Regulation of DOR-KOR heteromer formation in pain-sensing neurons

描述(申请人提供)：现在普遍认为G蛋白偶联受体(GPCRs)可以与相同的(同构体)或不同的(异构体)GPCRs伙伴形成多聚体复合体。通过异源表达系统进行的研究，我们已经了解了大量关于这些GPCR寡聚体的信息，在这些系统中，可以标记单个受体原型，并可以应用高分辨率显微镜技术以及严格的生化和细胞信号方法。通过对异源系统的研究，我们知道GPCR寡聚体可以被认为是独特的受体实体，因为它们具有不同于单个GPCR原型的不同的药理特性和不同的信号特征。然而，在天然系统中对GPCR寡聚体的研究相对较少，这在很大程度上是由于缺乏适用于在生理相关系统中研究这些石油聚合体的方法。我们对受体异构体在生理相关系统中的作用缺乏了解，这是我们知识中的一个关键差距，因为这些寡聚体受体可能是开发药物治疗的选择性药物的有价值的靶点。最近，我们发表了第一个证据，在一个生理相关的系统-成年大鼠外周痛觉神经元(伤害性感受器)-之间的功能阿片受体异构体之间的增量(DOR)和kappa(KOR)受体。我们相信，该系统包括体内行为疼痛分析和伤害性感受器的补充体外培养，为进一步研究DOR-KOR异构体在外周止痛机制中的作用提供了一个极好的机会来验证方法。因此，R21探索性应用的目标是使用这些伤害性感受器模型系统来验证在本地系统中研究DOR-KOR异构体的方法。其具体目的是：使用TM-TAT多肽干扰DOR和KOR在细胞表面的结合来干扰DOR-KOR异构体的形成。2)通过使用转基因小鼠敲除DOR或KOR表达来干扰DOR-KOR异构体的形成；以及3)使用DOR-KOR异构体选择性拮抗剂KDN-21来阻断DOR-KOR异构体的功能。该项目的结果将使我们能够研究在外周痛觉神经元中表达的DOR-KOR异构体的药理和信号机制，以及它们在这些神经元调节疼痛信号中的作用。此外，用于阻断DOR-KOR异构体的方法也可以应用于其他受体异构体的研究，以帮助理解这些新的寡聚受体的生理和药理相关性。

DESCRIPTION (provided by applicant): It is now generally accepted that G protein coupled receptors (GPCRs) can form multimeric complexes with the same (homomers) or different (heteromers) GPCR partners. We have learned a vast amount about these GPCR oligomers from studies conducted with heterologous expression systems where individual receptor protomers can be labeled and high-resolution microscopy techniques can be applied along with rigorous biochemical and cell signaling methods. As a result of studies in heterologous systems, we know that GPCR oligomers can be considered as unique receptor entities as they have distinct pharmacological properties and distinct signaling characteristics that can differ from those of the individual GPCR protomers. However, there are relatively few studies of GPCR oligomers in native systems, owing in large part to a lack of methods that are applicable to the study of these oliogomeric complexes in physiologically relevant systems. Our lack of understanding of roles for receptor heteromers in physiologically relevant systems represents a critical gap in our knowledge as these oligomeric receptors may be valuable targets for development of selective drugs for pharmacotherapy. Recently, we published the first evidence for functional opioid receptor heteromers between delta (DOR) and kappa (KOR) receptors in a physiologically relevant system - adult rat peripheral pain-sensing neurons (nociceptors). We believe this system, comprised of in vivo behavioral pain assays along with complementary ex vivo culture of nociceptors, provides an excellent opportunity to validate approaches to enable further study of the role of DOR-KOR heteromers in peripheral mechanisms of analgesia. Thus, the goal of this R21 exploratory application is to use these nociceptor model systems to validate approaches to study DOR-KOR heteromers in native systems. The specific aims are: to disrupt DOR-KOR heteromer formation using TM-TAT peptides that interfere with the association of DOR and KOR at the cell surface. 2) to disrupt DOR-KOR heteromer formation by knock-out of either DOR or KOR expression using genetically-modified mice and 3) to block DOR-KOR heteromer function using a DOR-KOR heteromer-selective antagonist, KDN- 21. Results from this project will allow for us to study the pharmacological and signaling mechanisms of DOR- KOR heteromers expressed in peripheral pain-sensing neurons and their role in regulating pain signaling by these neurons. Moreover, approaches used to disrupt DOR-KOR heteromers can be applied to the study of other receptor heteromers to aid in understanding the physiological and pharmacological relevance of these novel oligomeric receptors.