Novel Mechanisms of Smooth Muscle Beta2-receptor Regulation Relevant to Asthma

与哮喘相关的平滑肌β2受体调节新机制

• 批准号: 8661247
• 负责人: JEFFREY D HASDAY
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-19 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661247
• 关键词: Acute; Address; Adrenergic Receptor; Affect; Agonist; Allergens; Allergic; Animal Model; Arrestins; Asthma; Binding; Binding Sites; Biochemical; Bronchial Spasm; Bronchodilation; Cell Line; Cell model; Cells; Chronic Obstructive Airway Disease; Complex; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Down-Regulation; Drug Targeting; Drug usage; Elements; Event; Family; Feedback; Functional disorder; G protein coupled receptor kinase; Goals; Grant; Human; In Vitro; Individual; Inflammatory; Interleukin-13; Knowledge; Ligands; Link; Lung diseases; Mediating; Methods; MicroRNAs; Modeling; Molecular; Mus; Muscle function; Mutate; Organ; Ovalbumin; Pathway interactions; Phosphorylation; Phosphorylation Site; Physiological; Physiology; Receptor Activation; Receptor Cell; Receptor Signaling; Regulation; Relaxation; Reporting; Role; Seeds; Series; Shapes; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Specificity; Symptoms; System; Tachyphylaxis; Techniques; Testing; Transgenic Mice; Transgenic Organisms; Untranslated Regions; arrestin 2; base; effective therapy; human GPRK7 protein; improved; in vivo; insight; mRNA Expression; member; mouse model; mutant; novel; phosphoric diester hydrolase; protein expression; receptor; receptor binding; receptor downregulation; receptor expression; receptor function; receptor internalization; respiratory smooth muscle; response

DESCRIPTION (provided by applicant): Obstructive pulmonary diseases such as asthma and COPD are frequently treated with ¿-agonists. These drugs target the ¿2AR expressed on airway smooth muscle and are used for both long term control and rescue from acute bronchospasm. Of particular importance in human pathophysiology and treatment, ¿2AR signaling can be desensitized by b-agonist treatment, as well as the asthmatic inflammatory milieu. The mechanisms of this regulation are poorly understood, particularly in smooth muscle with associated physiologic function. These gaps in our knowledge have impaired our basic understanding of receptor-relaxation coupling and our ability to improve therapy for the treatment of bronchospasm. Mechanisms which will be explored in this grant include regulation by protein kinase A (PKA) and G-protein coupled receptor kinases (GRKs), and the let-7 family of microRNAs (miRNA). Our previous studies in airway smooth muscle point to a significant role for PKA phosphorylation of the receptor that alters GRK interaction with ¿2AR which had not been previously defined. Specific Aim 1 will delineate this mechanism of altered ¿2AR function with studies addressing phosphorylation, 2-arrestin recruitment, receptor internalization and phosphodiesterase (PDE) recruitment. Additionally, we have identified that ¿2AR baseline expression is directly regulated by let-7 miRNA. And, in an apparent feedback loop, let-7 miRNA is regulated by prolonged agonist activation of ¿2AR. In Specific Aim 2, transgenic mice will be generated with targeted expression on airway smooth muscle of WT ¿2AR and a mutant ¿2AR lacking the let-7 seed region in the 3"UTR. These mice will be studied to ascertain the significance of let-7 in establishing ¿2AR expression and its physiologic relevance in regulating bronchodilation. Additional studies will focus on the mechanisms and physiologic relevance of the apparent feedback loop, which may establish a new, and perhaps the most important, mechanism of ¿2AR downregulation by agonist. Loss of ¿2AR function in the asthmatic state has been reported in humans and animal models yet the mechanisms remain unknown, particularly with regard to linking cellular events to physiologic consequences. Evidence suggests several potential mechanisms that may involve PKA and GRK actions at the receptor, or altered let-7 miRNA expression. We have developed several transgenic mice that express mutated ¿2AR so that these potential mechanisms can be explored. In Specific Aim 3 transgenic mice lacking phosphorylation sites for PKA, GRK, PKA/GRK and the let-7 binding domains will be crossed into the inducible IL-13 allergic mouse model with confirmatory studies in the ovalbumin (OVA) allergen induced mouse model. Results from these various transgenic mice will define the specific mechanisms relevant to ¿2AR dysfunction in the asthmatic state. Taken together, these proposed studies will combine molecular, cellular and transgenic approaches to define mechanisms of ¿2AR function and regulation relevant to asthma.

描述（适用提供）：诸如哮喘和COPD之类的阻塞性肺部疾病经常用“激动剂治疗”。这些药物靶向在气道平滑肌上表达的2AR，用于长期控制和从急性支气管痉挛中救援。在人类病理生理学和治疗中特别重要的是，可以通过B-激动剂治疗以及哮喘性炎症环境脱敏2AR信号传导。该调节的机制知之甚少，尤其是在平滑肌和相关生理功能的平滑肌中。我们知识中的这些差距损害了我们对受体 - 释放耦合的基本理解，以及我们改善治疗支气管痉挛治疗的能力。将在此赠款中探索的机制包括蛋白激酶A（PKA）和G蛋白偶联受体激酶（GRKS）的调节，以及microRNAS（miRNA）的Let-7家族。我们先前在气道平滑肌方面的研究表明，受体的PKA磷酸化具有重要作用，从而改变了GRK与2AR的相互作用，但该受体的相互作用尚未定义。具体目标1将通过解决磷酸化，2- arrestin募集，受体内在化和磷酸二酯酶（PDE）募集的研究来描述这种改变的2AR功能的机制。此外，我们已经确定„ 2AR基线表达是由Let-7 miRNA直接调节的。并且，在明显的反馈回路中，Let-7 miRNA受到长时间的激动剂激活2AR的调节。在特定目标2中，将在3英寸UTR中缺乏let-7种子区域的wt-wt€2AR和一个突变体的靶向表达产生转基因小鼠。将研究这些小鼠在确定2AR表达及其在确定原本概念的机构方面的重要性。在人类中，循环可能建立了一种新的，也许是最重要的机制。2AR的损失是在哮喘状态下的2AR功能。表达突变2的转基因小鼠可以在特定目标中探索这些潜在的机制。在哮喘状态下，定义与2AR功能障碍相关的特定机制。