Glycosylation of NK1 Receptors: Chemistry and Function

描述（由申请人提供）： P物质（SP）是一种由11个氨基酸组成的肽，作为神经递质、自分泌剂和神经激素发挥多种生理和病理作用。SP参与多种功能，例如疼痛的传递、炎症的调节以及介导行为反应。SP的活性依赖于与其7跨膜G蛋白偶联受体NK 1受体（NK 1 R）的相互作用。NH 2末端有两个N糖基化的共有序列，有充分的证据表明NH 2末端是糖基化的;然而，对所连接的碳水化合物的化学结构及其功能知之甚少。该提案解决了我们知识中的这一空白，并专注于NK 1 R糖基化的化学和生物学后果。HT 29细胞（人结肠细胞）将用编码野生型人NK 1受体或具有一个、另一个或两个改变的N-糖基化位点的突变体受体的cDNA稳定转染。将开发基于组氨酸标记技术的受体纯化方法，以提取足够量和纯度的这些受体，从而允许对其碳水化合物部分进行质谱分析。将进行几项功能研究，以评估野生型和突变型受体的可能差异，包括比较a）配体与受体结合的缔合和解离速率; B）受体内化; c）和信号转导机制，包括cAMP的激活、磷酸肌醇水解和MARK途径的反式激活。这些试点实验可能会导致新的方向，了解糖基化在SP-NK 1 R介导的反应，可能是治疗的重要性的作用。

DESCRIPTION (provided by applicant): Substance P (SP) is an 11 amino acid peptide that plays multiple physiological and pathological roles as a neurotransmitter, an autocrine agent, and a neurohormone. SP participates in diverse functions such as the transmission of pain, regulation of inflammation, and in mediating behavioral responses. The activities of SP depend upon interaction with its 7-transmembrane G-protein coupled receptor, the NK1 receptor (NK1R). The NH2 terminus has two consensus sequences for N glycosylation and there is good evidence that the NH2 terminus is glycosylated; however little is known of the chemical structures of the attached carbohydrates nor their function. This proposal addresses this gap in our knowledge and focuses on the chemistry and biological consequences of NK1R glycosylation. HT29 cells (human colonic cells) will be stably transfected with cDNA encoding wild type human NK1 receptors or mutant receptors with one, the other or both N-glycosylation sites altered. Methods for purification of receptors based on his-tagged techniques will be developed to extract these receptors in sufficient quantity and purity to permit mass spectrometric analysis of their carbohydrate moities. Several functional studies will be done to assess possible differences in wild type and mutant receptors including comparing a) association and disassociation rates of ligand binding to receptors; b) receptor internalization; c) and signal transduction mechanisms including activation of cAMP, phosphoinositide hydrolysis and transactivation of the MARK pathway. These pilot experiments may lead to new directions for understanding the role of glycosylation in SP-NK1R mediated responses that could be of therapeutic importance.