G Protein-Coupled Receptor Regulationin Airway Myocytes

描述（由申请人提供）：激动剂是一线哮喘药物，靶向气道平滑肌（ASM）上的β -2-肾上腺素能受体（¿2ARs），以抑制引起气道狭窄和喘息的ASM收缩。近40年来，激动剂治疗的局限性一直是一个热议的话题。自本基金成立以来，我们的工作已经探索了多种气道细胞中GPCR调节的多种模式，并强调了¿2AR脱敏作为限制¿-激动剂治疗效果的重要机制的作用。我们现在发现阻断grk介导的ASM中¿2AR脱敏的益处受到第二信使激酶PKA调用的其他脱敏机制的限制。由于这项资助的长期目标是开发阻止- 2AR脱敏和提高-激动剂作为哮喘药物的疗效的方法，PKA作为ASM - 2AR的反馈调节器的重要作用提出了一个深远的挑战，因为PKA也被认为介导-激动剂的支气管松弛作用。本文提出的研究旨在：（目标1）确定pka依赖的ar脱敏机制及其与GRKs的协同作用；（目的2）解决关于PKA是否与cAMP效应物Epac相反是¿2AR介导的ASM松弛的效应的持续争论，同时建立PKA依赖的收缩抑制的特定机制；以及（目标3）确定区隔化cAMP信号发生的机制，并介导-激动剂在ASM中的抗收缩作用。实验将集中在人类ASM细胞和组织为基础的ASM信号传导和收缩模型，并利用不同的分子方法和分析工具。我们预计，这些研究结果将为生理细胞中介导激动剂特异性ar脱敏的机制的深度和复杂性提供新的见解。此外，我们将首次在ASM中描述区隔化的cAMP/PKA信号及其操纵手段，从而能够选择性地增强PKA功能，这对激动剂的治疗效果很重要。

DESCRIPTION (provided by applicant): ¿-agonists, a first line asthma drug, target the beta-2-adrenergic receptors (¿2ARs) on airway smooth muscle (ASM) to inhibit the ASM contraction that causes airway narrowing and wheezing. For almost 40 years, the limitations of ¿-agonist therapy has been a hotly debated topic. Our work since the inception of this grant has explored multiple modes of GPCR regulation in multiple airway cells, and has emphasized the role of ¿2AR desensitization as an important mechanism that limits the therapeutic efficacy of ¿-agonists. We have now discovered that the benefits of blocking GRK-mediated ¿2AR desensitization in ASM are limited by additional desensitization mechanisms invoked by the second messenger kinase PKA. Because a long term goal of this grant to develop means to thwart ¿2AR desensitization and improve the efficacy of ¿-agonists as asthma drugs, a significant role of PKA as a feedback regulator of ASM ¿2AR presents a profound challenge, given PKA is also presumed to mediate the bronchorelaxant effect of ¿-agonists. Studies proposed herein seek to: (Aim 1) identify PKA-dependent mechanisms of ¿2AR desensitization and their cooperativity with GRKs; (Aim 2) resolve the ongoing debate as to whether PKA, as opposed to the cAMP effector Epac, is the effector of ¿2AR- mediated ASM relaxation, while establishing the PKA-dependence of specific mechanisms of contraction inhibition; and (Aim 3) determine the mechanisms by which compartmentalized cAMP signaling occurs and mediates the anti-contractile effects of ¿-agonists in ASM. Experiments will focus on human ASM cell- and tissue- based models of ASM signaling and contraction, and utilize diverse molecular approaches and analytic tools. We anticipate that findings from the proposed studies will provide new insight into the depth and complexity of mechanisms that mediate agonist-specific ¿2AR desensitization in a physiological cell. Moreover, we will for the first time in ASM characterize compartmentalized cAMP/PKA signaling and the means to manipulate it, thereby enabling selective enhancement of PKA functions important to the therapeutic effect of ¿-agonists.