The role of endosomal sorting in regulating opioid receptor function

 描述(申请人提供)：阿片剂的生理效应，包括滥用药物，如海洛因或处方阿片类药物，是由G蛋白偶联受体(GPCRs)介导的。阿片类GPCRs的激活导致反馈机制，在反馈机制中，受体数量和信号活性通过内吞作用和内吞体内的分选来调节。目前的模型描述了阿片类GPCRs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(“循环”)，或在内体保持(“保留”)。这一过程在阿片类药物功能调节中的重要性通过观察到Mu-阿片受体(MOR)和增量-阿片受体DOR在内体以截然相反的方式分类：MOR循环，而DOR降解。虽然阿片受体的循环和降解所涉及的机制和蛋白质越来越为人所知，但内体滞留的作用仍然令人费解。内体滞留的中心悖论来自于实验数据，它与DOR(降解)和MoR(循环)的分类有关。这些数据提出了一个问题，即在已经描述的一般贩运结果的背景下，阿片受体何时以及如何发生内体滞留。事实证明，测试这些问题很困难，因为在过去的十年里，保留途径唯一已知的成分是GASP1(GPCR相关分类蛋白1)，而且它与受体的联系很难操纵。一个令人兴奋的最新进展是鉴定了一种新的蛋白质，命名为Beclin2，它与GASP1形成一个复合体，是GASP1功能所必需的。Beclin2更易于实验操作，为直接测试内体滞留在阿片受体调节中的作用打开了机会。这一假设得到了初步数据的支持，即阿片受体在内体的保留是再循环和降解途径的初始分选步骤：Beclin2-GASP1在内体持有阿片GPCR，直到受体的移交介导空间分离并进入再循环或降解途径。这一假设将得到严格检验，方法包括：(1)确定Beclin2在已定义的操作(针对受体和内体分选机械)中的作用；(2)通过流式细胞仪、显微镜、生化和质谱仪等技术，在原代神经元培养和非神经元循环GPCRs中确定Beclin2的相对功能位置；(2)确定Beclin2和内体循环模型细胞系的相对功能位置。这项建议的具体目标是：(1)复合体；(3)确立Beclin2分拣复合体的身份。这项建议从分子水平上解决阿片受体的调节问题，目的是更好地了解阿片类药物滥用和成瘾的细胞反应。

 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.