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

描述（由申请人提供）：阻塞性肺疾病，如哮喘和COPD，经常用β-受体激动剂治疗。这些药物靶向气道平滑肌上表达的2AR，用于长期控制和急性支气管痉挛的抢救。在人类病理生理学和治疗中特别重要的是，β-激动剂治疗以及哮喘炎症环境可以使Δ 2AR信号转导脱敏。这种调节的机制知之甚少，特别是在平滑肌相关的生理功能。这些知识上的空白损害了我们对受体-松弛偶联的基本理解，也损害了我们改善支气管痉挛治疗的能力。该基金将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let-7家族的microRNA（miRNA）。我们以前在气道平滑肌中的研究指出PKA受体磷酸化的重要作用，它改变了GRK与<$2AR的相互作用，这是以前没有定义的。具体目标1将通过研究磷酸化、2-arrestin募集、受体内化和磷酸二酯酶（PDE）募集来描述这种改变<$2 AR功能的机制。此外，我们已经确定，2AR基线表达直接受let-7 miRNA的调控。而且，在一个明显的反馈回路中，let-7 miRNA受到<$2AR激动剂激活时间延长的调节。在特定目标2中，将产生在气道平滑肌上靶向表达WT <$2AR和在3“UTR中缺乏let-7种子区的突变体<$2AR的转基因小鼠。将研究这些小鼠以确定let-7在建立2AR表达中的意义及其在调节支气管扩张中的生理相关性。进一步的研究将集中在表观反馈回路的机制和生理相关性上，这可能会建立一个新的，也许是最重要的，激动剂下调2AR的机制。损失2AR在哮喘状态下的功能已在人类和动物模型中报道，但其机制仍然未知，特别是关于将细胞事件与生理后果联系起来。有证据表明可能涉及PKA和GRK在受体上的作用或改变let-7 miRNA表达的几种潜在机制。我们已经开发了几个转基因小鼠，表达突变的2AR，使这些潜在的机制可以探索。在Specific Aim 3中，将缺乏PKA、GRK、PKA/GRK和let-7结合结构域的磷酸化位点的转基因小鼠与诱导型IL-13过敏小鼠模型杂交，并在卵清蛋白（OVA）过敏原诱导的小鼠模型中进行验证性研究。来自这些不同转基因小鼠的结果将定义与哮喘状态下的<$2AR功能障碍相关的特定机制。总之，这些拟议的研究将联合收割机分子，细胞和转基因的方法来确定机制的<$2AR功能和调节相关的哮喘。

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.