A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production

Kool, Jeroen; Marle, André van; Hulscher, Saskia; Selman, Maurice H. J.; Iperen, Dick J. van; Altena, Klaas van; Gillard, Michel; Bakker, Remko A.; Irth, H.; Leurs, Rob; Vermeulen, Nico

doi:10.1177/1087057107308881

Cited by 9 publications

(11 citation statements)

References 38 publications

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“…These receptor affinity detection (RAD) systems integrate liquid chromatography (LC) with affinity assessment and therefore allow the direct correlation between individual ligands and their affinity towards target proteins. Biochemical detection systems reported previously are based on fluorescence readout [12][13][14], fluorescent polarization [15] or mass spectrometry based reaction detection [16][17][18][19][20]. LC coupled to mass spectrometry (MS) is by far the most used tool in drug metabolism studies.…”

Section: Introductionmentioning

confidence: 99%

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

Vlieger

Kolkman

Ampt

et al. 2010

Journal of Chromatography B

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Section: Introductionmentioning

confidence: 99%

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

Vlieger

Kolkman

Ampt

et al. 2010

Journal of Chromatography B

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“…Fluorescence Polarization Assay for cAMP Dissociation-To determine if RegA interactions facilitate dissociation of cAMP from RI␣, we carried out a fluorescence polarization (FP) assay using 8-Fluo-cAMP saturated RI␣ (91-244) as described (25). This assay allows measurement of the rate of dissociation/competitive displacement of 8-FluocAMP from RI␣ by cAMP, because FP of bound fluorescent analog is greater than for analog free in solution.…”

Section: Methodsmentioning

confidence: 99%

“…4A). The maximal activity was seen under these conditions in the presence of 3 M or greater cAMP-free RI␣ (91-244) with an EC 50 for the activation equal to 0.13 M. PDE assays were then carried out with three concentrations of GST RegA (385-780) (25,50, and 100 nM) in the presence of 3 M cAMP-free RI␣ (91-244) and the relative activity showed a linear relationship with the concentration of GST RegA (385-780) used (data not shown). GST RegA (385-780) alone (50 nM) and in the presence of 3 M cAMP-free RI␣ (91-244) was then used to determine the kinetic parameters (K m and K cat ) by measuring activity at cAMP concentrations ranging from 10 M to 250 M (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…To test this further we used a fluorescent analog of cAMP, 8-Fluo-cAMP, which has been shown to have similar binding affinities to RI␣ as cAMP (44) and is highly resistant to PDEs as well (manufacturer's sheet, Biolog, Bremen, Germany) in a fluorescence polarization (FP) assay for measuring cAMP dissociation as described in Materials and Methods (25) (Fig. 8B, 8C).…”

Section: Deletion Mutagenesis Indicates That the Catalytic Domain Ofmentioning

confidence: 99%

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Phosphodiesterases Catalyze Hydrolysis of cAMP-bound to Regulatory Subunit of Protein Kinase A and Mediate Signal Termination

Moorthy

Gao

Anand

2011

Molecular & Cellular Proteomics

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Although extensive structural and biochemical studies have provided molecular insights into the mechanism of cAMP-dependent activation of protein kinase A (PKA), little is known about signal termination and the role of phosphodiesterases (PDEs) in regulatory feedback. In this study we describe a novel mode of protein kinase A-anchoring protein (AKAP)-independent feedback regulation between a specific PDE, RegA and the PKA regulatory (RI␣) subunit, where RI␣ functions as an activator of PDE catalysis. Our results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RI␣. Furthermore our results indicate that binding of RI␣ activates PDE catalysis several fold demonstrating a dual function of RI␣, both as an inhibitor of the PKA catalytic (C) subunit and as an activator for PDEs. Deletion mutagenesis has localized the sites of interaction to one of the cAMP-binding domains of RI␣ and the catalytic PDE domain of RegA whereas amide hydrogen/ deuterium exchange mass spectrometry has revealed that the cAMP-binding site (phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5AMP. cAMP-free RI␣ generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RI␣ to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RI␣ that has important consequences for PKA regulation and cAMP signal termination.

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“…Final testing, performed on plant extracts spiked with known inhibitors, showed that low levels of inhibitor could be detected. Kool et al [73] developed a flow through assay to measure the modulation of cAMP levels by G-protein coupled receptors based on fluorescence polarization (FP). The assay was coupled to the cAMP binding domain of protein kinase A and fluorescein-labeled cAMP and tested against other nucleotides for sensitivity.…”

Section: Hplc Coupled On-line To Ead and Rad Assays And Their Applmentioning

confidence: 99%

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

Malherbe

Beer

Joubert

2012

IJMS

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Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays.

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A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production

Cited by 9 publications

References 38 publications

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

Phosphodiesterases Catalyze Hydrolysis of cAMP-bound to Regulatory Subunit of Protein Kinase A and Mediate Signal Termination

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

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