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主要取决于它们在配体诱导的内吞作用后如何转运，但这一过程仍然知之甚少。逆转录酶（Retromer）是一种典型的参与核内体向反式高尔基网络逆行运输的蛋白质复合体，最近被发现也可用于从核内体直接向质膜回收几种gpcr，这是该复合体的一种新功能。逆转录酶被认为是一种涂层复合物，形成膜状管状结构，从核内体突出，挤压成囊泡，运送到不同的目的地。我推测逆转录复合体能够从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有关且快速循环的GPCR载体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，以及2)β -2肾上腺素能受体（β - 2ar），一种与奖励学习有关的儿茶酚胺受体。作为比较，gpcr样Wnt转运受体Wntless （Wls）将被用作逆行转运途径的模型货物。有趣的是，Wls也被确定为MOR的互动伙伴，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依赖于逆转录酶的回收和逆行运输货物的物理分支点；2)确定参与依赖于逆转录酶的回收和逆行运输的蛋白质是否对控制货物运输目的地具有特异性和足够性，并确定其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（一种驱动药物依赖行为必不可少的细胞类型）的模型培养细胞中，retromer依赖性转运机制是否存在差异。这些目标将解决使用各种技术，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GPCR通过内吞膜运输调节的特异性问题，这对我们理解药物滥用和成瘾的细胞基础至关重要。

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.