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A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

ABSTRACT: The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist.To overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the "mix and measure" format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity.Our study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.

SUBMITTER: Tsalkova T

PROVIDER: S-EPMC3262007 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

Tsalkova Tamara T Mei Fang C FC Cheng Xiaodong X

PloS one 20120119 1

<h4>Background</h4>The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important disco ...[more]

PMID: 22276201

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Project description:Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.

Dataset Information

A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

Publications

A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

Similar Datasets

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