G protein-coupled receptor regulation in airway myocytes

描述（由申请人提供）：收缩和增殖是气道平滑肌（ASM）的两种重要功能，这些功能的失调被认为在哮喘的发病机制中是重要的。已知G蛋白偶联受体（GPCR）是ASM功能的重要调节剂，能够促进（Gq偶联GPCR）或抑制（Gs偶联GPCR）ASM收缩和增殖。我们建议描述当暴露于其同源激动剂时这些竞争类GPCR如何脱敏，并确定特异性抑制Gs偶联受体β-肾上腺素能受体的脱敏是否可以使最常见的哮喘治疗β-激动剂成为ASM收缩和增殖的更有效拮抗剂。我们将采用最先进的生物化学、分子和遗传方法来表征GPCR激酶（GRK）的各种突变体抑制性构建体对Gq-和Gs-偶联GPCR的特异性，并建立这些构建体在调节受体信号传导和相关功能后果中的有效性。目的1和2将使用人和鼠ASM细胞培养物来确定GRK调节结构域肽或突变体以及siRNA介导的GRK敲低对ASM收缩和增殖重要的细胞信号传导事件的影响。目的3将通过评估ASM收缩（体内和体外）和增殖的变化来确定这种调节的功能后果。拟议的研究将首次确定脱敏机制如何优先影响ASM中的竞争性GPCR信号传导事件，并将这种调节与功能结果联系起来。这些研究的结果将确定靶向GRK介导的脱敏机制作为阻塞性气道疾病治疗的潜在有用性：与公共卫生的相关性：拟议的研究将测试β 2-肾上腺素能受体是否是气道平滑肌中最容易发生脱敏机制的受体，该机制导致反应性丧失，并研究选择性阻断这种脱敏作用的新策略是否能提高β-激动剂治疗哮喘的有益效果。

DESCRIPTION (provided by applicant): Contraction and proliferation are two important functions of airway smooth muscle (ASM), and dysregulation of these functions is believed to be important in the pathogenesis of asthma. G protein-coupled receptors (GPCRs) are known to be important regulators of ASM function, capable of either promoting (Gq-coupled GPCRs) or inhibiting (Gs-coupled GPCRs) ASM contraction and proliferation. We propose to characterize how these competing classes of GPCRs desensitize when exposed to their cognate agonists, and determine whether specific inhibition of desensitization of the Gs-coupled receptor (2-adrenergic receptor can render the most common asthma therapy, beta-agonist, a more effective antagonist of ASM contraction and proliferation. We will employ state of the art biochemical, molecular, and genetic approaches to characterize the specificity of various mutant inhibitory constructs of GPCR kinases (GRKs) for both Gq- and Gs- coupled GPCRs, and establish the effectiveness of these constructs in regulating receptor signaling and associated functional consequences. Aims 1 and 2 will use both human and murine ASM cell cultures to determine the effect of GRK regulatory domain peptides or mutants, and siRNA-mediated GRK knockdown, on cellular signaling events important to ASM contraction and proliferation. Aim 3 will determine the functional consequences of this regulation by assessing changes in ASM contraction (both in vivo and ex vivo) and proliferation. The proposed studies will identify for the first time how desensitization mechanisms preferentially influence competitive GPCR signaling events in ASM and link this regulation to functional outcomes. Results from these studies will establish the potential usefulness of targeting GRK-mediated desensitization mechanisms as a therapy for obstructive airway diseases: Relevance to Public Health: The proposed studies will test whether the (2-adrenergic receptor is the receptor in airway smooth muscle most prone to desensitization mechanisms that cause a loss of responsiveness, and examine whether a novel strategy to selectively block this desensitization could improve the beneficial effects of (-agonist therapy for asthma.