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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 triggering cAMP elevation. (A) Illustration of Beggiatoa PAC (bPAC) demonstrating how light illumination of the BLUF domain is coupled to activation of AC activity. (B) A truncated version of soluble AC (sACt) responds to elevated bicarbonate by catalyzing cAMP synthesis.
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Figure 2: Tools for triggering cAMP elevation. (A) Illustration of Beggiatoa PAC (bPAC) demonstrating how light illumination of the BLUF domain is coupled to activation of AC activity. (B) A truncated version of soluble AC (sACt) responds to elevated bicarbonate by catalyzing cAMP synthesis.

Mentions: Smaller PACs have subsequently been discovered in species other than Euglena, including Beggiatoa PAC (bPAC; Ryu et al., 2010; Stierl et al., 2011) (Figure 2A). bPAC comprises 350 residues, which facilitates transgenic delivery in comparison to PACα (1019 residues). bPAC also exhibits better responsiveness to blue light than PACα, and cyclase activity decreases faster for bPAC upon return to the dark (Ryu et al., 2010; Stierl et al., 2011). A different class of PAC has been identified in Microcoleus (mPAC) that relies on a blue light-responsive light oxygen voltage domain coupled to an AC domain (Raffelberg et al., 2013). mPAC compares favorably to bPAC when expressed in vivo, although its responsiveness to blue light is worse in vitro, suggesting that a cellular co-factor may support its proper function (Raffelberg et al., 2013). Both bPAC and mPAC require lower light intensity for cyclase activation than PACα (Ryu et al., 2010; Stierl et al., 2011; Raffelberg et al., 2013), so are better suited for application in tissue samples where light penetration is a challenge. A noteworthy alternative to PACs is to employ a C-terminally truncated version of soluble AC (sACt) that can be activated by addition of bicarbonate (Sample et al., 2012) (Figure 2B). This approach is compatible with simultaneous monitoring of cAMP concentration and PKA activity with FRET-based probes (Sample et al., 2012; see Combinatorial Applications).


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

Patel N, Gold MG - Front Pharmacol (2015)

Tools for triggering cAMP elevation. (A) Illustration of Beggiatoa PAC (bPAC) demonstrating how light illumination of the BLUF domain is coupled to activation of AC activity. (B) A truncated version of soluble AC (sACt) responds to elevated bicarbonate by catalyzing cAMP synthesis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Tools for triggering cAMP elevation. (A) Illustration of Beggiatoa PAC (bPAC) demonstrating how light illumination of the BLUF domain is coupled to activation of AC activity. (B) A truncated version of soluble AC (sACt) responds to elevated bicarbonate by catalyzing cAMP synthesis.
Mentions: Smaller PACs have subsequently been discovered in species other than Euglena, including Beggiatoa PAC (bPAC; Ryu et al., 2010; Stierl et al., 2011) (Figure 2A). bPAC comprises 350 residues, which facilitates transgenic delivery in comparison to PACα (1019 residues). bPAC also exhibits better responsiveness to blue light than PACα, and cyclase activity decreases faster for bPAC upon return to the dark (Ryu et al., 2010; Stierl et al., 2011). A different class of PAC has been identified in Microcoleus (mPAC) that relies on a blue light-responsive light oxygen voltage domain coupled to an AC domain (Raffelberg et al., 2013). mPAC compares favorably to bPAC when expressed in vivo, although its responsiveness to blue light is worse in vitro, suggesting that a cellular co-factor may support its proper function (Raffelberg et al., 2013). Both bPAC and mPAC require lower light intensity for cyclase activation than PACα (Ryu et al., 2010; Stierl et al., 2011; Raffelberg et al., 2013), so are better suited for application in tissue samples where light penetration is a challenge. A noteworthy alternative to PACs is to employ a C-terminally truncated version of soluble AC (sACt) that can be activated by addition of bicarbonate (Sample et al., 2012) (Figure 2B). This approach is compatible with simultaneous monitoring of cAMP concentration and PKA activity with FRET-based probes (Sample et al., 2012; see Combinatorial Applications).

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