Cyclic 3',5'-adenosine monophosphate (cAMP), the classical second messenger, regulates many diverse cellular functions. Although much has been learned about cAMP and cAMP-dependent protein kinase (PKA), there are still large gaps in our understanding of the spatial and temporal nature of cAMP signals and the molecular mechanisms that specifically couple cAMP and its effectors, including PKA and recently discovered exchange proteins directly activated by cAMP (Epac). The overall goal of our research is to elucidate the mechanisms and functional significance of cAMP compartmentation in achieving high specificity in cAMP signaling.
The specific aims are: 1) To further develop genetically encoded cAMP indicators and characterize the distinct cellular pools of cAMP generated by transmembrane and soluble adenylyl cyclases (AC). This proposed aim builds on preliminary data obtained with a fluorescent cAMP indicator that was recently developed in this laboratory and tests the involvement of AC and phosphodiesterase in establishing distinct pools of cAMP. A new generation of indicators will also be engineered. 2) To identify the functional effectors of mitochondrial cAMP. Imaging with the fluorescent cAMP indicator revealed rapid accumulation of cAMP in mitochondria following activation of beta adrenergic receptor, the functional role of which remains unknown. Epac will be tested as the functional effector of mitochondrial cAMP using fluorescent imaging, protein engineering, chemical biology and biochemical techniques. 3) To analyze the linkage between beta adrenergic receptor (beta-AR) and PKA and the effect of insulin on this linkage. Live-cell fluorescence imaging, biochemical assays, and pharmacological manipulation will be used to test the hypothesis that the linkage between beta-AR and PKA is disrupted by chronic insulin pretreatment. The studies proposed here should lead to a greater understanding of the molecular mechanisms that compartmentalize the production, degradation, and functional coupling of cAMP to the effectors. Impaired cAMP signaling has widespread implications for clinical conditions such as obesity and type II diabetes mellitus, particularly as it relates to normal adipocyte metabolism. A mechanistic understanding of cAMP signaling specificity is crucial to developing therapeutic strategies for these clinical conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK073368-04S2
Application #
7996294
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Silva, Corinne M
Project Start
2010-01-15
Project End
2010-12-31
Budget Start
2010-01-15
Budget End
2010-12-31
Support Year
4
Fiscal Year
2010
Total Cost
$100,000
Indirect Cost
Name
Johns Hopkins University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Ni, Qiang; Mehta, Sohum; Zhang, Jin (2018) Live-cell imaging of cell signaling using genetically encoded fluorescent reporters. FEBS J 285:203-219
Mehta, Sohum; Zhang, Yong; Roth, Richard H et al. (2018) Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. Nat Cell Biol 20:1215-1225
Hertel, Fabian; Mo, Gary C H; Dedecker, Peter et al. (2018) Observing the Assembly of Protein Complexes in Living Eukaryotic Cells in Super-Resolution Using refSOFI. Methods Mol Biol 1764:267-277
Ross, Brian L; Tenner, Brian; Markwardt, Michele L et al. (2018) Single-color, ratiometric biosensors for detecting signaling activities in live cells. Elife 7:
Rodriguez, Erik A; Campbell, Robert E; Lin, John Y et al. (2017) The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci 42:111-129
Mehta, Sohum; Zhang, Jin (2017) Illuminating the Cell's Biochemical Activity Architecture. Biochemistry 56:5210-5213
Gorshkov, Kirill; Mehta, Sohum; Ramamurthy, Santosh et al. (2017) AKAP-mediated feedback control of cAMP gradients in developing hippocampal neurons. Nat Chem Biol 13:425-431
Mo, Gary C H; Ross, Brian; Hertel, Fabian et al. (2017) Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat Methods 14:427-434
Nygren, Patrick J; Mehta, Sohum; Schweppe, Devin K et al. (2017) Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity. Elife 6:
Newman, Robert H; Zhang, Jin (2017) Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks. Methods Enzymol 589:133-170

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