Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins

Anal Chem. 2000 Dec 15;72(24):5918-24. doi: 10.1021/ac0006167.

Abstract

We describe herein fluorescent indicators for cyclic GMP (cGMP) in single living cells. cGMP-dependent protein kinase Ialpha (PKG Ialpha), a receptor for cGMP, was fused with blue- and red-shifted green fluorescent proteins (GFPs) to its N- and C-termini, respectively. Using PKG lalpha delta1-47, in which the dimerization domain was deleted, fluorescence resonance energy transfer between the GFPs was found to increase upon cGMP-induced conformational change in PKG Ialpha delta1-47. We demonstrated that thus-developed fluorescent indicators reversibly responded to cGMP that was produced in nitric oxide-stimulated HEK293 cells. The present genetically encoded fluorescent indicators open a way not only for understanding the dynamics of cGMP signaling in single cells and organisms but also for discovering pharmaceuticals such as isoform-specific inhibitors for phosphodiesterases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line
  • Cricetinae
  • Cyclic GMP / analysis*
  • Cyclic GMP-Dependent Protein Kinases / chemistry*
  • Fluorescent Dyes / chemistry*
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / chemistry

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Cyclic GMP-Dependent Protein Kinases
  • Cyclic GMP