Functional Rescue Of Obesity-Related Melanocortin-4 Receptor Mutations By Pharmacological Chaperone ML00253764

As the increasingly rich material and the pace of life gradually accelerated in the 21st century, obesity has been considered as a kind of disease by the medical community and become a global major public health problem. Obesity can lead to disease of cardiova-scular, endocrine metabolism disorder, osteoarthritis, and many chronic non-communicable diseases. This not only affect the patient’s physical and mental health and quality of life, but also will pay more serious adverse affects on social economic burden.Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor and plays a pivotal role in controlling energy balance and body weight. It has been shown that obesity caused by MC4R mutations accounts for 6% of the total obesity people, and intracellular retained MC4R mutants are the main form of early-onset obesity. This article took hMC4R as the research object, and site-directed mutagenesis was used to get the two mutants C84R and W174C which are defective in intracellular trafficking. Then through Calcium phosphate method we transfected pcDNA3.1-hMC4R-WT, hMC4R-C84R, hMC4R-W174C and the negative control pcDNA3.1 to HEK293 cell. FACS and Confocal microscopy were used to quantitative and qualitative analysis the cell surface expression level. As previously reported, C84R and W174C were poorly expressed on the cell surface of HEK293 cells. Also, they showed a significantly decrease in intracellular cAMP accumulation, which indicated the signal transduction of the two mutants is defected. When following the treatment of ML00253764, cell surface expression and cAMP accumulation of the two mutants are partially rescued compared to WT receptor without ML00253764 treatment. By treating each MC4R mutant with various concentrations of ML00253764, we found that it was able to increase cell surface expression of each mutant in a dose-dependent manner that was maximal at 10-4 M. By treating each mutant MC4R with ML00253764 (10-4 M) for different time periods, the results showed that ML00253764 was able to increase the cell surface expression of the two mutants in a time-dependent manner that was detectable after only 1 h of treatment and maximized (relative to WT level without ML00253764 treatment) following around 12 h of exposure. We next determined the constitutive activity of the two mutants and found that the basal level of cAMP accumulation and internalization was not significantly different among the WT and mutant MC4Rs following ML00253764 treatment. However, both WT and mutant receptors showed a significantly decreased basal activity following ML00253764 treatment compared with that of WT MC4R without ML00253764. Taken together, these results showed that the two mutants causes defect in intracellular trafficking of the receptors but did not affect signaling. For WT and C84R and W174C, treatment with ML00253764 caused the decrease of constitutive activity, confirming that ML00253764 acts as a MC4R inverse agonist.This study may provide a powerful foundation and basis that pharmacological chaperone is a possible therapeutic option for personalized treatment of early-onset obesity caused by inherited mutations in MC4R gene.