Novel single chain cAMP sensors for receptor-induced signal propagation

J Biol Chem. 2004 Sep 3;279(36):37215-8. doi: 10.1074/jbc.C400302200. Epub 2004 Jul 1.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cricetinae
  • Cyclic AMP / metabolism*
  • Fluorescence
  • Humans
  • Second Messenger Systems
  • Signal Transduction*

Substances

  • Cyclic AMP