Cyclic AMP (cAMP) Signaling

The cyclic adenosine 3'/ 5'-monophosphate (cAMP) signal pathway, is a G-protein mediate cascade of biochemical reactions associated with various hormone specificities and neurotransmitters. cAMP is a cyclic, second messenger, nucleotide that interacts directly with a handful of main targets; protein kinase A (PKA), cyclic nucleotide-gated ion channels (CNGCs), and associated exchange proteins (EPAC). These pieces are linked to a number of vital downstream signal pathways, including metabolism, calcium handling, gene expression, vascular ability, cell proliferation and apoptosis. 

Due to cAMPs broad modulatory ranges, including pro- and anti-inflammatory immune response, it's become apparent that this pathway has massive involvement in physiology. cAMP-regulated enzymes have the potential to be used as targets for immunotherapies for autoimmune and inflammatory diseases. Advancements in optical imaging techniques have allowed for clearer interpretation of the intricacies of cAMP pathway dynamics, however there is still a lot to learn. The cAMP pathway is far from linear, and understanding cellular sub compartments continue to show how cAMP is not only associated with local biochemical reactions, but also with distal events in the human microbiome.

cAMP Regulation

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Initiation by ACs

The cAMP signal pathway starts with the downstream activation of Adenylyl cyclase (AC) by the G-⍺ subunit on G-protein-coupled receptors (GPCRs). After activation by GPCRs in the plasma membrane, AC effectively converts adenosine triphosphate ATP into cAMP. Numerous cAMP molecules can be generated in the cytosol, thereby intensifying the outward signal onto target sites. In mammalian cells there are at least 10 known AC isoforms, each with similar architecture, but also with variable enzymatic ability and modulatory domains. Most however are related to Calcium (Ca) channel activity.

Application Notes:

Controlling fertilization and cAMP signaling in sperm by optogenetics

Termination by PDEs

Phosphodiesters (PDEs) terminate cAMP molecules by compartmentalization and degradation and are critical to the temporal and spatial regulatory dynamics of cAMP signaling. PDEs over the years have shown remarkable complexity, and over 100 different isoforms have been identified to date. Enzymatic differences arise due to the at least 21 different associated encoding genes, alternative splice sites and various transcription initiating points. Though structure may be similar, isotypes are functionally unique.

 

cAMP Targets and Subsequent effects

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Protein kinase A (PKA)

After cAMP binds to regulation sites on PKA, it may dissociate into 7 different regulatory and catalytic subunits that initiates an ensuing signal cascade by phosphorylation. Downstream effectors of PKA have been linked, including cardiomyocytes, endothelial cells, adipocytes, neurons and more. PKA phosphorylation also influences transcription factors in target genes, nuclear receptors and thyroid stimulating hormones. In the looping nature of the cAMP pathway, PKA as well interacts with EPACs and ion channels in the plasma membrane, further stimulating the flow of calcium, sodium, and potassium.

Datasets:

cAMP-dependent protein kinase (PKA) Inhibitors (IC50, Ki)

Exchange Proteins (EPAC) and GTPases

EPACs are guanine nucleotide exchange factors (GEF) that activate certain GTPases, Rap 1 and 2. These small G-proteins are involved in an array of cellular activities, including adhesion, exocytosis, macrophage rearrangement, cytoskeletal kinetics and restructuring. EPAC 1, after cAMP activation, tethers to the plasma membrane, while EPAC 2 interacts directly with activated G-proteins, namely Ras. EPACs have been shown to play a role in cell migration and proliferation in some types of cancer, making them a potentially ideal target for oncological therapies.

Application Notes:

Stiffness-Activated GEF-H1 Expression Exacerbates LPS-Induced Lung Inflammation

Cyclic nucleotide-gated ion channels (CNGCs)

CNGCs are directly influenced by the activity of cyclic nucleotides as nonspecific cation channels. Increased concentrations of cAMP (and cGMP) upregulates activity linked to the Ca signaling pathway, which stimulates calmodulin (CAM) and associated proteins, which reciprocally regulate the activity of AC and PDE. CGNCs function is crucial to maintaining cell homeostasis through regulation of de- and hyperpolarized events. CGNC activity has also shown significance in kidney function, olfactory and photoreceptor response.

Application Notes:

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma

cAMP and Pathophysiology

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Due to the broad range of function and association, cAMP concentration has many effects in regard to immunity in the body. It has been shown that intracellular cAMP levels have negative effects on production of monocytes and granulocytes, thereby decreasing inflammatory effects.

• In natural killer (NK) cells, a cell that suppresses tumor cell growth, cAMP levels regulate cytotoxicity.
• In endritic cells (DCs), cAMP suppresses and promotes pro- and anti-inflammatory mediators, respectively, to regulate T cell immunity.
• In B cells, cAMP stimulates antibody production.

Knowledge and understanding of the reach of cAMP in other signaling pathways can be used as therapeutic intervention for a range of autoimmune and inflammatory diseases. Though still in early stages of developments, AC-specific compounds like colforsin daropate hydrochloride, have been created with the goal of raising cAMP concentration to help fight associated cognitive, neurological and cardiac disorders.

Measuring cAMP

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Fluorescence resonance energy transfer (FRET)

FRET technology optically exploits the inner workings and complexities of the cAMP signaling pathway. The biosensors used contain a cAMP binding domain, PKA or an EPAC, sandwiched in between two fluorescent proteins. Even traditional optical microscopes provide the ability to visualize cAMP in real time, quantify concentration levels, and view interactions between GPCRs. Advancing FRET technologies have genetically modified biosensors to target cAMP at specific subunits, thereby providing an even more intuitive map of the signal process. FRET assays can provide readouts of fluctuating cAMP and Ca levels over time, and has recently been developed for high-throughput capabilities.

Enzyme linked immuno-assay (ELISA)

ELISA is based on the competition between labeled and unlabeled cAMP antibodies and can be used to detect AC activity within the cell. After a detector antibody has bound to the target protein, non-target substances are washed away. Using a microplate reader, cAMP activity can be monitored via absorbance. Several sample types, including blood, saliva or tissue, can also be utilized by ELISA and kits and protocols vary based on desired measurements.

 

Product Ordering Information

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References

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1. “Camp Signaling Pathway.” Cusabio Life Science-Your Biology Science Partner, https://www.cusabio.com/pathway/cAMP-signaling-pathway-2.html.
2. Zaccolo, Manuela, et al. “Subcellular Organization of the Camp Signaling Pathway.” Pharmacological Reviews, vol. 73, no. 1, 2020, pp. 278-309., https://doi.org/10.1124/pharmrev.120.000086.
3. Sassone-Corsi, P. “The Cyclic AMP Pathway.” Cold Spring Harbor Perspectives in Biology, vol. 4, no. 12, 2012, https://doi.org/10.1101/cshperspect.a011148.
4. Epstein, Paul M. “Different Phosphodiesterases (Pdes) Regulate Distinct Phosphoproteomes during Camp Signaling.” Proceedings of the National Academy of Sciences, vol. 114, no. 30, 2017, pp. 7741-7743., https://doi.org/10.1073/pnas.1709073114.
5. Raker, Verena Katharina, et al. “The Camp Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases.” Frontiers in Immunology, vol. 7, 2016, https://doi.org/10.3389/fimmu.2016.00123.
6. Milliken, Brandon, et al. “FRET Reporter Assays for Camp and Calcium in a 96-Well Format Using Genetically Encoded Biosensors Expressed in Living Cells.” BIO-PROTOCOL, vol. 10, no. 11, 5 June 2020, https://doi.org/10.21769/bioprotoc.3641.