Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

cAMP介导的信号传导调节生理条件下的无数重要的生物学过程。 条件和疾病状态，包括糖尿病、心力衰竭和癌症。在真核细胞中， cAMP由两种普遍表达的细胞内cAMP受体介导， A/cAMP依赖性蛋白激酶（PKA/cAPK）和最近发现的直接激活的交换蛋白 cAMP/cAMP调节的鸟嘌呤核苷酸交换因子（Epac/cAMP-GEF）。存在两 普遍表达的cAMP效应物提供了一种更精确和综合控制细胞凋亡的机制。 cAMP信号通路的空间和时间的方式。然而，关于其机制知之甚少。 Epac激活。本提案的目的是通过绘制 与Epac激活相关的构象变化。具体来说，我们计划用 以下具体目的：1）通过定点突变确定对Epac活化重要的特定残基， 诱变; 2）描绘与cAMP结合和Epac活化相关的构象变化 并使用增强的免疫荧光技术， 氘交换质谱（DXMS）和小角X射线散射;和3）解决晶体 使用X射线晶体学的Epac 2-cAMP的结构。我们研究的长期目标是了解 Epac调节的生理功能和机制。完成这项拟议的研究， 应用将大大推动该领域朝着这些目标前进。此外，医疗和 这项研究计划的药理学意义也是深远的。更好地了解cAMP- 介导的信号转导可能导致识别新的机制为基础的治疗 特别针对cAMP信号传导组分的策略。

cAMP-mediated signaling regulates a myriad of important biological processes under physiological conditions and disease states, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (Epac/cAMP-GEF). The existence of two ubiquitously expressed cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. However, little is known about the mechanism of Epac activation. The objective of this proposal is to fill the gap in our current knowledge by mapping the conformational changes associated with Epac activation. Specifically, we have planned experiments with the following Specific Aims: 1) To determine the specific residues important for Epac activation by site-directed mutagenesis; 2) to delineate the conformational changes associated with cAMP binding and Epac activation and to determine the protein interface between Epac and it's downstream effector, Rap1, using enhanced deuterium exchange-mass spectrometry (DXMS) and small-angle X-ray scattering; and 3) to solve the crystal structure of Epac2-cAMP using X-ray crystallography. The long-term goals of our research are to understand the physiological functions and mechanisms of Epac regulation. Accomplishing the proposed research in this application will significantly move the field forward towards these goals. Furthermore, the medical and pharmacological implications of this research program are also far-reaching. A better understanding of cAMP- mediated signal transduction could potentially lead to the identification of novel mechanism-based therapeutic strategies specifically targeting the cAMP-signaling components.