Perspectives on: Cyclic nucleotide microdomains and signaling specificity

The purpose of the Perspectives in General Physiology is to provide a forum where scientific uncertainties or controversies, or important problems, are discussed in an authoritative, yet open manner. The Perspectives are solicited by the editors—often based on recommendations by members of the

The purpose of the Perspectives in General Physiology is to provide a forum where scientific uncertainties or controversies, or important problems, are discussed in an authoritative, yet open manner. The Perspectives are solicited by the editors-often based on recommenda tions by members of the editorial advisory board. To frame the issue, two or more experts are invited to pre sent brief points of view on the problem; these are pub lished consecutively in the Journal. One or more experts and the organizer review the contributions, but the comments and opinions expressed in the Perspectives are those of the authors and not necessarily those of the editors or the editorial advisory board. The Perspectives are accompanied by a few editorial paragraphs that introduce the problem and invite the submission of com ments, in the form of letters to the editor, which usually are published four months after publication of the Per spectives. After the letters to the editor have been pub lished, further responses are limited to full manuscripts.
The prototypical second messenger, cAMP, was dis covered in 1957 (Sutherland, 1962), and many land mark discoveries since then have given us a basic biochemical description of cellular signaling events that are more widespread and more amplified in terms of their effects than membranedelimited events such as synaptic transmission (Beavo and Brunton, 2002;Conti and Beavo, 2007;Willoughby and Cooper, 2007). We have a wealth of information about the identities, struc tures, and functions of the different proteins involved in this signaling pathway. Yet, if one delves deeper than what the average undergraduate biology major takes for granted about this classic signaling pathway, we still lack answers to two related questions that an engineer would consider to be fundamental to the description of a communication device: (1) How is signaling specificity achieved? For cyclic nucleotides and other second mes sengers, there is much to be learned about how infor mation is relayed from a very large number of extracellular receptors for hormones, neurotransmitters, odorants, cytokines, etc., through a very small number of intracel lular signaling molecules; and (2) How is the signaling compartmentalized? The morphological and biochemical Correspondence to Jeffrey W. Karpen: j e f f r e y . k a r p e n @ c n u c o m . o r g Abbreviation used in this paper: PDE, phosphodiesterase.
basis for cellular microdomains that spatially segregate cyclic nucleotide signals remains largely a mystery. The concept of compartmentation emerged more than 30 years ago in studies of cardiac myocytes to help explain how a variety of extracellular stimuli that pri marily act through cAMP can have very different down stream effects on the cell (Corbin et al., 1977;Brunton et al., 1981;Steinberg and Brunton, 2001). For example, isoproterenol, a adrenergic agonist, triggers cAMP dependent activation of PKA and subsequent phos phorylation of proteins associated with excitability and the strength and timing of heart muscle contraction. Prostaglandins cause similar changes in total cellular cAMP and PKA activity but no changes in contractility. Glucagonlike peptide, also working through PKA, reg ulates metabolism in heart muscle cells (Bers and Ziolo, 2001;Vila Petroff et al., 2001). Simple diffusion theory predicts that in the absence of any interference, cAMP will traverse the cytoplasm of a 20µM cell in 0.2 s, and no appreciable accumulation of cAMP builds up around a single adenylyl cyclase molecule because it is a very slow enzyme (Rich et al., 2000). This is one way in which cAMP is very different from Ca 2+ , which can accumulate to high concentrations at the mouth of a Ca 2+ channel because of the high throughput rate. In essence, each cAMP diffuses away faster than the next one is pro duced. Thus, to explain the above observations, either there must be subcellular physical barriers that restrict the diffusion of cAMP, or the local variations in cAMP concentrations result from very high synthesis and deg radation rates.
In the past 15 years, progress in four areas has pro vided more direct evidence for cAMP compartmenta tion and information on some of the key molecular pro cesses that underlie this phenomenon: (1) the under standing of A kinase-anchoring proteins as molecular scaf folds that colocalize and organize signaling proteins has increased markedly (Jarnaess and Taskén, 2007;Dodge Kafka et al., 2008;Welch et al., 2010); (2) a series of intracellular sensors for cAMP and cGMP were devel oped that have allowed cyclic nucleotide signals to be of the cell); the resistance of nearmembrane cAMP sig nals to cellular washout in whole cell patchclamp ex periments; and new data showing that total stimulated PDE activity in neonatal cardiac myocytes is 100fold lower than the levels required to generate cAMP gradi ents in the absence of physical barriers. Saucerman et al. summarize the progress that has been made with com putational models of cAMP signaling over the last 13 years and reinforce the view that a combination of phys ical barriers and regulated PDE activity within the re gions of restricted diffusion create cAMP microdomains and shape cAMP signals. Several different mechanisms can cause diffusion restrictions, including buffering, or ganelles and intracellular membranes, cytoskeleton, local properties of cytosol, and cell shape. A major challenge for the future will be to assess in a quantitative way how these different mechanisms contribute to cyclic nucleo tide microdomains.
Letters to the editor related to these Perspectives should be received no later than Monday, March 3, 2014. The letters may be no longer than two printed pages (approximately six doublespaced pages) and will be subject to editorial review. They may contain no more than one figure, no more than 15 references, and no significant references to unpublished work. Letters should be prepared according to The Journal's Instructions and can be submitted electronically at http://www.jgp.org.
Olaf S. Andersen served as editor. 2012. Assessment of cellular mechanisms contributing to cAMP measured with better spatial and temporal precision (Rich et al., 2001;Nikolaev et al., 2004;Ponsioen et al., 2004;Nausch et al., 2008;Willoughby and Cooper, 2008); (3) the extensive study of the biochemical prop erties and potential modes of regulation of phosphodi esterases (PDEs) has led to an increased appreciation of the critical role PDEs play in shaping and controlling cAMP signals (Conti and Beavo, 2007;Baillie, 2009;Blackman et al., 2011); and (4) computational model ing of cellular compartments has ruled out several hy potheses and led to a greater understanding of possible mechanisms (Rich et al., 2000(Rich et al., , 2001Saucerman et al., 2006;Iancu et al., 2007;Oliveira et al., 2010;Feinstein et al., 2012;Sample et al., 2012). Despite these advances, however, our overall understanding of cAMP compart ments, or microdomains, remains fairly crude. It has seemed to some that we should be further along given the promise of the sensors and the wealth of molecular information that has been accumulated. The four Perspectives in this issue summarize recent developments and provide guidance for future research in each of the four areas described above. Countering the sentiment expressed above, these articles provide reasons to be optimistic that current technical barriers can be overcome and that longstanding controversies can be resolved. Kapiloff et al. take the available mo lecular information about A kinase-anchoring proteins and provide a conceptual framework for how these scaf fold proteins likely participate in feedback inhibition of cAMP levels and the shaping of cAMP signals in micro domains. Rich et al. critically evaluate the advantages and disadvantages of the available singlecell sensors for cAMP and cGMP, and they point out the pitfalls that can confound the interpretation of imaging data, such as overexpression and sensitivity to environmental vari ables like pH. They go on to describe recent improve ments in fluorescent sensors in terms of dynamic range and the highly promising developments in hyperspec tral imaging and automated analysis. These latter ap proaches should allow for improved signaltonoise ratio of fluorescence and FRET measurements, the simulta neous use of multiple probes, measurements in tissue preparations, and unbiased data analysis. All of the techniques are evaluated in terms of their ability to de tect a range of predicted cyclic nucleotide signals, which should make this a particularly useful guide for both future probe development and experiments. Conti et al. focus on the question of whether PDE activity is suffi cient to generate cAMP microdomains in the absence of physical diffusion barriers. They present several lines of evidence against this simple notion, including the highly isoformspecific regulation of PDEs by receptor stimulation and other intracellular signaling events; the absence of spreading of certain signals when PDEs are inhibited (PDE inhibition usually, but not always, allows for the eventual spread of cAMP signals to other parts