续的筛查方法来实现，该方法首先测量cAMP，然后是？-arrestin招募，然后是人类呼吸道的生理功能。来自这一筛选的化合物将成为哮喘临床前试验的候选药物。此外，它们的结构将教会我们工程激动剂发挥受体特定功能的分子基础。在目标2中，将确定β2AR与另一种促进支气管扩张的呼吸道受体-苦味受体(TAS2R)之间的相互作用。TAS2R激动剂正在开发中，用于治疗哮喘，预计TAS2R和β2AR激动剂将同时使用。然而，这两个受体似乎以异二聚体的形式在细胞表面缠绕在一起，其中一个受体的激活会改变另一个受体的功能。杂二聚体及其功能将用双分子荧光互补、免疫共沉淀、生物素化和共聚焦显微镜来研究。功能将通过测量每个单体成分(TAS2R的钙离子；β2AR的cAMP)的第二信使来确定。在目标3中，miRNA对β2AR基因的翻译抑制机制将决定β2AR蛋白在HASM上的静止水平是如何建立的。这一水平也是对？激动剂的急性反应的决定因素，而miRNAs本身似乎受到激动剂的调节。将通过在模型细胞和HASM中过表达、下调以及基因和蛋白质表达的测量，以let7、miR15和miR30 miRNA家族的选定成员来研究这些事件。总而言之，这些研究将确定新的治疗机制，这些新的治疗方法将在？-激动剂途径中用于改善哮喘的治疗。

 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.