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标

基本信息

批准号：
10465061
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
项目类别：
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/10465061
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 Coupled Coupling Cyclic AMP Cytometry Data Development 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 Muscle relaxation phase Mutate Outcome Pathway interactions Pharmaceutical Preparations Phosphorylation Phosphorylation Inhibition Phosphotransferases Physiological Physiology Process Property Protein Isoforms Proteins Receptor Signaling Relaxation Signal Transduction Site Slice Small Interfering RNA Smooth Muscle Myocytes Structure Tachyphylaxis Taste Buds Taste Perception Testing Therapeutic Agents Tongue Western Blotting analog asthmatic base beta-2 Adrenergic Receptors clinically relevant cofilin 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

项目摘要

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（TAS2R）在人类气道平滑肌（HASM）上表达，当激活时明显放松肌肉并扩张气道。该途径的利用与β-不同作用于β2-肾上腺素受体（β2AR）的激动剂将提供一类新的直接支气管扩张剂或防止哮喘中支气管痉挛。 TAS2R14亚型在HASM中高度表达，并且是Prime 开发新型热剂的目标。但是，我们关于 hasm tas2rs的分子/细胞生物学和生理学，包括它们如何放松，由于短期（受体磷酸化）和长期（下调的下调受体表达）事件，以及对接收器信号传导偏向哮喘的有利信号的潜力治疗。该项目的广泛，长期目标是提高我们对HASM TAS2R的理解与治疗哮喘中气道收缩有关的生物学。在我们的知识中填补这些空白，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细胞中进行从非哮喘和哮喘供体肺中，后者很重要，因为有潜力疾病以改变受体功能。研究将包括基于siRNA的cofilin敲除和上游将接收器连接的提议途径的组成部分：G蛋白：效应子复合物与Cofilin。在AIM 2中， GRK对Tas2R14的激动剂促进的磷酸化将使用全细胞磷酸化和受体纯化实验。为了定义GRK磷酸化的精确残留物，Tas2r14将是突变以用ALA代替潜在的SER/THR磷酸化磷酸化位点，从而定义了βarRestin的条形码结合。磷酸化对βarrestin构象和细胞内接收器的后果然后将确定信号传导和HASM松弛。在AIM 3中，TAS2R14激动剂面板将是用于确定TAS2R激动剂可以偏离有害的机制在哮喘治疗方面的结果和优势结果。这项工作将提供基于激动剂的接收器的“调整”的基础是在支气管扩张和抑制HASM方面高效扩散，但几乎没有短期或长期激动剂促进的脱敏或下调，以至于临床速度不明显。这三个目标将利用平行的生理测量使用非哮喘和哮喘HASM细胞的收缩和放松，以及炎症精度切割人类肺切片模型，以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可以单独使用，也可以与β-激动剂组合使用，以治疗哮喘。