Eludicating the Retromer-dependent recycling of opiod and catecholamine receptors

描述(申请人提供)：G蛋白偶联受体(GPCRs)是许多处方药和非法药物的靶标，参与调节药物依赖行为。GPCRs维持或停止信号传递的能力在很大程度上取决于它们在配体诱导的内吞作用后如何传递，但这个过程仍然知之甚少。最近发现，经典的参与货物从内体到跨高尔基网络的逆行运输的蛋白质复合体，逆转录聚体，也被用于将几个GPCRs从内体直接循环到质膜，这是该复合体的一种新功能。逆转聚体被认为是一种衣物复合体，形成膜状管状结构，这些结构从内小体伸出，掐断成小泡，然后输送到不同的目的地。我推测，逆转录复合体能够在物理和生物化学上区分货物，以便将它们运送到不同的目的地。我建议使用两种与药物依赖行为和快速循环有关的GPCR货物来严格检验这一假设：1)阿片肽和药物的靶标--MU型阿片受体(MOR)，以及2)β-2-肾上腺素能受体(β-2AR)，一种与基于奖赏的学习有关的儿茶酚胺受体。作为比较，与GPCR样的Wnt转运受体Wnless(WLS)将被用于逆行转运途径的模型货物。有趣的是，WLS也被确定为MOR的互动伙伴，所以我也将调查MOR的存在和激活如何影响WLS的贩运。本项目的具体目标是：1)确定模型培养细胞中逆转录依赖回收和逆行运输货物的物理分支点；2)确定参与逆转录依赖回收和逆行运输的蛋白质是否特异且足以控制货物贩运目的地，并确定它们在模型培养细胞中的作用机制；3)确定在原代纹状体培养细胞中发现的逆转录依赖运输机制是否存在差异，纹状体原代培养神经元是一种对驱动药物依赖行为至关重要的细胞类型。这些目标将使用包括显微镜、荧光流式细胞术和生物化学在内的各种技术来实现。拟议的项目解决了内细胞膜运输调节GPCRs的特异性问题，这是我们理解药物滥用和成瘾的细胞基础的基础。

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.