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There is compelling evidence for human melanocortin-4-receptor (hMC4R) playing a critical function regulating energy balance; yet signal transduction pathways contributing to this are unclear. The hMC4R activates multiple signaling pathways, including induced increases in cAMP and mobilization of intracellular calcium ([Ca2+]i). Recent evidence showed cAMP signaling was not a good predictor for hMC4R variant-associated obesity. We hypothesize that hMC4R mobilization of [Ca2+]i plays an important role in regulating energy balance. To test this, we developed a robust high-throughput Fura-2 ratiometric fluorescent assay to quantitatively measure [Ca2+]i in vitro. We compared basal and α-melanocyte stimulating hormone (α-MSH) activation of [Ca2+]i for hMC4R-wildtype (WT) and hMC4R-variants stably expressed in HEK293 cells. The loss-of-function variants studied were two obesity-associated variants (R7H and R18L) known to exhibit cAMP signaling similar to WT, two obesity-associated variants (H76R and L250Q) known to exhibit cAMP-constitutive activity (CA) compared to WT, and one overweight-associated variant (H158R) known to exhibit cAMP-CA compared to WT. The gain-of-function variants (V103I and I251L) studied are known to exhibit cAMP signaling similar to WT. The data for basal [Ca2+]i were pooled from three independent experiments performed with WT and all variants in each assay. Data (mean ± SEM) were analyzed using one-way ANOVA with Dunnett’s multiple comparisons. The data (mean ± SEM) for α-MSH activation of hMC4R were pooled from three independent experiments and analyzed using non-parametric sum of squares F-test for maximum best-fit values and EC50. The α-MSH activated assays were performed with each hMC4R variant alongside WT. WT hMC4R and non-CA loss-of-function variants exhibited similar basal and α-MSH activated [Ca2+]i (WT: EC50 = 1.44 nM; R7H: EC50 = 1.40 nM; R18L: EC50 = 1.12 nM). The CA loss-of-function variants exhibited significantly (p &amp;lt; 0.0001) increased basal [Ca2+]i compared with WT (WT = 97.6 ± 0.9 nM; H76R = 114.2 ± 1.7 nM; L250Q = 112.1 ± 2.6 nM; H158R = 110.7 ± 1.8 nM) and significantly lower EC50’s compared with WT (H76R: EC50 = 0.07 nM; p = 0.0019; L250Q: EC50 = 0.09 nM; p = 0.0066; H158R: EC50 = 0.14 nM; p = 0.0009). The gain-of-function hMC4R variants exhibited significantly (p &amp;lt; 0.0001) decreased basal [Ca2+]i compared with WT (WT = 97.6 ± 0.9 nM; V103I: = 86.4 ± 0.9 nM; I251L = 87.5 ± 1.0 nM) and significantly (p = 0.0001) increased α-MSH stimulated maximum [Ca2+]i compared with WT (WT = 224.5 ± 13.6 nM; V103I = 288.2 ± 31.5 nM; I251L = 295.6 ± 20.0 nM). To summarize, we show three distinct patterns of hMC4R-associated calcium signaling; (1) WT and non-CA loss-of-function, (2) CA loss-of-function and (3) non-CA gain-of-function. Future studies are required to understand how hMC4R mobilization of [Ca2+]i might contribute to the regulation of energy balance.

Distinct Calcium Signaling for Wildtype, Loss-of-Function and Gain-of-Function Human MC4R Variants