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The genetically encoded tool set for investigating cAMP: more than the sum of its parts.

Patel N, Gold MG - Front Pharmacol (2015)

Bottom Line: We chart the evolution of sequences for selectively modifying protein-protein interactions that support cAMP signaling, and for driving cAMP sensors and manipulators to different subcellular locations.Importantly, these different genetically encoded tools can be applied synergistically, and we highlight notable instances that take advantage of this property.Finally, we consider prospects for extending the utility of the tool set to support further insights into the role of cAMP in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London London, UK.

ABSTRACT
Intracellular fluctuations of the second messenger cyclic AMP (cAMP) are regulated with spatial and temporal precision. This regulation is supported by the sophisticated arrangement of cyclases, phosphodiesterases, anchoring proteins, and receptors for cAMP. Discovery of these nuances to cAMP signaling has been facilitated by the development of genetically encodable tools for monitoring and manipulating cAMP and the proteins that support cAMP signaling. In this review, we discuss the state-of-the-art in development of different genetically encoded tools for sensing cAMP and the activity of its primary intracellular receptor protein kinase A (PKA). We introduce sequences for encoding adenylyl cyclases that enable cAMP levels to be artificially elevated within cells. We chart the evolution of sequences for selectively modifying protein-protein interactions that support cAMP signaling, and for driving cAMP sensors and manipulators to different subcellular locations. Importantly, these different genetically encoded tools can be applied synergistically, and we highlight notable instances that take advantage of this property. Finally, we consider prospects for extending the utility of the tool set to support further insights into the role of cAMP in health and disease.

No MeSH data available.


Related in: MedlinePlus

Tools for monitoring cyclic AMP (cAMP) signaling. Two cAMP sensors and an A-kinase activity reporter are illustrated. (A) Cartoon showing mechanism for cAMP sensing by ‘Ci/C-Epac2-camps,’ which is based on the second cAMP binding domain of Epac2. Elevation of cAMP leads to separation of citrine and cerulean fluorescent proteins that can be detected as a decrease in FRET (B) cAMP sensor based on cyclic nucleotide-gated channel 2 (CNG2), which is modified with removal of residues 61–90 to prevent Ca2+/calmodulin regulation, and the substitutions C460W/E583M for high affinity and specificity for cAMP. Elevation of cAMP leads to increased flux of Ca2+ which can be detected using Ca2+ dyes or by measuring current across the membrane. (C) AKAR4 contains a central FHA1 domain. PKA phosphorylation of threonine within the motif ‘LRRATLVD’ leads to association of phosphothreonine with FHA1, which can be detected as an increase in FRET between the terminal fluorescent proteins.
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Figure 1: Tools for monitoring cyclic AMP (cAMP) signaling. Two cAMP sensors and an A-kinase activity reporter are illustrated. (A) Cartoon showing mechanism for cAMP sensing by ‘Ci/C-Epac2-camps,’ which is based on the second cAMP binding domain of Epac2. Elevation of cAMP leads to separation of citrine and cerulean fluorescent proteins that can be detected as a decrease in FRET (B) cAMP sensor based on cyclic nucleotide-gated channel 2 (CNG2), which is modified with removal of residues 61–90 to prevent Ca2+/calmodulin regulation, and the substitutions C460W/E583M for high affinity and specificity for cAMP. Elevation of cAMP leads to increased flux of Ca2+ which can be detected using Ca2+ dyes or by measuring current across the membrane. (C) AKAR4 contains a central FHA1 domain. PKA phosphorylation of threonine within the motif ‘LRRATLVD’ leads to association of phosphothreonine with FHA1, which can be detected as an increase in FRET between the terminal fluorescent proteins.

Mentions: The discovery of Epac1 and Epac2 (de Rooij et al., 1998; Kawasaki et al., 1998) opened the door for development of unimolecular cAMP sensors based upon these cAMP-dependent GTPase activators (DiPilato et al., 2004; Nikolaev et al., 2004; Ponsioen et al., 2004). Unimolecular sensors based on Epacs exhibit higher FRET efficiency (increase in FRET signal from maximal to minimal cAMP) of ∼20–30% compared to ∼8% for multimolecular PKA-based sensors. Epac probes also show better temporal resolution (Nikolaev et al., 2004), and there are no concerns regarding balancing expression of the donor and acceptor fluorophores in unimolecular probes. For these reasons, Epac probes are now the most popular option for sensing cAMP fluctuations. The latest optimized Epac-based probes include pH-insensitive Ci/Ce Epac2-camps (Everett and Cooper, 2013) (Figure 1A) and the Epac1-based probes ICUE3 (DiPilato and Zhang, 2009) and TEpacVV (Klarenbeek et al., 2011; Li et al., 2015) although the original probes exhibit good FRET efficiency and are still popular. CNGC-based cAMP sensors excel in temporal resolution of cAMP fluctuations (Rich et al., 2000; Fagan et al., 2001). The first example of this approach exploited rat CNG2 expression in human embryonic kidney-293 (HEK-293) cells (Rich et al., 2000). Measurement of Ca2+ flux through the channels as a proxy for cAMP elevation using electrophysiology supported the existence of cellular cAMP microdomains. Mutations can be incorporated into the CNG2 to tailor it for sensing cAMP: C460W improves cAMP sensitivity, E583M improves cAMP specificity over cGMP, and removal of residues 61-90 abrogates channel regulation by Ca2+/calmodulin (Rich et al., 2001) (Figure 1B). A C460W/E583M double CNG2 mutant was used as a sensor to reveal that both G protein coupled receptor kinases (GRKs) and PKA stimulate PDE degradation of cAMP following β2-AR simulation of HEK-293 cells (Xin et al., 2008). A common way to apply CNG2 is to combine expression of the channel with the Ca2+ dye Fura-2, allowing measurement of Ca2+ influx by imaging rather than electrophysiology (Fagan et al., 2001; Rich et al., 2001, 2007; Rochais et al., 2004). For example, the E583M CNG2 variant was applied in this way to establish the necessity of PKA anchoring for negative feedback through PKA activation of type IV PDEs (Willoughby et al., 2006). CNGCs can also be adapted as FRET sensors (Nikolaev et al., 2006). The hyperpolarization-activated CNG2 (HCN2) has a higher sensitivity than CNG2, and exhibits as wide a dynamic range as a cAMP FRET sensor when YFP and CFP are fused either side of a single HCN2 cAMP binding domain (Nikolaev et al., 2006).


The genetically encoded tool set for investigating cAMP: more than the sum of its parts.

Patel N, Gold MG - Front Pharmacol (2015)

Tools for monitoring cyclic AMP (cAMP) signaling. Two cAMP sensors and an A-kinase activity reporter are illustrated. (A) Cartoon showing mechanism for cAMP sensing by ‘Ci/C-Epac2-camps,’ which is based on the second cAMP binding domain of Epac2. Elevation of cAMP leads to separation of citrine and cerulean fluorescent proteins that can be detected as a decrease in FRET (B) cAMP sensor based on cyclic nucleotide-gated channel 2 (CNG2), which is modified with removal of residues 61–90 to prevent Ca2+/calmodulin regulation, and the substitutions C460W/E583M for high affinity and specificity for cAMP. Elevation of cAMP leads to increased flux of Ca2+ which can be detected using Ca2+ dyes or by measuring current across the membrane. (C) AKAR4 contains a central FHA1 domain. PKA phosphorylation of threonine within the motif ‘LRRATLVD’ leads to association of phosphothreonine with FHA1, which can be detected as an increase in FRET between the terminal fluorescent proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526808&req=5

Figure 1: Tools for monitoring cyclic AMP (cAMP) signaling. Two cAMP sensors and an A-kinase activity reporter are illustrated. (A) Cartoon showing mechanism for cAMP sensing by ‘Ci/C-Epac2-camps,’ which is based on the second cAMP binding domain of Epac2. Elevation of cAMP leads to separation of citrine and cerulean fluorescent proteins that can be detected as a decrease in FRET (B) cAMP sensor based on cyclic nucleotide-gated channel 2 (CNG2), which is modified with removal of residues 61–90 to prevent Ca2+/calmodulin regulation, and the substitutions C460W/E583M for high affinity and specificity for cAMP. Elevation of cAMP leads to increased flux of Ca2+ which can be detected using Ca2+ dyes or by measuring current across the membrane. (C) AKAR4 contains a central FHA1 domain. PKA phosphorylation of threonine within the motif ‘LRRATLVD’ leads to association of phosphothreonine with FHA1, which can be detected as an increase in FRET between the terminal fluorescent proteins.
Mentions: The discovery of Epac1 and Epac2 (de Rooij et al., 1998; Kawasaki et al., 1998) opened the door for development of unimolecular cAMP sensors based upon these cAMP-dependent GTPase activators (DiPilato et al., 2004; Nikolaev et al., 2004; Ponsioen et al., 2004). Unimolecular sensors based on Epacs exhibit higher FRET efficiency (increase in FRET signal from maximal to minimal cAMP) of ∼20–30% compared to ∼8% for multimolecular PKA-based sensors. Epac probes also show better temporal resolution (Nikolaev et al., 2004), and there are no concerns regarding balancing expression of the donor and acceptor fluorophores in unimolecular probes. For these reasons, Epac probes are now the most popular option for sensing cAMP fluctuations. The latest optimized Epac-based probes include pH-insensitive Ci/Ce Epac2-camps (Everett and Cooper, 2013) (Figure 1A) and the Epac1-based probes ICUE3 (DiPilato and Zhang, 2009) and TEpacVV (Klarenbeek et al., 2011; Li et al., 2015) although the original probes exhibit good FRET efficiency and are still popular. CNGC-based cAMP sensors excel in temporal resolution of cAMP fluctuations (Rich et al., 2000; Fagan et al., 2001). The first example of this approach exploited rat CNG2 expression in human embryonic kidney-293 (HEK-293) cells (Rich et al., 2000). Measurement of Ca2+ flux through the channels as a proxy for cAMP elevation using electrophysiology supported the existence of cellular cAMP microdomains. Mutations can be incorporated into the CNG2 to tailor it for sensing cAMP: C460W improves cAMP sensitivity, E583M improves cAMP specificity over cGMP, and removal of residues 61-90 abrogates channel regulation by Ca2+/calmodulin (Rich et al., 2001) (Figure 1B). A C460W/E583M double CNG2 mutant was used as a sensor to reveal that both G protein coupled receptor kinases (GRKs) and PKA stimulate PDE degradation of cAMP following β2-AR simulation of HEK-293 cells (Xin et al., 2008). A common way to apply CNG2 is to combine expression of the channel with the Ca2+ dye Fura-2, allowing measurement of Ca2+ influx by imaging rather than electrophysiology (Fagan et al., 2001; Rich et al., 2001, 2007; Rochais et al., 2004). For example, the E583M CNG2 variant was applied in this way to establish the necessity of PKA anchoring for negative feedback through PKA activation of type IV PDEs (Willoughby et al., 2006). CNGCs can also be adapted as FRET sensors (Nikolaev et al., 2006). The hyperpolarization-activated CNG2 (HCN2) has a higher sensitivity than CNG2, and exhibits as wide a dynamic range as a cAMP FRET sensor when YFP and CFP are fused either side of a single HCN2 cAMP binding domain (Nikolaev et al., 2006).

Bottom Line: We chart the evolution of sequences for selectively modifying protein-protein interactions that support cAMP signaling, and for driving cAMP sensors and manipulators to different subcellular locations.Importantly, these different genetically encoded tools can be applied synergistically, and we highlight notable instances that take advantage of this property.Finally, we consider prospects for extending the utility of the tool set to support further insights into the role of cAMP in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London London, UK.

ABSTRACT
Intracellular fluctuations of the second messenger cyclic AMP (cAMP) are regulated with spatial and temporal precision. This regulation is supported by the sophisticated arrangement of cyclases, phosphodiesterases, anchoring proteins, and receptors for cAMP. Discovery of these nuances to cAMP signaling has been facilitated by the development of genetically encodable tools for monitoring and manipulating cAMP and the proteins that support cAMP signaling. In this review, we discuss the state-of-the-art in development of different genetically encoded tools for sensing cAMP and the activity of its primary intracellular receptor protein kinase A (PKA). We introduce sequences for encoding adenylyl cyclases that enable cAMP levels to be artificially elevated within cells. We chart the evolution of sequences for selectively modifying protein-protein interactions that support cAMP signaling, and for driving cAMP sensors and manipulators to different subcellular locations. Importantly, these different genetically encoded tools can be applied synergistically, and we highlight notable instances that take advantage of this property. Finally, we consider prospects for extending the utility of the tool set to support further insights into the role of cAMP in health and disease.

No MeSH data available.


Related in: MedlinePlus