CHARACTERIZATION OF GLYCOSYLATION PATTERN OF NK1 RECEPTOR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。神经激肽1受体(NK1-R)上碳水化合物部分的结构特征和功能的阐明正在被研究中。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。这个几乎完整的论文项目是对神经激肽1受体(NK-1R)糖基化的功能和结构分析。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。结合在NK-1R上的碳水化合物部分的结构和功能正在被探索，特别是通过以下实验方法：a)建立制备和纯化野生型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，其数量和纯度足以对碳水化合物部分进行MS分析；以及b)通过包括MALDI和ES-MS在内的质谱法确定糖部分的序列和分支模式的结构。该项目的另一个分支集中于野生型或突变型受体缺乏N-连接糖基化的共同序列的细胞中的功能研究，涉及三个部分：a)125I？SP和125I？nka与野生型和突变型受体结合特性的比较；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。基于其他受体模型中的证据和本实验室以前的工作，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.