Pharmacological characterization and functional rescue of the giant panda and human melanocortin-4 receptors
Type of DegreePhD Dissertation
General Veterinary Medicine
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The melanocortin-4 receptor (MC4R) is a member of family A rhodopsin-like G protein-coupled receptors. It is well established that MC4R is critically involved in regulating food intake and energy expenditure in mammals. As the most common monogenic form of human obesity, more than 170 naturally occurring MC4R mutations have been identified from different patient cohorts. Functional characterization of mutant MC4Rs is indispensable for confirming the causal role of MC4R mutations in obesity pathogenesis. In this study, giant panda (Ailuropoda melanoleuca) MC4R was cloned, expressed, and functionally characterized. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequence levels to human, mouse, rat, pig, cat, cattle, and dog MC4Rs. With human MC4R (hMC4R) as a control, four agonists, including [Nle4, D-Phe7]-alpha-melanocyte stimulating hormone (NDP-MSH), alpha-MSH, beta-MSH, and a small molecule agonist, THIQ, were used in the binding and signaling assays to investigate the pharmacological properties of amMC4R. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, alpha-MSH, and beta-MSH, with the same ranking as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent accumulation of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of MC4R in regulating food intake and energy expenditure in giant panda. In addition, we reported systematic functional characterization of nine novel human MC4R mutations including L23R, K73R, T101N, T112K, M161T, L207V, M215L, R310K, and I316S. Mutants with defects in the receptor expression, ligand binding, and/or cAMP signaling were identified and classified based on a classification scheme that we proposed earlier. Consistent with previous reports, intracellular retention was demonstrated to be the most common defect in the nine mutants studied herein. Mutant M161T had normal cAMP production in stimulation with NDP-MSH but not alpha-MSH, MTII, or THIQ. In addition, we demonstrated that defective ERK1/2 signaling might be a cause of obesity in patients harboring Class V mutant MC4Rs. Five mutant receptors were identified as biased receptors in the cAMP and ERK1/2 signaling pathways. Furthermore, we showed that two MC4R inverse agonists, Ipsen 5i and ML00253764, acted as pharmacological chaperones to rescue the cell surface expression and function of thirteen intracellularly retained mutant MC4Rs. In summary, we provided detailed functional data and a potential therapeutic approach for the treatment of obese patients harboring these mutations.