Abstract
The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]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 [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]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 Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin 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 [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.
Highlights
Mutations in human melanocortin-4 receptor (MC4R) (hMC4R) are the single most common gene mutation associated with human obesity (Farooqi et al 2000, Vaisse et al 2000)
Because α-MSHinduced [Ca2+]i in HEK293 cells is not associated with an increase in inositol triphosphate but is significantly attenuated by cholera toxin and dependent on Gαs (Mountjoy et al 2001), the signalling pathway monitored by nuclear factor of activated T cell (NFAT) luciferase reporter in HEK293 cells may or may not involve α-melanocytestimulating hormone (α-MSH)-induced [Ca2+]i
Fura-2/AM loading into HEK293 cells was compared between buffers, Dulbecco’s modified Eagle medium (DMEM) and calcium loading bufferhigh glucose (CLB-HG)
Summary
Mutations in hMC4R are the single most common gene mutation associated with human obesity (Farooqi et al 2000, Vaisse et al 2000). Using microscopy analysis, Class V obesity-associated hMC4R variants (i.e. those that are obesity associated but appear similar to WT on cAMP assays and protein expression studies) were shown to mobilise [Ca2+]i with similar efficacy to WT hMC4R (Sharma et al 2020). Application of this method is limited as it is neither of high throughput nor quantitative. A high-throughput nuclear factor of activated T cell (NFAT) luciferase reporter gene assay was recently used to show that Class V obesityassociated hMC4R variants exhibit impaired activation of phospholipase C-β (PLC) compared with WT hMC4R (Clement et al 2018). While the intracellular signalling pathway responsible for α-MSH-induced [Ca2+]i in HEK293 cells remains unknown, there is a need to develop a highthroughput assay to measure MC4R-induced mobilisation of [Ca2+]i in vitro
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