The role of endosomal sorting in regulating opioid receptor function

内体分选在调节阿片受体功能中的作用

• 批准号: 9032352
• 负责人: Braden Lobingier
• 金额: $ 6.07万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2018-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9032352
• 关键词: Address; Biochemistry; Cell Line; Cell surface; Cells; Complex; Culture Media; Data; Development; Down-Regulation; Drug Addiction; Drug abuse; Endocytosis; Endosomes; Feedback; Flow Cytometry; G-Protein-Coupled Receptors; G-substrate; Genetic; Goals; Heroin; Human Cell Line; Individual; Life; Link; Location; Lysosomes; Mass Spectrum Analysis; Mediating; Microscopy; Modeling; Molecular; Movement; Names; Nervous system structure; Neurons; Opiates; Opioid; Opioid Receptor; Outcome; Pathway interactions; Pharmaceutical Preparations; Physiological; Process; Proteins; Receptor Cell; Receptor Signaling; Recycling; Regulation; Research; Role; Signal Transduction; Sorting - Cell Movement; Techniques; Testing; Work; base; beta-2 Adrenergic Receptors; delta opioid receptor; drug of abuse; mu opioid receptors; novel; operation; opioid abuse; prescription opiate; prescription opioid; protein complex; public health relevance; receptor; receptor function; receptor recycling; response; restoration; trafficking

 DESCRIPTION (provided by applicant): The physiological effects of opiates, including drugs of abuse such as heroin or prescription opiate medications, are mediated by G protein-coupled receptors (GPCRs). Activation of opioid GPCRs results in feedback mechanisms in which receptor number and signaling activity is regulated through endocytosis and sorting at the endosome. The current model describes three sorting operations for opioid GPCRs at the endosome: degrade at the lysosome, reinsert at the cell surface ('recycling'), or hold at the endosome ('retention'). The importance of this process in regulation of opioid function is underscored by the observation that the mu-opioid receptor (MOR) and the delta-opioid receptor DOR are sorted in diametrically opposed manners at the endosome: MOR recycles while DOR degrades. While the mechanisms and proteins involved in recycling and degradation of opioid receptors are becoming well understood, the role of the endosomal retention remains puzzling. The central paradox of endosomal retention comes from experimental data linking it to the sorting of DOR (which degrades) but also MOR (which recycles). These data raise the question of when and how endosomal retention occurs for opioid receptors in context of the general trafficking outcomes already described. Testing these questions has proven difficult, as for the last decade the only known component of the retention pathway was the protein GASP1 (GPCR-associated sorting protein 1), and its connection with the receptor is challenging to manipulate. An exciting recent development has been the identification of a new protein, named Beclin2, which forms a complex with GASP1 and is necessary for GASP1 function. Beclin2 is more amenable to experimental manipulation, opening the opportunity to directly test the role of endosomal retention in the regulation of opioid receptors. The hypothesis of this proposal, supported by preliminary data, is that the endosomal retention of opioid receptors functions as the initial sorting step for both the recycling and degrading pathways: Beclin2- GASP1 hold opioid GPCRs at endosomes until a handoff of the receptor mediates spatial separation and entry into the recycling or degrading pathways. This hypothesis will be rigorously tested using a combination of Determine the role of Beclin2 in sorting of defined manipulations (to the receptors and endosomal sorting machinery) with techniques including flow cytometry, microscopy, biochemistry, and mass spectrometry in primary neuronal cultures and non-neuronal recycling GPCRs; (2) Define the relative functional locations of Beclin2 and the endosomal recycling model cell lines. The specific aims of this proposal: (1) complex; (3) Establish the identity of the Beclin2 sorting complex. This proposal addresses opioid receptor regulation on the molecular level with the goal of better understanding the cellular response to opiate drug abuse and addiction.

 描述（由申请人提供）：阿片类药物（包括滥用药物，如海洛因或处方阿片类药物）的生理效应由G蛋白偶联受体（GPCR）介导。阿片样物质GPCR的激活导致反馈机制，其中受体数量和信号传导活性通过内吞作用和内体分选来调节。目前的模型描述了阿片类GPCR在内体的三种分选操作：在溶酶体降解，在细胞表面重新插入（“再循环”），或在内体保持（“保留”）。μ-阿片样物质受体（莫尔）和δ-阿片样物质受体DOR在内体以完全相反的方式分类的观察结果强调了该过程在阿片样物质功能调节中的重要性：莫尔聚集而DOR降解。虽然阿片受体的再循环和降解的机制和蛋白质越来越清楚，内体保留的作用仍然令人费解。核内体滞留的中心悖论来自实验数据，这些数据将其与DOR的分选（降解）以及莫尔的分选（降解）联系起来。这些数据提出了一个问题：在已经描述的一般贩运结果的背景下，阿片受体的内体保留何时以及如何发生。测试这些问题已被证明是困难的，因为在过去十年中，保留途径的唯一已知组分是蛋白质GASP 1（GPCR相关分选蛋白1），并且其与受体的连接具有挑战性。最近一个令人兴奋的进展是鉴定出一种新的蛋白质，名为Beclin 2，它与GASP 1形成复合物，并且是GASP 1功能所必需的。Beclin 2更易于实验操作，从而有机会直接测试内体保留在阿片受体调节中的作用。初步数据支持的这一提议的假设是，阿片受体的内体保留作为回收和降解途径的初始分选步骤：Beclin 2-GASP 1将阿片GPCR保持在内体，直到受体的传递介导空间分离并进入回收或降解途径。该假设将使用以下技术的组合进行严格测试：（1）确定Beclin 2在原代神经元培养物和非神经元再循环GPCR中对定义操作（受体和内体分选机制）的分选中的作用，包括流式细胞术、显微镜、生物化学和质谱;（2）定义Beclin 2和内体再循环模型细胞系的相对功能位置。本提案的具体目的：（1）复合体;（3）确定Beclin 2分选复合体的身份。该提案在分子水平上解决阿片受体调节，目的是更好地理解阿片类药物滥用和成瘾的细胞反应。