CHARACTERIZATION OF GLYCOSYLATION PATTERN OF NK1 RECEPTOR

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 正在对神经激肽1受体（NK 1-R）上的碳水化合物部分进行结构表征并阐明其功能。 糖基化的功能作用正在使用两种不同的放射性配体，神经激肽A和P物质，都结合NK 1-R检查。 本论文旨在对神经激肽1受体（NK-1 R）的糖基化进行功能和结构分析。神经肽和MS实验室多年来一直在共同研究结合口袋，并绘制了P物质（SP）/NK-1 R相互作用和神经激肽A（NKA）/NK-1 R相互作用的各种附着位点。具体地，通过以下实验方法探索了附接至NK-1 R的碳水化合物部分的结构和功能：a）开发用于制备和纯化野生型NK-1 R和缺乏糖基化共有位点之一或两者的突变体受体的方法。在用缺乏N-连接糖基化的一个、另一个或两个共有序列的野生型或突变型受体转染的细胞中的功能研究涉及三个部分：a）125 I SP和125 I NKA在野生型和突变型受体上的结合特征的比较，B）由P物质和神经激肽A结合诱导的信号传导途径的激活（例如MAPK p42/p44、JNK/SAPK和p38）和c）受体内化的比较。 基于其他受体模型的证据和本实验室以前的工作，结果表明糖基化在神经激肽-1受体-配体相互作用及其下游效应中起着关键作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structural characterization of the carbohydrate moieties on the neurokinin 1 receptor (NK1-R) and elucidation of their function are being carried out. The functional role of glycosylation is being examined using two different radioligands, neurokinin A and substance P, that both bind NK1-R. This thesis project was a functional and structural analysis of the glycosylation of the Neurokinin 1 receptor (NK-1R). The neuropeptide and MS laboratories have worked together over the years to examine the binding pocket and mapped the various sites of attachment for the substance P (SP)/NK-1R interaction, and the neurokinin A (NKA)/NK-1R interaction. The structures and functions of the carbohydrate moieties attached to the NK-1R were explored, specifically, through the following experimental approaches: a) Development of methodology for preparation and purification of wild-type NK-1R and mutant receptors lacking either, or both, glycosylation consensus sites. Functional studies in cells transfected with wildtype or mutant receptors lacking one, the other, or both consensus sequences for N-linked glycosylation involved three parts: a) Comparison of binding characteristics of 125I SP and 125I NKA on the wildtype and mutant receptors, b) Activation of signaling pathways induced by Substance P and Neurokinin A binding (e.g. MAPK p42/p44, JNK/SAPK, and p38) and c) Comparison of receptor internalization. Based on evidence in other receptor models and previous work in this laboratory, the results suggest that glycosylation plays a critical role in Neurokinin-1 receptor-ligand interactions and its downstream effects.