Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16513-8. doi: 10.1073/pnas.0405973101. Epub 2004 Nov 15.

Abstract

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cell Line
  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HeLa Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Molecular Sequence Data
  • PC12 Cells
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Second Messenger Systems
  • Spectrometry, Fluorescence
  • Subcellular Fractions / metabolism

Substances

  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Guanine Nucleotide Exchange Factors
  • Luminescent Proteins
  • Peptide Fragments
  • RAPGEF3 protein, human
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases