NEURONAL REGULATION OF OPIOID RECEPTOR TRAFFICKING

描述（由申请人提供）：G蛋白偶联受体（GPCR）运输的动态细胞控制影响蛋白质的许多功能方面。越来越多的证据表明，GPCR的内吞作用和细胞内转运不仅在受体的脱敏和再敏过程中至关重要，而且这些过程还参与产生替代信号。阿片受体的贩运也受到严格的细胞控制，并且似乎在细胞对慢性阿片受体激动剂治疗的反应中起作用。我们实验室和其他实验室的先前报告表明，mu和delta阿片受体虽然在结构上是同源的，但具有不同的细胞内运输模式。激动剂诱导的内化的mu-阿片受体可以再敏化并循环回到细胞表面，而delta-阿片受体则被引导到溶酶体降解途径。受体嵌合体研究表明，这两种受体的羧基尾部结构域负责这种运输模式。然而，正如我们的初步数据所示，在神经细胞模型神经母细胞瘤Neuro2A （N2A）细胞中，羧基尾部结构域参与但不足以指导阿片受体的运输。delta-阿片受体细胞内第三环的二亮氨酸基序与羧基尾之间的相互作用是引导受体到溶酶体的必要条件。因此，我们假设除了线性受体序列外，由受体共价修饰（如磷酸化和泛素化）产生的多个三维序列也参与指导阿片受体的运输。修饰后的受体在内吞途径中的蛋白质支架将决定受体运输的方向。因此，在目前的研究中，我们建议确定：(a)参与阿片受体细胞内运输的线性受体序列；(b)泛素化和磷酸化在受体细胞内运输中产生三维序列的作用；(c)在受体的循环和溶酶体运输中起关键作用的tmns-内吞蛋白。将对阿片受体序列进行截断、缺失、单氨基酸分析和随机突变分析，以确定所涉及的主要序列。酵母双杂交筛选和使用质谱的蛋白质组学方法将用于鉴定所涉及的反式内吞蛋白。这些蛋白的显性阴性突变体将用于证明它们在受体运输中的作用。通过建立/，-和0-阿片受体的行程，并通过识别N2A细胞中各种途径中的内吞蛋白复合物，我们将能够阐明细胞控制的动态性质，并阐明受体运输在神经元中阿片受体激动剂功能中的重要性。

DESCRIPTION (provided by applicant): The dynamic cellular control of G protein-coupled receptor (GPCR) trafficking impacts many functional aspects of the protein. Accumulating evidences have suggested the endocytosis and intracellular trafficking of GPCR not only are critical in the desensitization and resensitization of the receptor but also these processes participate in generating alternative signals. The trafficking of the opioid receptors is also under stringent cellular control, and appears to have a role in the cellular responses to the chronic opioid agonist treatment. Previous reports from our laboratory and others have indicated that mu and delta-opioid receptors, though structurally homologous, have different intracellular trafficking patterns. The agonist-induced internalized mu-opioid receptor can be resensitized and recycled back to the cell surface, while the delta-opioid receptor is directed to the lysosomal degradation pathway. Receptor chimeras studies suggest that the carboxyl tail domains of these two receptors are responsible for such trafficking patterns. However, as demonstrated in our Preliminary Data, carboxyl tail domains participate but not sufficient in directing the trafficking of the opioid receptors in a neuronal cell model, neuroblastoma Neuro2A (N2A) cells. An interaction between a dileucine motif within the 3 rd intracellular loop of the delta-opioid receptor with the carboxyl tail is needed for directing the receptors to lysosomes. Thus, we hypothesize that in addition to the linear receptor sequence, multiple three-dimensional sequences generated by covalent modifications of the receptors such as phosphorylation and ubiquitination are involved in directing the opioid receptor traffic. The scaffolding of the proteins in the endocytic pathways with the modified receptors will determine the direction of the receptor traffic. Hence, in the current studies, we propose to identify: (a) the linear receptor sequences that participate in the intracellular trafficking of the opioid receptor; (b) the role of ubiquitination and phosphorylation on generating the three-dimensional sequence in the intracellular trafficking of the receptor; and (c) the tmns-endocytic proteins that are critical in the recycling and lysosomal trafficking of the receptor. Truncation, deletion, single amino acid and random mutational analyses of the mu and delta-opioid receptor sequences will be carried out to delineate the primary sequences involved. Yeast two-hybrid screens and proteomic approaches using mass spectrometry will be used to identify the trans-endocytic proteins involved. Dominant negative mutants of these proteins will be used to demonstrate their roles in receptor trafficking. By establishing the itineraries of the/,- and 0-opioid receptors, and by identifying the endocytic protein complexes in various pathways in N2A cells, we will be able to illuminate the dynamic nature of the cellular control and elucidate the significance of the receptor trafficking in the opioid agonist function in neurons.