Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

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DESCRIPTION (provided by applicant): Exchange protein directly activated by cAMP (Epac) or cAMP-activated guanine nucleotide exchange factor (cAMP-GEF) is a novel intracellular cAMP receptor, which directly activates Rap1, a Ras-like small G protein. The discovery of Epac opens up a new dimension in the study of cAMP-mediated signaling. In particular, the finding of a second intracellular cAMP receptor in addition to PKA suggests that some, or even the majority of cAMP actions described in the vast cAMP literature, do not act through the activation of PKA alone, as previously believed. Recent studies also suggest that Epac is a multifunctional protein that is capable of mediating cAMP actions differentially from PKA. The major goal of this proposal is to dissect the cAMP-signaling pathways, with particular emphasis on the structure and function of the novel cAMP sensor protein Epac. Specifically we will: 1) study subcellular targeting of Epac, particularly the mitochondrial localization, and its regulation that is important for Epac's cellular functions using immunofluorescence microscopy and GFP (green fluorescent protein) fusion protein; 2) test the hypothesis that tubulin/microtubule, acting either as an upstream regulator or downstream effector, is a bona fide Epac cellular partner by examining the biochemical and functional consequences of Epac and tubulin/microtubule interaction; 3) map the conformational changes associated with the activation of Epac by cAMP and Epac- Rap1 and Epac-tubulin interactions that are important to Epac functions using chemical protein footprinting and a sensitive MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mapping technique. Our long-term goal is to understand how the disparate functions of cAMP binding and guanine nucleotide exchange activity are assembled and coordinated to mediate cAMP signaling at the molecular and structural levels by dissecting Epac-mediated cellular and biochemical interactions. These proposed studies are essential for further understanding cAMP signaling pathways and elucidating the actions of cAMP in cellular regulation under normal and pathologic states.