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Regulation of human melanocortin-5 receptor by melanocortin-2 receptor accessary proteins and functional characterization of hMC5R mutants


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dc.contributor.advisorTao, Ya-Xiong
dc.contributor.authorJiang, Shanshan
dc.date.accessioned2024-08-06T14:40:31Z
dc.date.available2024-08-06T14:40:31Z
dc.date.issued2024-08-06
dc.identifier.urihttps://etd.auburn.edu//handle/10415/9458
dc.description.abstractMelanocortin-5 receptor (MC5R) is the most recently discovered melanocortin receptor (MCR). It is widely distributed in both central nervous system and peripheral tissues and has unique tissue expression patterns, pharmacological properties, and physiological functions. MC5R influences exocrine gland function and regulates energy metabolism by affecting lipolysis, fatty acid oxidation, and glucose uptake in adipocytes. Preliminary clinical studies also indicated that hMC5R is associated with obesity and type 2 diabetes. Melanocortin-2 receptor accessory proteins (MRAPs) are important regulators of MCRs, which can regulate their transport, binding, and signal transduction, thereby affecting various physiological processes. There are two members of MRAP family: MRAP1 and MRAP2. Many studies have shown that MRAP1 and MRAP2 primarily interact with MC2R, MC3R, and MC4R. However, the interaction between different isoforms of MRAPs and MC5R is still unclear and needs further study. To investigate the effects of MRAPs on trafficking, ligand binding and signaling properties of human (h) MC5R, HEK293T cells were transiently co-transfected with plasmids encoding MC5R and different isoforms of MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c). The results showed that hMRAP1a, hMRAP2a and hMRAP2c increased the cell surface expression of hMC5R. All MRAPs have no effect on affinity to the superpotent analog of a-melanocyte stimulating hormone (a-MSH), NDP-MSH, while hMRAP2c significantly increased maximal binding. All MRAPs had no effect on a-MSH-stimulated cAMP production (Gas-cAMP pathway). Additionally, a-MSH induced ERK1/2 activation of hMC5R. Cells co-transfected with hMC5R and hMRAP1a, hMRAP2a, hMRAP2b, or hMRAP2c had significantly increased pERK1/2 level upon stimulation with a-MSH. In summary, the two MRAP1s and three MRAP2s had differential effects on MC5R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of MC5R by MRAP1s and MRAP2s. Melanocortin-4 and -5 receptors (MC4R and MC5R) play important roles in regulating energy homeostasis. MRAP2 regulates MC4R and MC5R trafficking, ligand binding, and signaling. Loss of MRAP2 function results in reduced MC4R activity, leading to obesity and metabolic disorders. In this study, we selected five hMRAP2a mutants (G31V, F62C, N77S, K102*, and P195L) and investigated their effects on hMC4R and hMC5R pharmacology. In hMC4R, F62C and P195L reduced cell surface expression and maximal response to a-MSH. Additionally, hMRAP2a and the remaining three mutants (G31V, N77S, and K102*) reduced the maximal response to a-MSH as well as basal activity. For hMC5R, K102* reduced total and cell surface expression. Furthermore, F62C and P195L reduced cell surface expression of hMC5R, whereas N77S increased it. F62C reduced the maximal response to a-MSH, while P195L impaired both the maximal binding and the maximal response to a-MSH. Like MC4R, MC5R exhibits basal cAMP signaling, which was reduced by hMRAP2a and all five mutants. In conclusion, hMRAP2a and its identified obesity-associated mutants can modulate the pharmacology of hMC4R and hMC5R. Different hMRAP2a mutants exhibited distinct regulatory effects on hMC4R and hMC5R, revealing a complex regulatory mechanism within the melanocortin receptor family. Recent genomics studies have identified hundreds of naturally occurring MC5R mutations. Human MC5R is primally coupled to Gas, resulting in increased intracellular cAMP. Studies have shown that some missense polymorphisms in hMC5R affect its ligand binding activity and downstream functions, particularly those involving conserved amino acids in the transmembrane domain (TMD). Herein, we studied pharmacological properties of 12 hMC5R mutants at highly conserved residues by measuring their expression, ligand binding and signaling. Results showed only Y295H had decreased total expression while I294T had increased cell surface expression. D119Y, P253L, P292S, I294T, and I294M were defective in ligand binding as well as a-MSH-stimulated cAMP production. The remaining seven mutants (P70A, Y141F, R158H, R158L, D291H, Y295C, Y295H) had similar binding affinities for a-MSH as wild-type hMC5R. P70A, Y141F, and R158H had significantly decreased maximal binding and signaling. D291H, Y295H and Y295C were defective in a-MSH-stimulated Gas-cAMP signaling pathway. Y141F and R158H also had decreased a-MSH potency. In summary, we did not identify any mutant with misfolding defect. Five mutants had defects in binding and consequent signaling. Three mutants had binding but no signaling, with a clear defect in G protein binding and/or activation.en_US
dc.rightsEMBARGO_GLOBALen_US
dc.subjectGeneral Veterinary Medicineen_US
dc.titleRegulation of human melanocortin-5 receptor by melanocortin-2 receptor accessary proteins and functional characterization of hMC5R mutantsen_US
dc.typePhD Dissertationen_US
dc.embargo.lengthMONTHS_WITHHELD:24en_US
dc.embargo.statusEMBARGOEDen_US
dc.embargo.enddate2026-08-06en_US
dc.contributor.committeeHuang, Chen-Che “Jeff”
dc.contributor.committeeGriffett, Kristine
dc.contributor.committeeJeganathan, Ramesh
dc.contributor.committeeWest, Rachel

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