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Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation

Part of the Methods in Molecular Biology book series (MIMB,volume 2173)

Abstract

cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatiotemporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with nanoluciferase (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.

Key words

  • cAMP
  • Adenylyl cyclase
  • Optogenetics
  • bPAC
  • Nanoluciferase

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Acknowledgments

This research was supported by National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH), and the Molecular Pharmacology Training Program of the University of Pittsburgh under grant Awards Number R01-GM09975, R01-GM130612, T32-GM00842419/20/21, and the Wistar Morris’s Cotswold Foundation Fellowship.

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Naim, N., Reece, J.M., Zhang, X., Altschuler, D.L. (2020). Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_14

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