MOLECULAR BIOPHYSICS OF GLUCAGON RECEPTOR FUNCTION

G蛋白没有高分辨率的结构信息- 偶联受体(GPCRs)，一个极其重要的细胞表面家族 感受器。我们的长期目标是描绘出 GPCR多肽激素受体的信号转导机制 一家人。我们选择了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于现场的结构信息 使用跨学科的方法研究受体。结构特征 决定配体结合亲和力的胰高血糖素受体，G蛋白 耦合、下行信令将被确定。具体目标1是 确定胰高血糖素结合亲和力的结构基础和 胰升糖素受体的专一性。我们将准备突变受体 和胰升糖素类似物，并表征它们的分子性质。 有关激素结合的化学特征的详细信息 对后续的激活机制的研究具有重要意义 感受器。具体目标2是确定以下结构决定因素 介导胰升糖素依赖的信号通路的胰升糖素受体。 胰高血糖素导致cAMP和细胞内钙浓度 增加。解释这一双重信号通路的确凿实验 而要确定介导钙离子流动的G蛋白还有待于 已执行。因此，我们建立了稳定的细胞系，我们有 开发了一种可靠的测定钙通量的方法来解决 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功能的受体的方法 表达系统，或稳定的细胞系，将被开发。我们会研究 用荧光和电子顺磁共振技术研究受体 用光谱学监测配基的构象变化 结合和受体激活。这项跨学科研究的成果 这种方法将促进我们对发生的分子事件的了解 在通过胰升糖素受体进行细胞信号传递的过程中，一个模型系统 多肽与激素结合的GPCRs的研究。

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.