CHARACTERIZATION OF GLYCOSYLATION PATTERN OF NK1 RECEPTOR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 神经激肽1受体(NK1-R)上的碳水化合物部分的结构特征和功能的阐明正在进行中。糖基化的功能作用正在使用两种不同的放射性配基--神经激动素A和P物质--来研究，这两种配基都与NK1-R结合。本论文对神经激肽1受体(NK-1R)的糖基化进行了功能和结构分析。神经肽和MS实验室多年来一直在合作检查结合口袋，并绘制了P物质(SP)/NK-1R相互作用和神经激肽A(NKA)/NK-1R相互作用的不同附着部位。具体地，通过以下实验方法探索了与NK-1R结合的碳水化合物部分的结构和功能：a)制备和纯化野生型NK-1R和缺乏一个或两个糖基化共识位点的突变受体的方法学。对野生型或突变型受体的细胞功能研究包括三个部分：a)125I SP和125I NKA对野生型和突变型受体的结合特性的比较；b)P物质和神经激肽A结合诱导的信号通路的激活(如MAPK p42/p44、JNK/SAPK和p38)；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.