Alejandra V. Rodríguez Rondón1, Ana Gebara Garcia de Lima1, Patric J.D. Delhanty1, JennyA. Visser1
(1) Dept. of Internal Medicine and Obesity Center CGG, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands
G protein-coupled receptors (GPCRs) in hypothalamic neurons, such as melanocortin-4 receptor (MC4R) and the serotonin receptors, HTR1B and HTR2C, are important mediators of appetite and energy regulation. Loss of function (LoF) variants in MC4R are associated with monogenic obesity in humans. Interactions between GPCRs modulate their signaling mechanisms. Since MC4R, HTR1B and HTR2C are co-expressed in hypothalamic neurons, we aimed to discover whether serotonin receptors modulate signaling of wild type (WT) MC4R and obesity-associated MC4R variants.
HEK293 cells were transiently transfected with expression plasmids encoding WT or variant MC4R and empty vector, WT HTR1B or HTR2C, and stimulated with α-MSH. The effects of the serotonin receptors on MC4R cell surface expression and α-MSH-stimulated cAMP response were measured using bioluminescent assays. We assessed WT and three obesity-associated MC4R variants (T150I, F262L, and V255A), previously categorized as complete-LoF, partial-LoF, and WT-like, respectively, based on their cAMP responses to α-MSH.
HTR1B suppressed maximal cAMP responses of WT MC4R and variant V255A by 69±10% and 62±8% (p<0.001), respectively. HTR1B decreased WT MC4R cell surface expression by 29±3% (p<0.001) but not of the variants. Unlike HTR1B, HTR2C potentiated maximal cAMP responses of WT MC4R and variants F262L and V255A by 82±23%, 31±5% and 114.2±34% respectively (p<0.05), but not of T150I. However, HTR2C increased the cell surface expression of T150I by 60±16% (p<0.05) to levels of WT MC4R, but had no effect on WT MC4R, or variants F262L and V255A.
Results:
Our results show that serotonin receptors HTR1B and HTR1C oppositely modulate MC4R signaling. These findings suggest that MC4R may heterodimerize with serotonin receptors impacting cell surface expression and possibly the interaction with downstream signaling proteins. Furthermore, our results show that these serotonin receptors alter signaling of MC4R variants. Unraveling the underlying mechanism may aid to understand the impact of MC4R variants in monogenic obesity.