Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state

Rehmann, Holger; Das, Joost H.G.; Knipscheer, Puck; Wittinghofer, Alfred; Bos, Johannes L.

doi:10.1038/nature04468

Cited by 186 publications

(311 citation statements)

References 20 publications

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“…1A; it utilizes the enhanced variant of YFP, citrine (36), and Renilla luciferase as the BRET pair with human Epac1 inserted in between. Spectra of the expressed sensor protein revealed strong resonance energy transfer that was significantly reduced in response to cAMP, as expected from the FRET sensors and known structural changes in the molecule (21,37,38).…”

Section: An Epac-based Bret Sensor For Camp-camyel-severalmentioning

confidence: 89%

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Jiang

Collins

Davis

et al. 2007

Journal of Biological Chemistry

296

344

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Regulation of intracellular cyclic adenosine 3,5-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G s -dependent receptors for isoproterenol and prostaglandin E 2 . Whereas two ligands, uridine 5-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G q /calcium and G i , the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G s -coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P 2 receptor and the heterotrimeric G 13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), a ubiquitous second messenger, mediates a wide range of cellular functions including cell metabolism (1), cell proliferation and differentiation (1), immune responses (2, 3), memory formation (4), and cardiac contractility (5). Canonically, the concentration of intracellular cAMP is regulated by two distinct families of enzymes. The transmembrane adenylyl cyclases (ACs) 3 synthesize cAMP from adenosine triphosphate (6, 7), whereas the cAMP-specific phosphodiesterases metabolize cAMP to biologically inactive adenosine 5Ј-monophosphate (8, 9). ACs are primarily activated by G␣ s but their activities can also be differentially regulated by G␣ i , G␤␥, or Ca 2ϩ (10, 11). The activities of various phosphodiesterases can be regulated by protein kinase A (PKA), extracellular-regulated kinase (ERK), phosphoinositide 3-kinase, and the concentration of cAMP itself (12-16). Thus integration of signaling by stimuli that can regulate the intracellular concentration of cAMP will depend strongly on the various pathways and the subtypes of ACs and phosphodiesterases expressed in individual cells at any given time.Assessment of the regulation of intracellular cAMP in vivo has only become possible recently. Zaccolo et al. (17) first described a FRET sensor for cAMP based on the cAMP binding domain of PKA. Subsequently, several reports have described FRET sensors for cAMP based on binding of the nucleotide to the Epac proteins (18 -21). While these FRET sensors have been effective for measuring changes and localization of cAMP in single cells, measurements are tedious. Furthermore, the requirement for excitation of donor molecules pro...

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Section: An Epac-based Bret Sensor For Camp-camyel-severalmentioning

confidence: 89%

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Jiang

Collins

Davis

et al. 2007

Journal of Biological Chemistry

296

344

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“…As shown in Fig. 5, when the regions with decreased solvent accessibility in response to ESI-07 binding were mapped onto the crystal structure of apo-EPAC2, they defined a continuous area in three dimensions spanning the interface between the two CBDs that are arranged in a faceto-face configuration to form a continuous structural lobe in the apo-EPAC2 crystal structure (29)(30)(31). However, cAMP binding to EPAC2 protected additional flanking areas on both CBDs (29).…”

Section: Resultsmentioning

confidence: 99%

Isoform-specific antagonists of exchange proteins directly activated by cAMP

Tsalkova

Mei

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

127

148

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The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC-and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.cAMP-regulated guanine nucleotide exchange factor | high-throughput screening | hydrogen/deuterium exchange mass spectrometry

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“…Epac2, a Sos homolog that activates the Ras-related protein Rap1, is autoinhibited by regulatory domains that prevent Rap1 binding to the active site (22). Interestingly, the helical hairpin in the Cdc25 domain of inactive Epac2 is pivoted inward relative to that of active Sos, blocking the active site (Fig.…”

Section: Structural Basis For the Allosteric Activation Of Sos By Rasmentioning

confidence: 99%

A Ras-induced conformational switch in the Ras activator Son of sevenless

Freedman

Sondermann

Friedland

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

129

152

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The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.Cdc25 ͉ nucleotide-exchange factor ͉ crystal structure ͉ Ras exchanger motif (Rem) domain ͉ Ras guanine nucleotide-releasing factor 1

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Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state

Cited by 186 publications

References 20 publications

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Isoform-specific antagonists of exchange proteins directly activated by cAMP

A Ras-induced conformational switch in the Ras activator Son of sevenless

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