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

项目 2 - 气道平滑肌苦味受体作为新型支气管扩张剂的靶点

• 批准号: 10683126
• 负责人: Stephen B Liggett
• 金额: $ 38.89万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10683126
• 关键词: Acetylcholine; Actins; Adrenergic Agents; Affect; Agonist; Antibodies; Arrestins; Asthma; Bar Codes; Binding; Biochemical; Biology; Bronchial Spasm; Bronchodilation; Bronchodilator Agents; Caring; Cell Proliferation; Cell physiology; Cells; Characteristics; Clinical; Code; Complex; Coupling; Cyclic AMP; Cytometry; Data; Development; Dimensions; Disease; Down-Regulation; Effectiveness; Endothelin-1; Event; F-Actin; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Histamine; Human; In Vitro; Individual; Inflammatory; Knock-out; Knowledge; LIMK1 gene; Link; Lung; Magnetism; Measurement; Measures; Mechanics; Mediating; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Molecular and Cellular Biology; Monitor; Muscle relaxation phase; Mutate; Outcome; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Inhibition; Physiological; Physiology; Polymers; Process; Proliferating; Property; Protein Isoforms; Proteins; Receptor Down-Regulation; Receptor Signaling; Relaxation; Signal Transduction; Site; Slice; Small Interfering RNA; Smooth Muscle Myocytes; Structure; Tachyphylaxis; Taste Buds; Taste Perception; Testing; Therapeutic Agents; Tongue; Transfection; Western Blotting; analog; asthmatic; beta-2 Adrenergic Receptors; clinically relevant; cofilin; constriction; desensitization; experimental study; improved; in vivo; knock-down; knockout gene; mutant; novel; novel therapeutics; polymerization; prevent; receptor; receptor downregulation; receptor expression; receptor function; receptor internalization; recruit; respiratory smooth muscle; response; therapy outcome

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.

项目摘要 苦味受体(TAS2Rs)在人呼吸道平滑肌(HASM)上表达，当被激活时 显著放松肌肉，扩张呼吸道。利用这条不同于β的途径-- 作用于β2-肾上腺素能受体(β2AR)的激动剂将为治疗提供一类新的直接支气管扩张剂 或预防哮喘的支气管痉挛。TAS2R14亚型在HASM中高表达，是一个质数 开发一种新型治疗剂的目标。然而，我们对这一问题的了解存在差距 HASM TAS2Rs的分子/细胞生物学和生理学，包括它们如何与松弛、 由于短期(受体磷酸化)和长期(下调 受体表达)事件，以及使受体信号偏向哮喘有利信号的可能性 治疗。该项目的广泛、长期目标是提高我们对HASM TAS2R的了解 与治疗哮喘的呼吸道收缩有关的生物学。为了填补我们知识的这些空白，在目标1中，我们将 定义TAS2Rs引起松弛的机制，我们假设这是通过抑制 肌动蛋白切断蛋白cofilin的磷酸化。研究将在培养的HASM细胞中进行 来自非哮喘和哮喘供体肺，后者很重要，因为 疾病改变了受体的功能。研究将包括基于siRNA的cofilin基因敲除，以及上游 连接受体：G-蛋白：效应器复合体和粘附素的拟议途径的组成部分。在目标2中， 激动剂诱导的GRKs对TAS2R14的磷酸化将使用全细胞磷酸化和 受体纯化实验。为了定义被GRKs磷酸化的精确残基，TAS2R14将被 突变，以Ala取代潜在的丝氨酸/苏氨酸磷酸受体位点，从而定义了β排列的条形码 有约束力的。磷酸化对βarrestin构象和细胞内受体的影响 信令和HASM松弛将在那时确定。在AIM 3中，一组TAS2R14激动剂将 用来确定TAS2R激动剂远离有害物质的机制 结果和在哮喘治疗方面的有利结果。这一努力将为 以激动剂为基础的受体调谐在支气管扩张和抑制HASM方面高效的基础 增殖，但几乎没有短期或长期的激动剂促进的脱敏或下调，以至于 临床上的快速反应并不明显。这三个目标都将利用平行的生理测量 使用非哮喘和哮喘HASM细胞的收缩和松弛，以及炎性精密切割 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，这些研究将为开发一类新型的直接支气管扩张剂提供基础 它可以单独使用，也可以与β激动剂联合用于治疗哮喘。