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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 R_min for quantitation of [Ca²⁺]_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 [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.

The human melanocortin-4 receptor plays a critical role regulating energy homeostasis. Studies on obesogenic hMC4R variants have not yet revealed how human melanocortin-4 receptor maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q human melanocortin-4 receptor variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase, cyclic adenosine monophosphate response element-driven transcription, and calcium mobilization, but not phosphorylated extracellular signal-regulated kinase 1/2 activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription, but not impaired α-melanocyte-stimulating-hormone-induced adenylyl cyclase, calcium, or phosphorylated extracellular signal-regulated kinase 1/2. This profile was not observed for transfected H158R, a constitutively active human melanocortin-4 receptor variant associated with overweight, but not obesity. We concluded that there is potential for α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription in HEK293 cells transfected with obesogenic human melanocortin-4 receptor variants to be the key predictive tool for determining whether they exhibit loss-of-function. Furthermore, in vivo α-melanocyte-stimulating-hormone-induced human melanocortin-4 receptor cyclic adenosine monophosphate response element-driven transcription may be key for maintaining body weight.