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

痛觉神经元中 DOR-KOR 异聚体形成的调节

基本信息

批准号：
8824055
负责人：
WILLIAM P CLARKE
金额：
$ 22.43万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8824055
关键词：
Absence of pain sensation Adult Adverse effects Afferent Neurons Analgesics Animals Behavioral Behavioral Assay Biochemical Biological Assay Biological Models Body Regions Cell Culture System Cell Culture Techniques Cell surface Cells Characteristics Complex Development Fluorescence G-Protein-Coupled Receptors Genetic Transcription Goals Individual Knock-out Knowledge Label Learning Mediating Methods Microscopy Modeling Molecular Mus Neurons Nociceptors Opioid Receptor Pain Peptides Peripheral Pharmaceutical Preparations Pharmacotherapy Physiological Primary Cell Cultures Property Protomer Publishing Rattus Regulation Resolution Role Signal Transduction System Techniques Testing Time Trans-Activators Transmembrane Domain Wild Type Mouse body system efficacy testing in vitro Assay in vivo novel public health relevance receptor research study response tool

项目摘要

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.

描述（由申请人提供）：现在普遍认为G蛋白偶联受体（GPCR）可以与相同（同聚物）或不同（异聚物）GPCR配偶体形成多聚体复合物。我们从异源表达系统进行的研究中了解了大量关于这些 GPCR 寡聚体的信息，在异源表达系统中，可以标记单个受体原聚体，并且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传导方法。作为异源系统研究的结果，我们知道 GPCR 寡聚体可以被视为独特的受体实体，因为它们具有与单个 GPCR 原聚体不同的独特药理学特性和独特的信号传导特征。然而，对天然系统中 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寡聚体复合物的方法。我们对受体异聚体在生理相关系统中的作用缺乏了解，这代表了我们知识中的一个关键差距，因为这些寡聚受体可能是开发用于药物治疗的选择性药物的有价值的靶标。最近，我们发表了第一个证据，证明在生理相关系统——成年大鼠外周疼痛感知神经元（伤害感受器）中，δ（DOR）和κ（KOR）受体之间存在功能性阿片受体异聚体。我们相信，该系统由体内行为疼痛测定以及伤害感受器的补充体外培养物组成，为验证方法提供了绝佳的机会，从而能够进一步研究 DOR-KOR 异聚体在外周镇痛机制中的作用。因此，R21 探索性应用的目标是使用这些伤害感受器模型系统来验证在天然系统中研究 DOR-KOR 异聚体的方法。具体目标是：使用 TM-TAT 肽干扰 DOR 和 KOR 在细胞表面的结合，破坏 DOR-KOR 异聚体形成。 2) 通过使用基因修饰小鼠敲除 DOR 或 KOR 表达来破坏 DOR-KOR 异聚体形成，以及 3) 使用 DOR-KOR 异聚体选择性拮抗剂 KDN-21 阻断 DOR-KOR 异聚体功能。该项目将使我们能够研究外周疼痛感觉神经元中表达的 DOR-KOR 异聚体的药理学和信号传导机制及其在调节这些神经元疼痛信号传导中的作用。此外，用于破坏 DOR-KOR 异聚体的方法可应用于其他受体异聚体的研究，以帮助理解这些新型寡聚受体的生理和药理学相关性。