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.
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