Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.
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.
Sato
M
M
Department of Chemistry, School of Science, The  University of Tokyo, Japan.
Hida
N
N
Ozawa
T
T
Umezawa
Y
Y
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006152:Cyclic GMP / Q000032:analysis*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP