Airway Smooth Muscle Bitter Taste Receptors as Targets for Novel Bronchodilators

项目概述（见说明书）：支气管扩张剂用于哮喘治疗，用于支气管痉挛的急性缓解和长期控制。目前，只有一类直接支气管扩张剂被使用，即作用于气道平滑肌P2-肾上腺素能受体的p激动剂。由于许多哮喘患者没有得到充分的疾病控制，需要额外的治疗，包括通过新机制作用的直接支气管扩张剂。我们在人气道平滑肌（HASM）上发现了多种苦味受体（TAS2R）亚型。苦味配体激活TAS2Rs可在体内、体外及各种哮喘模型中引起气道平滑肌明显松弛。tas2r介导的支气管扩张与p激动剂一样有效，并且是通过一个完全不同的机制，涉及专门的Ca^ ^信号传导。有数千种潜在的TAS2R激动剂从植物中提取或合成用于其他目的，但在我们的发现之前，这些激动剂没有被考虑用于哮喘治疗。在本项目中，我们将研究HASM TAS2Rs的特性，并着眼于激动剂作为治疗哮喘的新型支气管扩张剂。在目标1中，我们将重点关注急性脱敏的可能性，这可能会限制治疗效果。我们将在HASM细胞、精确切割的人肺切片支气管和重组表达的模型细胞中检测激动剂促进的3个最丰富的HASM TAS2Rs的脱敏。g蛋白偶联、磷酸化、内化、B蛋白相互作用和偏配体性质将被建立。哮喘素质对这些特性的影响也将使用2个模型来确定，以便确定哮喘治疗的适用性。在Aim 2中，我们将利用位点定向诱变和重组表达，建立相关HASM TAS2RS的特定GRK磷酸化位点，从而确定信号猝灭的精确机制。当长期暴露于激动剂时，一些TAS2Rs似乎经历上调，这种表型与p2 -肾上腺素能受体相反，后者在慢性p激动剂治疗时下调。TAS2R上调将是一个非常有利的情况，对限制快速反应起作用。目的3将确定哪些HASM TAS2Rs表现出上调，这一过程的分子机制，以及偏向配体促进这种效果的基础。总的来说，这些研究将确定HASM TAS2Rs的分子和药理学特性，为未来开发一种新型支气管扩张剂治疗哮喘所必需。

PROJECT SUMMARY (See Instructions): Bronchodilators are used in the treatment of asthma for acute relief of bronchospasm and for long-term control. Currently, only one class of direct bronchodilators, p-agonists acting at airway smooth muscle P2- adrenergic receptors, are in use. With many asthmatics not achieving adequate disease control, there is a need for additional therapeutics, including direct bronchodilators acting by novel mechanisms. We have discovered multiple bitter taste receptor (TAS2R) subtypes on human airway smooth muscle (HASM). Activation of TAS2Rs by bitter ligands causes marked airway smooth muscle relaxation in vitro and in vivo, and in various models of asthma. TAS2R-mediated bronchodilation is as efficacious as p-agonists, and is via a completely different mechanism involving specialized Ca^"^ signaling. There are thousands of potential TAS2R agonists derived from plants or synthesized for other purposes, yet prior to our findings these were not considered for asthma therapy. In this Project, we will examine the properties of HASM TAS2Rs with an eye towards agonists being a new class of bronchodilators for treating asthma. In Aim 1, we will focus on the potential for acute desensitization, which can limit therapeutic efficacy. Agonist-promoted desensitization of the 3 most abundant HASM TAS2Rs will be examined in HASM cells, bronchi from precision-cut human lung slices and recombinantly expressing model cells. G-protein coupling, phosphorylation, internalization, B arrestin interactions, and biased ligand properties will be established. The influence ofthe asthma diathesis on these properties will also be determined using 2 models, so that applicability to asthma treatment can be ascertained. In Aim 2, we will establish the specific GRK phosphorylation sites for the relevant HASM TAS2RS, using site-directed mutagenesis and recombinant expression, so that a precise mechanism ofthe signal quenching is determined. Some TAS2Rs appear to undergo upregulation when exposed chronically to agonists, a phenotype that is opposite to P2-adrenergic receptors which downregulate upon chronic p-agonist treatment. TAS2R upregulation would be an extremely favorable profile, acting to limit tachyphylaxis. Aim 3 will determine which ofthe HASM TAS2Rs display upregulation, the molecular mechanism ofthe process, and the basis for biased ligands to promote the effect. Collectively, these studies will define the molecular and pharmacologic properties of HASM TAS2Rs required for future development of a novel class of bronchodilators for treating asthma.