Novel single chain cAMP sensors for receptor-induced signal propagation.
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 between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) 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 us to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed ( approximately 40 microm/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.
Nikolaev
Viacheslav O
VO
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, D-97078 Würzburg, Germany.
Bünemann
Moritz
M
Hein
Lutz
L
Hannawacker
Annette
A
Lohse
Martin J
MJ
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D015290:Second Messenger Systems; D015398:Signal Transduction
D000242:Cyclic AMP