Quantitative high-throughput assay to measure MC4R-induced intracellular calcium

The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca(2+)](i)). However, very little is known about the physiological significance of MC4R-induced [Ca(2+)](i) since few studies measure MC4R-induced [Ca(2+)](i). High-throughput, read-out assays for [Ca(2+)](i) have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca(2+)](i). Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca(2+)](i) and to maximise ligand-activated MC4R-induced [Ca(2+)](i). Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine R(min) for quantitation of [Ca(2+)](i). Therefore, we developed a method to determine R(min) in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca(2+)](i), for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca(2+)](i) in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.