Project 2 - Airway Smooth Muscle Bitter Taste Receptors as Targets for Novel Bronchodilators

项目摘要 苦味受体（TAS 2 R）在人气道平滑肌（HASM）上表达，并且当激活时， 显著放松肌肉和扩张气道。利用这一途径，这是不同于β- 作用于β2-肾上腺素能受体（β 2AR）的激动剂，将为治疗哮喘提供一类新的直接支气管扩张剂。 或预防哮喘中的支气管痉挛。TAS 2 R14亚型在HASM中高度表达，并且是HASM的主要亚型。 用于开发新型治疗剂的靶点。然而，我们对这一问题的认识还存在空白。 HASM TAS 2 R的分子/细胞生物学和生理学，包括它们如何与放松耦合， 由于短期（受体磷酸化）和长期（下调 受体表达）事件，以及将受体信号传导偏向哮喘有利信号传导的可能性 治疗该项目的广泛的长期目标是提高我们对HASM TAS 2 R的理解 与治疗哮喘气道收缩相关的生物学。为了填补这些知识空白，在目标1中，我们将 定义了TAS 2 Rs引起松弛的机制，我们假设这是通过抑制 肌动蛋白切割蛋白cofilin的磷酸化。研究将在培养的HASM细胞中进行， 从非哮喘以及哮喘供体肺，后者是重要的，因为潜在的 疾病改变受体功能。研究将包括基于siRNA的cofilin基因敲除，以及上游基因敲除。 将受体：G蛋白：效应子复合物与cofilin连接的拟议途径的组分。在目标2中， 将使用全细胞磷酸化研究激动剂促进的GRKs对TAS 2 R14的磷酸化， 受体纯化实验。为了确定GRK磷酸化的精确残基，将TAS 2 R14与GRK磷酸化的TAS 2 R14进行比较。 突变为用Ala取代潜在的Ser/Thr磷酸受体位点，从而定义β抑制蛋白的条形码 约束力磷酸化对β抑制蛋白构象和细胞内受体的影响 然后将确定信号传导和HASM松弛。在目标3中，将使用一组TAS 2 R14激动剂。 用于确定TAS 2 R激动剂可以偏离有害的 在哮喘治疗方面取得了良好的效果。这一奋进将提供 基于激动剂的受体“调节”在支气管扩张和抑制HASM中高度有效的基础 增殖，但几乎不显示短期或长期激动剂促进的脱敏或下调， 临床快速耐受性不明显。所有这三个目标将利用并行的生理测量， 使用非哮喘和哮喘HASM细胞的收缩和舒张，以及炎症精确切割， 人肺切片模型，以便将生化事件与临床相关的生理反应联系起来。 总的来说，这些研究将为开发新型直接支气管扩张剂提供基础 其可单独使用或与β-激动剂组合使用以治疗哮喘。

Project Summary Bitter taste receptors (TAS2Rs) are expressed on human airway smooth muscle (HASM) and when activated markedly relax the muscle and dilate the airway. Utilization of this pathway, which is distinct from that of β- agonists acting at β2-adrenergic receptors (β2ARs), will provide a new class of direct bronchodilators for treating or preventing bronchospasm in asthma. The TAS2R14 subtype is highly expressed in HASM and is a prime target for developing a novel therapeutic agent. However, there are gaps in our knowledge about the molecular/cellular biology and physiology of HASM TAS2Rs, including how they couple to relaxation, the potential for tachyphylaxis due to short-term (receptor phosphorylation) and long-term (downregulation of receptor expression) events, and the potential to bias receptor signaling towards favorable signaling for asthma treatment. The broad, long-term objective of the Project is to improve our understanding of HASM TAS2R biology relevant to treating airway contraction in asthma. To fill these gaps in our knowledge, in Aim 1 we will define the mechanism by which TAS2Rs evoke relaxation, which we hypothesize is via inhibiting phosphorylation of the actin severing protein cofilin. Studies will be performed in cultured HASM cells derived from nonasthmatic as well as asthmatic donor lungs, the latter being important because of the potential for the disease to modify receptor function. Studies will include siRNA-based knockouts of cofilin, and the upstream components of the proposed pathway that link the receptor:G-protein:effector complex to cofilin. In Aim 2, agonist-prompted phosphorylation of TAS2R14 by GRKs will be studied using whole cell phosphorylation and receptor purification experiments. To define the precise residues phosphorylated by GRKs, TAS2R14 will be mutated to substitute potential Ser/Thr phospho-acceptor sites with Ala, thus defining a bar-code for βarrestin binding. The consequences of phosphorylation on βarrestin conformation and intracellular receptor signaling, and HASM relaxation, will then be determined. In Aim 3, a panel of TAS2R14 agonists will be utilized to determine the mechanisms by which a TAS2R agonist can be biased away from deleterious outcomes and towards advantageous outcomes in regards to asthma therapy. This endeavor will provide the basis for agonist-based “tuning” of the receptor to be highly efficacious in bronchodilating and inhibiting HASM proliferation, but display little short- or long-term agonist-promoted desensitization or downregulation, such that clinical tachyphylaxis is not apparent. All three aims will utilize parallel physiological measurements of contraction and relaxation using nonasthmatic and asthmatic HASM cells, and an inflammatory precision-cut human lung slice model, in order to link biochemical events to clinically relevant physiological responses. Collectively, these studies will provide the basis for development of a novel class of direct bronchodilators which can be utilized alone, or in combination, with β-agonists for the treatment of asthma.