Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

描述（由申请人提供）：cAMP介导的信号传导在生理条件和疾病状态下调节无数重要的生物过程，包括糖尿病、心力衰竭和癌症。在真核细胞中，cAMP的作用由两种广泛表达的细胞内cAMP受体介导，经典的蛋白激酶A/cAMP依赖性蛋白激酶（PKA/cAPK）和最近发现的由cAMP/cAMP调节的鸟嘌呤核苷酸交换因子直接激活的交换蛋白（Epac/cAMP-GEF）。两种普遍表达的cAMP效应物的存在提供了以空间和时间方式更精确和综合控制cAMP信号传导途径的机制。然而，对Epac激活的机制知之甚少。这个建议的目的是填补差距，在我们目前的知识，通过映射的构象变化与Epac激活。具体而言，我们计划了具有以下特定目的的实验：1）通过定点诱变确定对Epac活化重要的特定残基; 2）描绘与cAMP结合和Epac活化相关的构象变化，并使用增强的氘交换质谱（DXMS）和小角X射线散射确定Epac与其下游效应物Rap 1之间的蛋白质界面; 3）用X射线晶体学解析Epac 2-cAMP的晶体结构。我们的长期研究目标是了解Epac调节的生理功能和机制。完成本申请中提出的研究将大大推动该领域朝着这些目标前进。此外，这项研究计划的医学和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会导致识别新的机制为基础的治疗策略，特别是针对cAMP信号成分。 公共卫生相关性：环腺苷酸介导的信号传导调节生理条件和疾病状态下的无数重要生物过程，包括糖尿病，心力衰竭和癌症。cAMP信号级联的成分与异常细胞生长和药物作用有关，并成功地用于癌症和其他疾病的诊断和化疗。更好地理解cAMP介导的信号转导可能会导致新的药物靶点的鉴定和新的或改进的治疗剂的开发。

DESCRIPTION (provided by applicant): 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 its 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. PUBLIC HEALTH RELEVANCE: Cyclic AMP-mediated signaling regulates a myriad of important biological processes under both physiological conditions and disease states, including diabetes, heart failure and cancer. Components of the cAMP-signaling cascade have been implicated in abnormal cell growth and drug actions and successfully targeted for diagnosis and chemotherapy of cancer and other diseases. A better understanding of cAMP mediated signal transduction could potentially lead to the identification of novel drug targets and the development of new or improved therapeutic agents.