CHARACTERIZATION OF GLYCOSYLATION PATTERN OF NK1 RECEPTOR

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。神经激肽1受体（NK1-R）上碳水化合物部分的结构特征及其功能的阐明正在研究中。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物质进行检测，这两种配体都能结合NK1-R。这个即将完成的论文项目是神经激肽1受体（NK-1R）糖基化的功能和结构分析。神经肽和质谱实验室多年来一直在一起研究结合袋，并绘制了P物质(SP)/NK-1R相互作用和神经动素A (NKA)/NK-1R相互作用的各种附着位点。具体来说，通过以下实验方法，正在探索与NK-1R相连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-1R和突变型受体的方法，这些受体缺乏糖基化共识位点中的一个或两个，其数量和纯度足以允许对碳水化合物部分进行质谱分析；b)用质谱法（MALDI和ES-MS）测定碳水化合物部分的结构序列和分支模式。该项目的另一个分支侧重于在转染了野生型或突变型受体的细胞中进行功能研究，这些受体缺乏n -链糖基化的一种、另一种或两种共识序列，包括三个部分：a) 125I ？SP和125I？NKA对野生型和突变型受体的影响；b) P物质和神经激肽A结合诱导的信号通路激活（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之前的工作，我们认为糖基化在神经激肽-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 is being investigated. The functional role of glycosylation is being examined using two different radioligands, neurokinin A and substance P, that both bind NK1-R. This nearly complete thesis project is 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 are being 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, in sufficient quantity and purity to permit MS analysis of the carbohydrate moieties; and b) Structural determination of sequence and branching patterns of the carbohydrate moieties by mass spectrometry, including MALDI and ES-MS. Another branch of this project focuses on functional studies in cells transfected with wildtype or mutant receptors lacking one, the other, or both consensus sequences for N-linked glycosylation and involves 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, we suggest that glycosylation plays a critical role in Neurokinin-1 receptor-ligand interactions and its downstream effects.