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

基本信息

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
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/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

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.

项目概要苦味受体 (TAS2R) 在人类气道平滑肌 (HASM) 上表达，当被激活时利用该途径可显着放松肌肉并扩张气道，这与β-途径不同。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中的支气管痉挛 TAS2R14 亚型在 HASM 中高表达，是主要的亚型。开发新型治疗剂的目标然而，我们对此的了解存在差距。 HASM TAS2R 的分子/细胞生物学和生理学，包括它们如何与放松、由于短期（受体磷酸化）和长期（受体磷酸化下调）可能导致快速耐受受体表达）事件，以及使受体信号偏向对哮喘有利信号的潜力该项目的广泛、长期目标是提高我们对 HASM TAS2R 的理解。与治疗哮喘气道收缩相关的生物学为了填补我们的知识空白，我们将在目标 1 中进行研究。定义 TAS2R 引起放松的机制，我们通过抑制来实现这一机制肌动蛋白切断蛋白丝切蛋白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，因为有潜力研究将包括基于 siRNA 的 cofilin 及其上游基因敲除。在目标 2 中，将受体：G 蛋白：效应物复合物与丝切蛋白连接起来的拟议途径的组成部分。 GRKs 激动剂促进的 TAS2R14 磷酸化将使用全细胞磷酸化和受体纯化实验。为了确定GRK磷酸化的精确残基，TAS2R14将被确定。突变以用 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。然后将确定目标 3 中的 TAS2R14 激动剂组。用于确定 TAS2R 激动剂可以远离有害物质的机制哮喘治疗方面的成果和有利成果。基于激动剂的受体“调节”在支气管扩张和抑制 HASM 方面非常有效的基础增殖，但几乎没有表现出短期或长期激动剂促进的脱敏或下调，因此临床快速耐受并不明显。所有三个目标都将利用平行的生理测量。使用非哮喘和哮喘 HASM 细胞进行收缩和放松，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，这些研究将为开发一类新型直接支气管扩张剂提供基础它可以单独使用，或与β-激动剂联合用于治疗哮喘。