Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

描述（由申请人提供）：camp介导的信号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。在真核细胞中，cAMP的作用是由两种普遍表达的细胞内cAMP受体介导的，即经典的蛋白激酶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（Epac/cAMP- gef）直接激活的交换蛋白。两种普遍表达的cAMP效应物的存在为在空间和时间上更精确和综合地控制cAMP信号通路提供了一种机制。然而，Epac的激活机制尚不清楚。本提案的目的是通过绘制与Epac激活相关的构象变化来填补我们目前知识的空白。具体来说，我们计划的实验有以下具体目的：1)通过位点定向诱变确定对Epac激活重要的特定残基；2)利用增强的氘交换质谱（DXMS）和小角度x射线散射技术，描述cAMP结合和Epac活化相关的构象变化，并确定Epac与其下游效应物Rap1之间的蛋白质界面；3)利用x射线晶体学分析Epac2-cAMP的晶体结构。我们的长期研究目标是了解Epac的生理功能和调控机制。完成本应用程序中提出的研究将显着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基于机制的治疗策略，特别是针对cAMP信号传导成分。

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.