Eludicating the Retromer-dependent recycling of opiod and catecholamine receptors

阐明阿片和儿茶酚胺受体的逆转录酶依赖性回收

基本信息

批准号：
8725500
负责人：
Katherine Celina Varandas
金额：
$ 3.5万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8725500
关键词：
Address Adrenergic Receptor Affect Automobile Driving Behavior Biochemical Biochemistry Catecholamine Receptors Cell Line Cell membrane Cells Complex Corpus striatum structure Culture Media Cultured Cells Destinations Development Drug Addiction Drug Exposure Drug abuse Endocytosis Endosomes Evaluation Flow Cytometry Fluorescence Microscopy G-Protein-Coupled Receptors Habits Hela Cells Illicit Drugs Individual Learning Ligands Mediating Membrane Protein Traffic Microscopy Modeling Neurons Opioid Peptide Opioid Receptor Pathway interactions Pharmaceutical Preparations Process Protein Family Proteins Recycling Regulation Rewards Role Signal Transduction Sorting - Cell Movement Specificity Stimulus Structure Techniques Testing Translating Tubular formation Vesicle base beta-2 Adrenergic Receptors cell type insight nexin novel protein complex protein transport public health relevance receptor response retrograde transport trafficking trans-Golgi Network

项目摘要

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the targets of many prescription and illicit drugs and are involved in mediating drug dependent behaviors. The ability of GPCRs to maintain or halt signaling critically depends on how they traffic after ligand-induced endocytosis, but this process remains poorly understood. The protein complex classically involved in retrograde transport of cargos from endosomes to the trans-Golgi network, Retromer, was recently discovered to also be utilized in the recycling of several GPCRs from endosomes directly to the plasma membrane, a novel function for this complex. Retromer is thought to act as a coat-complex, forming membranous tubular structures which protrude from endosomes to pinch off into vesicles that traffic to different destinations. I hypothesize that the Retromer complex is able to physically and biochemically distinguish cargos in order to transport them to distinct destinations. I propose to rigorously test this hypothesis using two GPCR cargos that are implicated in drug-dependent behavior and that rapidly recycle: 1) the mu-type opioid receptor (MOR), the target for opioid peptides and drugs, and 2) the beta-2-adrenergic receptor (beta-2AR), a catecholamine receptor implicated in reward-based learning. As a comparison, the GPCR-like Wnt transport receptor, Wntless (Wls), will be used model cargo for the retrograde transport pathway. Interestingly, Wls has also been identified as an interaction partner for MOR, so I will also investigate how the presence and activation of MOR affects the trafficking of Wls. The specific aims of my project are to: 1) determine the physical branch-point of Retromer-dependent recycling and retrograde transport cargos in model cultured cells, 2) determine if proteins involved in Retromer-dependent recycling and retrograde transport are specific for and sufficient to control cargo trafficking destination and define their mechanisms of action in model cultured cells and, 3) determine if there are differences in Retromer-dependent trafficking mechanisms found in model cultured cells in primary striatal medium spiny neurons, a cell type essential to driving drug-dependent behavior. These aims will be addressed using a variety of techniques including microscopy, fluorescence flow cytometry, and biochemistry. The proposed project addresses the problem of specificity of GPCR regulation by endocytic membrane trafficking, which is fundamental to our understanding of the cellular basis of drug abuse and addiction.

描述（由申请人提供）：G 蛋白偶联受体 (GPCR) 是许多处方药和非法药物的靶标，并参与调节药物依赖性行为。 GPCR 维持或停止信号传导的能力关键取决于它们在配体诱导的内吞作用后如何运输，但这一过程仍然知之甚少。蛋白质复合物Retromer通常参与从内体到跨高尔基体网络的逆行运输，最近发现它也可用于将几种GPCR从内体直接回收到质膜，这是该复合物的一个新功能。逆转录酶被认为是一种外壳复合物，形成膜管状结构，从内体突出，夹入囊泡，运输到不同的目的地。我假设 Retromer 复合体能够在物理和生化上区分货物，以便将它们运输到不同的目的地。我建议使用两种与药物依赖性行为有关并快速回收的 GPCR 货物来严格检验这一假设：1）mu 型阿片受体（MOR），阿片肽和药物的靶标，2）β- 2-肾上腺素能受体 (beta-2AR)，一种儿茶酚胺受体，与基于奖励的学习有关。作为比较，类似 GPCR 的 Wnt 转运受体 Wntless (Wls) 将用作逆行转运途径的模型货物。有趣的是，Wls 也被确定为 MOR 的交互伙伴，因此我还将研究 MOR 的存在和激活如何影响 Wls 的贩运。我的项目的具体目标是：1）确定模型培养细胞中Retromer依赖性回收和逆行运输货物的物理分支点，2）确定参与Retromer依赖性回收和逆行运输的蛋白质是否是特异性的和充分的控制货物运输目的地并定义其在模型培养细胞中的作用机制，3）确定在原代纹状体介质多棘神经元（一种驱动药物所必需的细胞类型）的模型培养细胞中发现的逆转录酶依赖性运输机制是否存在差异依赖的行为。这些目标将通过多种技术来实现，包括显微镜、荧光流式细胞术和生物化学。拟议的项目解决了内吞膜运输对 GPCR 调节的特异性问题，这对于我们理解药物滥用和成瘾的细胞基础至关重要。