Novel single chain cAMP sensors for receptor-induced signal propagation

Nikolaev VO, Bunemann M, Hein L, Hannawacker A and Lohse MJ
Source: J Biol Chem
Publication Date: (2004)
Issue: 279(36): 37215-8
Research Area:
Cells used in publication:
Neuron, hippocampal, mouse
Species: mouse
Tissue Origin: brain
Cyclic adenosine monophosphate (cAMP) is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer (FRET) between variants of green fluorescent protein (ECFP and EYFP) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties and interactions with other proteins and thereby allow to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP-signals were observed to travel with high speed (40 m/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.