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

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.