Abstract
Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.
Highlights
Cyclic guanosine monophosphate is a second messenger involved in the regulation of many physiological processes including cardiovascular homeostasis, smooth muscle tone, blood pressure, platelet aggregation, memory, learning, and sensory transduction [1,2,3]. cGMP is synthesised from guanosine triphosphate (GTP) by two forms of guanylate cyclase (GC): soluble GCs, which are cytosolic enzymes activated by nitric oxide (NO) and carbon monoxide [4]; and particulate GCs, which are membrane receptors activated by natriuretic peptides and some intestinal peptides [5]. cGMP signals via protein kinase G (PKG) and cyclic nucleotide-gated ion channels, and modulates the activity of some phosphodiesterases (PDEs) [6]. cGMP is degraded by PDE-mediated hydrolysis [6]
End point cGMP assays usually require the addition of PDE inhibitors to stop the breakdown of cGMP, leading to the accumulation and measurement of total cGMP, which can produce a type of “observational bias”, whereby the dynamics of cGMP are not detected, leading to an incomplete or misleading idea of the signalling patterns induced by different ligands [17]
Several fluorescence resonance energy transfer (FRET)-based biosensors have been engineered and used to detect cGMP in single cells, including CGY [20], cygnet [18], and cGi [23], which use truncated forms of PKG as the “sensor” element, and cGES-DE5, which uses the cGMP-binding domain from human PDE5A as the “sensor” [19]. cGES-DE5 was chosen as the basis to generate a bioluminescence resonance energy transfer (BRET)-based biosensor due to its superior temporal resolution and high selectivity for cGMP over cAMP, compared to other FRET-based sensors [19]
Summary
Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of many physiological processes including cardiovascular homeostasis, smooth muscle tone, blood pressure, platelet aggregation, memory, learning, and sensory transduction [1,2,3]. cGMP is synthesised from guanosine triphosphate (GTP) by two forms of guanylate cyclase (GC): soluble GCs (sGCs), which are cytosolic enzymes activated by nitric oxide (NO) and carbon monoxide [4]; and particulate GCs (pGCs), which are membrane receptors activated by natriuretic peptides and some intestinal peptides [5]. cGMP signals via protein kinase G (PKG) and cyclic nucleotide-gated ion channels, and modulates the activity of some phosphodiesterases (PDEs) [6]. cGMP is degraded by PDE-mediated hydrolysis [6]. CGMP is usually measured in populations of cells in a plate-based format using traditional end point signalling assays that involve the detection of cGMP in cell lysates. Such assays may be sensitive, they are laborious, as cells must be lysed at individual time points. We report here the conversion of the cGES-DE5 FRET-based cGMP biosensor [19] to a BRET-based biosensor (cyclic GMP sensor using YFP-PDE5-Rluc; CYGYEL), cloning of the novel biosensor into a lentiviral vector for stable expression in mammalian cells, validation and characterisation of the biosensor in live cells in a real-time multi-well plate-based format, and investigation of the applicability of the sensor for the detection of sGC- and pGC-mediated cGMP activity in human primary vascular cells
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