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

• 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
• 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

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）：现在人们普遍认为G蛋白偶联受体（GPCR）可以与相同（同聚体）或不同（异聚体）的GPCR伴侣形成多聚体复合物。我们已经从异源表达系统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，并且可以沿着严格的生物化学和细胞信号方法应用高分辨率显微镜技术。作为在异源系统中的研究的结果，我们知道GPCR寡聚体可以被认为是独特的受体实体，因为它们具有不同的药理学性质和不同的信号传导特征，这些特征可以不同于单个GPCR原聚体。然而，有相对较少的研究GPCR寡聚体在本地系统，在很大程度上是由于缺乏方法，适用于研究这些低聚体复合物在生理相关的系统。我们缺乏对受体异聚体在生理相关系统中的作用的理解，这代表了我们知识中的一个关键空白，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点。最近，我们发表了第一个证据的δ（DOR）和κ（KOR）受体之间的功能性阿片受体异聚体的生理相关系统-成年大鼠外周痛觉感受神经元（伤害感受器）。我们相信，这个系统，包括在体内的行为疼痛测定沿着与互补的离体培养的伤害感受器，提供了一个很好的机会，以验证的方法，使进一步研究的作用DOR-KOR异聚体外周机制的镇痛。因此，该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：使用干扰DOR和KOR在细胞表面结合的TM-TAT肽破坏DOR-KOR异聚体形成。2)使用遗传修饰的小鼠通过敲除DOR或KOR表达来破坏DOR-KOR异聚体形成，和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。该项目的结果将使我们能够研究DOR-KOR异聚体在外周疼痛感受神经元中表达的药理学和信号传导机制及其在这些神经元调节疼痛信号传导中的作用。此外，用于破坏DOR-KOR异聚体的方法可以应用于其他受体异聚体的研究，以帮助理解这些新的寡聚体受体的生理和药理学相关性。