Eludicating the Retromer-dependent recycling of opiod and catecholamine receptors

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点，并参与介导药物依赖行为。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的交通方式，但这一过程仍然知之甚少。最近发现，典型地参与从内体到trans-Golgi网络的货物逆行运输的蛋白质复合物Retromer也被用于从内体直接到质膜的几种GPCR的再循环，这是该复合物的新功能。Retromer被认为是一种外套复合物，形成膜状管状结构，从内体突出，夹入囊泡中，运输到不同的目的地。我假设Retromer复合体能够在物理和生物化学上区分货物，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行为有关并快速循环的GPCR货物来严格测试这一假设：1）μ型阿片受体（莫尔），阿片肽和药物的靶点，以及2）β-2-肾上腺素能受体（β-2 AR），一种与基于奖励的学习有关的儿茶酚胺受体。作为比较，GPCR样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也被确定为莫尔的相互作用伙伴，因此我也将研究莫尔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逆转录酶依赖性再循环和逆行运输货物的物理分支点，2）确定参与逆转录酶依赖性再循环和逆行运输的蛋白质是否特异于并足以控制货物运输目的地，并确定它们在模型培养细胞中的作用机制，3）确定在原代纹状体中棘神经元（一种对驱动药物依赖性行为至关重要的细胞类型）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。这些目标将使用各种技术，包括显微镜，荧光流式细胞术和生物化学来解决。拟议的项目解决了GPCR调节的特异性问题，内吞膜贩运，这是我们的药物滥用和成瘾的细胞基础的理解的基础。

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.