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dc.contributor.advisorTao, Ya-Xiong
dc.contributor.authorMo, Xiu-Lei
dc.date.accessioned2013-07-15T16:11:57Z
dc.date.available2013-07-15T16:11:57Z
dc.date.issued2013-07-15
dc.identifier.urihttp://hdl.handle.net/10415/3739
dc.description.abstractThe melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that are suggested to be essential in ligand binding and signaling. Several MC4R mutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all 26 TM3 mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling, and six mutants had normal ligand binding, but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the extracellular signal-regulated kinase (ERK) 1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of wild type (WT) receptor, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in an inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways. The MC4R is constitutively active in vivo and its basal activity has been suggested to be important in obesity pathogenesis. Theoretically, it is expected that constitutively active MC4R mutants are associated with a lean phenotype, even anorexia nervosa. However, there are several constitutively active MC4R mutants identified from obese patients, and the mechanism of how these mutations associate with obesity still needs further investigation. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, ERK1/2. Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the Gs-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i, and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2 fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the Gs-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.en_US
dc.rightsEMBARGO_NOT_AUBURNen_US
dc.subjectVeterinary Anatomy, Physiology, and Pharmacologyen_US
dc.titleHuman melanocortin-4 receptor: structure-function relationship of transmembrane domain 3 and biased signalingen_US
dc.typedissertationen_US
dc.embargo.lengthNO_RESTRICTIONen_US
dc.embargo.statusNOT_EMBARGOEDen_US


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