Dynamics of the Opioid Receptor Signaling Complex

 描述(由申请人提供)：需要资金进行核磁共振研究，以加深对G蛋白偶联受体(GPCRs)及其信号复合体的动态行为的了解，从而能够构建更好的作用机制模型。GPCRs是真核生物重要的信号转导守门人，识别多种细胞外刺激，包括光子、离子、小分子、多肽和蛋白质。它们通过耦合到不同的细胞内蛋白来传递产生的细胞外信号，这些蛋白随后激活下游效应器，并触发细胞和生理反应的级联反应。这具有深远的治疗意义，使这些受体成为目前约30%的处方药的靶标。因此，更好地了解它们是如何发挥作用的，有望有助于开发针对它们的新的更好的药物。尽管GPCRs与拮抗剂、激动剂(包括偏向配体、反向激动剂和变构调节剂)结合的结构数量急剧增加，但激活的分子机制仍然知之甚少。拟议的研究的前提是，可以通过更好地理解系统的动态行为来实现所需的清晰度。我们计划描述阿片受体家族(A类，亚家族)所有成员的动态行为。在这个家族中有四个成员--经典的阿片受体-，-，(MOR，KOR，DOR)和伤害素受体(NOP)，所有这些受体都主要与异源三聚体GI/GO蛋白偶联。口服避孕药是由阿片类药物作用的，阿片类药物在慢性和急性疼痛控制和成瘾方面都有很长的历史。这项资金申请是为了支持对科索沃特派团的研究，以及开发可应用于家庭其他成员的新知识和方案。我们将通过三个具体目标更好地理解包括激活过程在内的作用机制：(1)了解KOR在活动和非活动状态下的动态构象；(2)了解KOR信号复合体的动态行为；以及(3)了解KOR-强啡肽在“低”和“高”亲和力状态下的相互作用。在这个提案中，我们将使用我们最新开发的19F核磁共振方法来表征KOR的动态行为，并使用标准的核磁共振技术对强啡肽及其信号伙伴进行2H/13C/15N核磁共振研究，以表征它们与KOR结合时的动态行为的变化。研究将使我们能够探索各种问题，这些问题的答案将帮助我们建立一个更好的模型来描述激活过程。此外，研究中产生的方案和试剂应有助于建立一个新的平台，用于开发副作用减少或最小的针对阿片受体的新药。

 DESCRIPTION (provided by applicant): Funds are requested to carry out NMR studies to develop a deeper understanding of the dynamic behavior of G-Protein Coupled Receptors (GPCRs) and their signaling complexes to enable the construction of a better model of their mechanism of action. GPCRs are critical eukaryotic signal transduction gatekeepers which recognize a variety of extracellular stimuli, including photons, ions, small molecules, peptides and proteins. They transmit the resulting extracellular signal across the membrane by coupling to different intracellular proteins which then activate downstream effectors and trigger cascades of cellular and physiological responses. This has profound therapeutic implications, making these receptors the targets of ~30% of currently prescribed drugs. Developing a better understanding of how they work is, therefore, expected to help in developing new and better drugs targeting them. Although there has been a dramatic growth in the number of structures of GPCRs bound to antagonists, agonists including biased ligands, inverse agonists, and allosteric modulators, the molecular mechanisms of activation remain poorly understood. The proposed study rests on the premise that needed clarity can be achieved with a better understanding of the dynamic behavior of the system. We plan to characterize the dynamic behavior of all the members of the opioid family of receptors (Class A, -subfamily). There are four members in this family - the classical opioid receptors (OR) -, -, , (MOR, KOR, DOR) and the nociceptn receptor (NOP), all of which are coupled predominantly to heterotrimeric Gi/Go proteins. ORs are acted on by opioids which have had a long history in both chronic and acute pain control and addiction. This funding request is to support studies on KOR and the development of new knowledge and protocols that could be applied to the rest of the family. We will achieve a better understanding of the mechanism of action including the activation process through three specific aims: (1) Develop an understanding of the dynamic conformational landscape of KOR in the active and inactive state, (2) Develop an understanding of the dynamic behavior of the KOR signaling complex, and (3) Develop an understanding of the KOR-dynorphin interaction in the "low" and "high" affinity states. In this proposal, we will use our newly developed 19F NMR approach to characterize the dynamic behavior of KOR and standard NMR techniques for 2H/13C/15N NMR studies of the peptide dynorphin and the signaling partners to characterize changes to their dynamic behavior on binding to KOR. Studies will allow us to explore various questions, answers to which will help us build a better model describing the activation process. In addition, protocols and reagents generated in the study should help establish a new platform for developing new drugs targeting opioid receptors with reduced or minimal side-effects.