Abstract
Intracellular 3′-5′-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming—all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control—something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.
Highlights
The small molecule 3′-5′-cyclic adenosine monophosphate is an established second messenger, involved in signal transduction in most living organisms
We provide some guiding landmarks that would be of some assistance to the reader facing an uneasy task of selection of the most appropriate tool for cyclic adenosine monophosphate (cAMP) probing for a particular experiment involving living cells
Inability to register significant flow of cAMP molecules between the cells once cAMP breakdown machinery was fully active and not silenced with inhibitors [such as 3isobutyl-1-methylxanthine (IBMX) limits the resolution of the method, as the readout captured in the absence of PDE activity reflects severely distorted patterns of cAMP kinetics and intracellular distribution
Summary
The small molecule 3′-5′-cyclic adenosine monophosphate (cAMP) is an established second messenger, involved in signal transduction in most living organisms. Most of these tools are genetically encoded proteins, tailored for sensing and modulation of cAMP in living systems These engineered proteins provide excellent spatial resolution down to desired subcellular domains, can respond to genuine oscillations of cAMP levels in real time and are designed to uncover cAMP signaling partners, and as such have enabled a paradigm-shift in cyclic nucleotide research. Besides being genetically-encoded proteins and applicable to studies in living cells, the modern biosensors do not have much in common, as they strive to probe different aspects of cAMP signaling, are governed by distinct biological phenomena and rely on diverse biophysical techniques Considering this heterogeneity and in order to make this review more sound and cohesive, we decided to categorize the biosensors into two major groups: tools for direct measurement of cAMP and tools for indirect cAMP probing. Combinatorial use of these cutting-edge instruments with biosensors for cAMP creates the most powerful research platform, which opens new avenues for fruitful studies of the life cycle and signaling properties of this cyclic nucleotide
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