MOLECULAR BIOPHYSICS OF GLUCAGON RECEPTOR FUNCTION

胰高血糖素受体功能的分子生物物理学

• 批准号: 2727237
• 负责人: THOMAS P SAKMAR
• 金额: $ 11.34万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-15 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2727237
• 关键词: G protein; binding sites; biological signal transduction; biophysics; calcium flux; cell line; cyclic AMP; glucagon; hormone receptor; molecular biology; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; receptor binding; receptor expression; tissue /cell culture

High resolution structural information is not available for G protein- coupled receptors (GPCRs), an exceedingly important family of cell surface receptors. Our long-range objective is to delineate the molecular mechanism of signal transduction by peptide hormone receptors of the GPCR family. We have chosen the glucagon receptor as a model system for study, and we will attempt to obtain site-specific structural information about the receptor using an interdisciplinary approach. The structural features of the glucagon receptor that dictate ligand-binding affinity, G protein coupling, and downstream signaling will be determined. Specific Aim 1 is to identify the structural basis for glucagon binding affinity and specificity for the glucagon receptor. We will prepare mutant receptors and glucagon analogues and characterize their molecular properties. Detailed information about the chemical features of the hormone binding site are important for the subsequent study of the activation mechanism of the receptor. Specific Aim 2 is to identify the structural determinants of the glucagon receptor that mediate glucagon-dependent signaling pathways. Glucagon causes both cAMP and intracellular calcium concentrations to increase. Definitive experiments to explain this dual signaling pathway and to identify the G proteins that mediate the calcium flux remain to be performed. Therefore, we have established stable cell lines and we have developed a reliable assay to measure calcium flux to address the mechanism of the glucagon-dependent calcium response. Specific Aim 3 is to determine how glucagon binding leads to receptor activation using biochemical and biophysical approaches. To accomplish this aim, a procedure to isolate sufficient functional receptor from an insect cell expression system, or stable cell line, will be developed. We will study the receptors using fluorescence and electron paramagnetic resonance spectroscopy to monitor conformational changes that accompany ligand binding and receptor activation. Results from this interdisciplinary approach will advance our knowledge of the molecular events that occur during cellular signaling by glucagon receptor, a model system for the study of peptide-hormone-binding GPCRs.

G 蛋白没有高分辨率结构信息 偶联受体（GPCR），一个极其重要的细胞表面家族 受体。我们的长期目标是描绘分子 GPCR肽激素受体信号转导机制 家庭。我们选择胰高血糖素受体作为研究的模型系统， 我们将尝试获取特定站点的结构信息 使用跨学科方法的受体。结构特点 决定配体结合亲和力的胰高血糖素受体，G 蛋白 耦合和下游信令将被确定。具体目标 1 是 确定胰高血糖素结合亲和力的结构基础和 胰高血糖素受体的特异性。我们将准备突变受体 和胰高血糖素类似物并表征它们的分子特性。 有关激素结合化学特征的详细信息 对于后续研究其激活机制具有重要意义 受体。具体目标 2 是确定结构决定因素 介导胰高血糖素依赖性信号传导途径的胰高血糖素受体。 胰高血糖素导致 cAMP 和细胞内钙浓度 增加。解释这种双重信号通路的确定性实验 并鉴定介导钙流的 G 蛋白仍有待进一步研究 执行。因此，我们已经建立了稳定的细胞系，并且我们已经 开发了一种可靠的测定法来测量钙通量，以解决 胰高血糖素依赖性钙反应的机制。具体目标 3 是 确定胰高血糖素结合如何导致受体激活 生物化学和生物物理方法。为了实现这一目标，一个 从昆虫细胞中分离足够的功能受体的程序 将开发表达系统或稳定细胞系。我们将学习 使用荧光和电子顺磁共振的受体 光谱监测伴随配体的构象变化 结合和受体激活。这个跨学科的结果 方法将增进我们对发生的分子事件的了解 在胰高血糖素受体的细胞信号传导过程中，胰高血糖素受体的模型系统 肽激素结合 GPCR 的研究。