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）：现在普遍认为，G蛋白耦合受体（GPCR）可以与相同（同源物）或不同（异源）GPCR伴侣形成多聚体配合物。我们已经从通过异源表达系统进行的研究中学到了这些GPCR低聚物，可以将单个受体素的毒物标记为标记，并且可以将高分辨率显微镜技术与严格的生化和细胞信号传导方法一起应用。由于在异源系统中进行研究，我们知道GPCR低聚物可以被视为独特的受体实体，因为它们具有独特的药理特性和不同的信号传导特性，这些特性与单个GPCR原始物的不同。但是，在天然系统中对GPCR低聚物的研究相对较少，这在很大程度上是由于缺乏适用于研究这些寡聚体复合物在生理相关系统中的方法。我们缺乏对生理相关系统中受体异构体的作用的理解，这在我们的知识上是一个关键的差距，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宝贵目标。最近，我们在生理相关的系统 - 成年大鼠外周外疼痛神经元（Nocptors）中发表了Delta（DOR）和Kappa（Kor）受体之间功能性阿片受体异构体的第一个证据。我们认为，该系统由体内行为疼痛分析以及伤害感受器的体内培养物组成，为验证方法提供了一个绝佳的机会，以进一步研究Dor-kor异源在镇痛周围机制中的作用。因此，该R21探索性应用的目的是使用这些伤害感受器模型系统来验证研究天然系统中的Dor-Kor异构体的方法。具体目的是：使用干扰细胞表面DOR和KOR缔合的TM-TAT肽破坏Dor-Kor异构体的形成。 2）通过使用遗传改性的小鼠敲除DOR或KOR表达来破坏Dor-kor异构体形成，3）使用Dor-kor异构体 - 选择性拮抗剂KDN-21阻止Dor-kor异构体功能。它们在调节这些神经元的疼痛信号传导中的作用。此外，用于破坏Dor-kor异构体的方法可以应用于其他受体异构体的研究，以帮助理解这些新型寡聚受体的生理和药理相关性。