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dc.contributor.advisorTao, Ya-Xiong
dc.contributor.authorYang, Likun
dc.date.accessioned2019-07-19T16:27:53Z
dc.date.available2019-07-19T16:27:53Z
dc.date.issued2019-07-19
dc.identifier.urihttp://hdl.handle.net/10415/6850
dc.description.abstractThe neural melanocortin receptors, including melanocortin-3 and -4 receptors (MC3R and MC4R), are two important factors in regulation of energy homeostasis. MC3R primarily regulates feed efficiency and nutrient partitioning, whereas MC4R plays an essential role in controlling both food intake and energy expenditure. In several other G protein-coupled receptors, the DRYxxI motif and intracellular loop 2 (ICL2) have been shown to play a significant role in receptor activation. To gain a better understanding of functions of this region in human MC4R (hMC4R), we performed systematic functional study using alanine-scanning mutagenesis in terms of DRYxxI motif and ICL2 of hMC4R. We identified several residues important for receptor cell surface expression (T150), receptor and ligand interaction (D146, Y148, Y153, Q156, Y157, M161, and T162), constitutive cAMP signaling (R147, FI151, L155, Y157), ligand-induced cAMP (R147, I151, and L155) and ERK1/2 signaling pathways (R147, F149, H158, and I160), as well as biased signaling between cAMP and ERK1/2 pathways (Y148, F149, T150, H158, I160, and M161). The investigation of structure-function relationship of hMC4R provides a better understanding towards the role of MC4R in obesity pathogenesis and will be valuable for the structure-based drug design of molecules with the capacity to modulate MC4R activity as potential therapeutics. Currently, the approaches for improving growth and feed efficiency of fish are limited to the time-consuming selective breeding methods. Therefore, understanding of endocrine regulation of energy metabolism in economically important species can potentially lead to novel approaches to achieve better economic return. In this investigation, we attempted to functionally investigate channel catfish (Ictalurus punctatus) MC3R (ipMC3R). Five agonists, including adrenocorticotropin, alpha-melanocyte stimulating hormone (alpha-MSH), beta-MSH, [Nle4, D-Phe7]-alpha-MSH, and D-Trp8-gamma-MSH, were used in the pharmacological studies. Our results showed that ipMC3R bound beta-MSH with a higher affinity and D-Trp8-gamma-MSH with a lower affinity compared with human MC3R. All agonists were demonstrated to stimulate ipMC3R and increase intracellular cAMP production with sub-nanomolar potencies. The ERK1/2 activation was also be triggered by ipMC3R. The ipMC3R exhibited constitutive activities in both cAMP and ERK1/2 pathways, and the Agouti-related protein served as an inverse agonist at ipMC3R, potently inhibiting the high basal cAMP level. Moreover, we showed that melanocortin receptor accessory protein 2 preferentially modulated ipMC3R in cAMP production rather than ERK1/2 activation. Our study leads to further investigation of the physiological roles of the ipMC3R, especially in energy homeostasis, in channel catfish. We have also cloned swamp eel (Monopterus albus) mc4r (mamc4r), consisting of a 981 bp open reading frame encoding a protein of 326 amino acids. The sequence of maMC4R was homologous to those of several teleost MC4Rs. Phylogenetic and chromosomal synteny analyses showed that maMC4R was closely related to piscine MC4Rs. qRT-PCR revealed that mc4r transcripts were highly expressed in brain and gonads of swamp eel. The maMC4R was further demonstrated to be a functional receptor by pharmacological studies. Four agonists, alpha-MSH, beta-MSH, [Nle4, D-Phe7]-alpha-MSH, and adrenocorticotropin, could bind to maMC4R and induce intracellular cAMP production dose-dependently. Small molecule agonist THIQ allosterically bound to maMC4R and exerted its effect. Similar to other fish MC4Rs, maMC4R also exhibited significantly increased basal activity compared with that of hMC4R. The high basal activity of maMC4R could be decreased by inverse agonist ML00253764, suggesting that maMC4R was indeed constitutively active. The availability of maMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in swamp eel. In summary, we studied DRYxxI motif and ICL2 of hMC4R in a systemic manner, and identified some critical residues for receptor functions. We also investigated functionality of MC3R and MC4R in two economically important fish species, laying a foundation for future physiological studies.en_US
dc.rightsEMBARGO_NOT_AUBURNen_US
dc.subjectGeneral Veterinary Medicineen_US
dc.titleStructure-function relationship of human melanocortin-4 receptor and characterization of fish neural melanocortin receptorsen_US
dc.typePhD Dissertationen_US
dc.embargo.lengthMONTHS_WITHHELD:25en_US
dc.embargo.statusEMBARGOEDen_US
dc.embargo.enddate2021-08-03en_US


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