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.
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