MOLECULAR BIOPHYSICS OF GLUCAGON RECEPTOR FUNCTION

G蛋白的高分辨率结构信息不可用- 偶联受体（GPCR），一个非常重要的细胞表面家族， 受体。我们的长期目标是描绘出 GPCR肽激素受体信号转导机制 家人我们选择胰高血糖素受体作为研究的模型系统， 我们将尝试获得特定地点的结构信息， 受体使用跨学科的方法。的结构特点 决定配体结合亲和力的胰高血糖素受体，G蛋白 耦合和下游信号将被确定。具体目标1是 鉴定胰高血糖素结合亲和力的结构基础， 胰高血糖素受体的特异性。我们将准备变异受体 和胰高血糖素类似物，并表征它们的分子性质。 关于激素结合的化学特征的详细信息 对后续的活化机制研究具有重要意义。 受体。具体目标2是确定 介导胰高血糖素依赖性信号通路的胰高血糖素受体。 胰高血糖素引起cAMP和细胞内钙浓度， 增加解释这种双重信号通路的连续实验 并确定G蛋白介导的钙流仍然是 执行。因此，我们已经建立了稳定的细胞系， 开发了一种可靠的测定方法来测量钙通量， 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活化， 生物化学和生物物理方法。为了实现这一目标，A 从昆虫细胞分离足够功能性受体的方法 表达系统或稳定的细胞系。我们将研究 用荧光和电子顺磁共振技术 光谱学以监测伴随配体构象变化 结合和受体活化。这一跨学科的结果 这种方法将推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， 肽激素结合GPCR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.