Cyclic nucleotide analogs as probes of signaling pathways

Poppe, H; Rybalkin, Sergei D.; Rehmann, Holger; Hinds, Thomas R.; Tang, Xiao; Christensen, Anne Elisabeth; Schwede, Frank; Genieser, Hans‐Gottfried; Bos, Johannes L.; Døskeland, Stein Ove; Beavo, Joseph A.; Butt, Elke

doi:10.1038/nmeth0408-277

Cited by 222 publications

(268 citation statements)

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“…These results are consistent with a previous study showing that Rp-PET can inhibit cGMPactivated cGKI in intact cells under certain conditions (24). Considering the 46-fold higher lipophilicity (14) and the 4-fold higher concentration of Rp-PET versus 8-Br-cGMP used in our experiment, it appears that the inhibitory potential of Rp-PET in intact cells is quite moderate and that it must be present in large excess to inhibit the effect of a strong agonist such as 8-Br-cGMP.…”

Section: Resultssupporting

confidence: 93%

“…In intact cells, Rp-PET may either inhibit or activate cGKImediated pathways depending on the intracellular cGMP level and the prevalence of the cGKI␣ or cGKI␤ isoform. Other studies have shown that Rp-PET can also exert cGKI-independent effects (40,41), perhaps via modulation of PDEs (3,14). The unpredictable behavior of Rp-PET complicates the interpretation of intact cell studies.…”

Section: Resultsmentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. 1 cGMP receptors and can elevate the cAMP level by inhibiting the cAMP-degrading PDE3 (8,13,14). Moreover, there is evidence that one of the most frequently used cGKI inhibitors, KT5823, does not at all inhibit cGKI activity both in vitro and in intact cells and may in fact inhibit other protein kinases (15)(16)(17).…”

mentioning

confidence: 99%

“…1B) competitively inhibits activation of purified cGKI by cGMP (24). Rp-PET is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies (14,19).…”

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confidence: 99%

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The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Valtcheva

Nestorov

Beck

et al. 2009

Journal of Biological Chemistry

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cGMP is a cyclic-nucleotide second messenger with multiple targets and functions. Small-molecule modulators of cGMP generators and effectors are important biochemical tools as well as established and prospective drugs for the treatment of human diseases, such as erectile dysfunction, pulmonary hypertension, and various cardiovascular disorders (1-3). cGMP is generated by nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and can bind to and modulate the activity of at least three classes of cGMP effector proteins: cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs), 2 cyclic nucleotide-gated cation channels, and cGMP-dependent protein kinases (cGKs, also known as protein kinase G or PKG) (4).Based on pharmacological and genetic studies, the cGK type I (cGKI) is considered the major mediator of cGMP signaling in many tissues including the cardiovascular system (5-8). The mammalian cGKI is a cytosolic Ser/Thr protein kinase comprising an N-terminal regulatory domain with two cGMPbinding sites and a C-terminal catalytic domain. It exists in two isoforms termed cGKI␣ and cGKI␤. The isozymes have identical cGMP-binding sites and catalytic domains but differ in their N-terminal ϳ100 amino acids, which contribute to homodimerization, sensitivity to cGMP activation, and interaction with anchoring and substrate proteins. Recent in vivo studies with transgenic mice demonstrated that both isoforms can induce smooth muscle relaxation and vasodilation (9), but the respective molecular mechanisms behind these effects are controversial (6, 10). Similarly, opposing effects of cGMP/cGKI signaling have been reported on the growth and phenotype of vascular smooth muscle cells (VSMCs) (11,12). The inconsistency of the results concerning the function of cGKI might in part be related to unexpected effects of the pharmacological cGKI activators and inhibitors that are commonly used to distinguish between cGKI-dependent and cGKI-independent signaling. For instance, cGMP analogues can bind to multiple * This work was supported by grants from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: PDE, phosphodiesterase; cGKI, cGMP-dependent protein kinase type I; PET, 8-Br-PET-cGMP; Rp-PET, Rp-8-Br-PETcGMPS; 8-Br-PET-cGMP, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphate; Rp-8-pCPT-cGMPS, 8-(4-chlorophenylthio)-guanosine-3Ј, 5Ј-cyclic monophosphorothioate, Rp-isomer; Rp-8-Br-PET-cGMPS, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ESI-MS, electrospray ionization-mass spectrometry; ...

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Valtcheva

Nestorov

Beck

et al. 2009

Journal of Biological Chemistry

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“…However, the sulfur-containing Epacselective agonist may inhibit some PDEs and could lead to elevation of endogenous cAMP, stimulating PKA in the absence of a PKA-selective agonist (30,31). Indeed, when 8-pCPT-2Ј-O-Me-cAMP was infused in place of Sp-8-pCPT-2Ј-O-MecAMPS, it did not rescue retrieval at 2 g alone but fully rescued retrieval at 0.5 g when combined with Sp-6-Phe-cAMPS (Fig.…”

mentioning

confidence: 99%

Epac signaling is required for hippocampus-dependent memory retrieval

Ouyang

Zhang

Zhu

et al. 2008

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

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Previously we uncovered a critical role for norepinephrine and ␤1-adrenergic signaling in hippocampus-dependent memory retrieval. Because the ␤1 receptor couples to Gs, we examine here whether cAMP is also required for contextual memory retrieval. Using pharmacologic and genetic approaches to manipulate cAMP and downstream signaling, we demonstrate that cAMP and two of its targets, protein kinase A (PKA) and exchange protein activated by cAMP (Epac), are both required for retrieval. These findings demonstrate that cAMP signaling through Epac (as well as PKA) plays an essential role in cognition.norepinephrine ͉ protein kinase A ͉ context ͉ ␤-adrenergic ͉ cyclic AMP

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Crystal structure of cGMP‐dependent protein kinase Iβ cyclic nucleotide‐binding‐B domain : Rp‐cGMPS complex reveals an apo‐like, inactive conformation

et al. 2016

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The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B), considered a ‘gatekeeper’ for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iβ CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo- or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B.

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Cyclic nucleotide analogs as probes of signaling pathways

Cited by 222 publications

References 4 publications

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Epac signaling is required for hippocampus-dependent memory retrieval

Crystal structure of cGMP‐dependent protein kinase Iβ cyclic nucleotide‐binding‐B domain : Rp‐cGMPS complex reveals an apo‐like, inactive conformation

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