A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells

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.

Detecting and measuring of GPCR signaling – comparison of human induced pluripotent stem cells and immortal cell lines

“…Lentiviral vectors are advantageous because they can infect both dividing and non-dividing cells, have a large gene fragment capacity, and generate long expression times for target genes. For example, lentiviral vectors were used to detect cGMP activity as one of the most important downstream signaling components for various cell types ( 130 ). Adeno-associated virus (AAV)-mediated transfection is considered safe and tissue-specific, making it ideal for transfecting primary cells from a wide range of cell types, as exemplified by the successful lacZ reporter gene transfection of primary cardiomyocytes (CMs) using AAV5 ( 131 ).…”

“…HEK293 cells are particularly attractive for GPCR research as they naturally express 75 different GPCRs together with many G protein signaling regulators (i.e., β-arrestin1 and 2, and GRK3-5). In addition, HEK293 cells are easy to transfect by either viral ( 130 ) or non-viral methods ( 198 ) and possess an almost complete set of G protein subunits constituting an attractive environment for overexpressing additional GPCRs of interest ( 199 ). HEK293 cells have been utilized to answer a multitude of different questions in the past: For example, the Gα q/11 -knockout HEK293 cells were used to validate dimerization and signal crosstalk of EP2 and OTR ( 4 ) while the contributions of endogenous Gα q/11 , Gαs, Gα12/13, and Gβγ proteins to histamine receptor signaling has been studied utilizing unlabeled DMR detection technique ( 200 ).…”