cAMP-mediated signaling regulates a myriad of important biological processes under physiologicalconditions and disease states, including diabetes, heart failure and cancer. In eukaryotic cells, the effects ofcAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinaseA/cAMP-dependent protein kinase (PKA/cAPK) and the recently discovered exchange protein directly activatedby cAMP/cAMP-regulated guanine nucleotide exchange factor (Epac/cAMP-GEF). The existence of twoubiquitously expressed cAMP effectors provides a mechanism for a more precise and integrated control of thecAMP signaling pathways in a spatial and temporal manner. However, little is known about the mechanism ofEpac activation. The objective of this proposal is to fill the gap in our current knowledge by mapping theconformational changes associated with Epac activation. Specifically, we have planned experiments with thefollowing Specific Aims: 1) To determine the specific residues important for Epac activation by site-directedmutagenesis; 2) to delineate the conformational changes associated with cAMP binding and Epac activationand to determine the protein interface between Epac and it's downstream effector, Rap1, using enhanceddeuterium exchange-mass spectrometry (DXMS) and small-angle X-ray scattering; and 3) to solve the crystalstructure of Epac2-cAMP using X-ray crystallography. The long-term goals of our research are to understandthe physiological functions and mechanisms of Epac regulation. Accomplishing the proposed research in thisapplication will significantly move the field forward towards these goals. Furthermore, the medical andpharmacological 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 therapeuticstrategies specifically targeting the cAMP-signaling components.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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University of Texas Health Science Center Houston
Schools of Medicine
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Almahariq, Muayad; Mei, Fang C; Cheng, Xiaodong (2016) The pleiotropic role of exchange protein directly activated by cAMP 1 (EPAC1) in cancer: implications for therapeutic intervention. Acta Biochim Biophys Sin (Shanghai) 48:75-81
Singhmar, Pooja; Huo, XiaoJiao; Eijkelkamp, Niels et al. (2016) Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1. Proc Natl Acad Sci U S A 113:3036-41
Wang, Hui; Robichaux, William G; Wang, Ziqing et al. (2016) Inhibition of Epac1 suppresses mitochondrial fission and reduces neointima formation induced by vascular injury. Sci Rep 6:36552
Banerjee, Upasana; Cheng, Xiaodong (2015) Exchange protein directly activated by cAMP encoded by the mammalian rapgef3 gene: Structure, function and therapeutics. Gene 570:157-67
Almahariq, Muayad; Chao, Celia; Mei, Fang C et al. (2015) Pharmacological inhibition and genetic knockdown of exchange protein directly activated by cAMP 1 reduce pancreatic cancer metastasis in vivo. Mol Pharmacol 87:142-9
Almahariq, Muayad; Mei, Fang C; Wang, Hui et al. (2015) Exchange protein directly activated by cAMP modulates regulatory T-cell-mediated immunosuppression. Biochem J 465:295-303
Zhu, Yingmin; Chen, Haijun; Boulton, Stephen et al. (2015) Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 ""therapeutic window"". Sci Rep 5:9344
Ye, Na; Zhu, Yingmin; Chen, Haijun et al. (2015) Structure-Activity Relationship Studies of Substituted 2-(Isoxazol-3-yl)-2-oxo-N'-phenyl-acetohydrazonoyl Cyanide Analogues: Identification of Potent Exchange Proteins Directly Activated by cAMP (EPAC) Antagonists. J Med Chem 58:6033-47
Schwede, Frank; Chepurny, Oleg G; Kaufholz, Melanie et al. (2015) Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion. Mol Endocrinol 29:988-1005
Almahariq, Muayad; Mei, Fang C; Cheng, Xiaodong (2014) Cyclic AMP sensor EPAC proteins and energy homeostasis. Trends Endocrinol Metab 25:60-71

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