Eukaryotic Expression And Pharmacological Characterization Of Chicken,Duck,Goose And Pig Melanocortin-5 Receptor And Mutants

The melanocortin-5 receptor(MC5R)is a G protein-coupled receptor(GPCR).The receptor has been shown to play an important role in lipid production,skeletal muscle fatty acid oxidation and fat cell metabolism.However,current studies about MC5R mainly focus on human and mice,only few studies have been conducted on poultry and pigs.Four animal MC5R genes were cloned and eukaryotic expression vectors were constructed,in order to further analysis the functions of c/d/g/pMC5R and the signal pathway changes during the interaction between receptors and ligands,and to provide preliminary data for drug screening or genetic breeding in the next step.According to the amino acid sites found in previous studies that are critical for ligand binding,three mutants were constructed by single point mutation technology:c/d/gMC5R-D119A/F254A/H257A、pMC5R-D204A/F339A/H342A.After MC5R and mutants of the four animals were transfected into Human Embryonic Kidney T cells(HEK293T),dual-luciferase reporter gene assay and Western Blot were used to determine the level of Cyclic adenosine monophosphate(cAMP)and the phosphorylation level of Extracellular regulated protein kinases 1/2(ERK1/2)induced by ligand,in order to illuminate the differences in the reactivity and signaling pathways of MC5R and mutants expressed in vitro to different ligands.The results are as follows:1.The coding region of MC5R were cloned and inserted into pcDNA3.1(+)to construct a eukaryotic expression vector plasmid of pcDNA3.1(+)-c/d/g/pMC5R.Three mutants in each of four species were constructed by site-directed mutagenesis and there were four wild-types and twelve mutants totally.2.The wild-types and mutants eukaryotic expression vectors of MC5R in four species were 4transfected together with Firefly and Renilla luciferase reporter plasmids to HEK293T respectively,to detect the intracellular basal cAMP level.The results showed that the basal activities of c/d/gMC5R-F254A and pMC5R-H342A were significantly increased,and the basal activities of other mutants were not significantly different from those of the wild-type.Then,intracellular cAMP changes were detected by stimulation with different ligands at different concentrations.The results showed that c/d/gMC5R-D119A and pMC5R-D204A lost reactivity against all agonists and antagonists.The reactivity of c/d/gMC5R-F254A and pMC5R-F339A decreased significantly.The c/d/gMC5R-H257A and pMC5R-H342A showed decreased or loss of reactivity in response to a-MSH and NDP-a-MSH.The signal window of c/d/gMC5R-H257A to SHU9119 significantly increased.The pMC5R-H342A shows a decrease in signal window and an increase in EC50 on SHU9119.Under the stimulation antagonist AgRP,the reactivity of the mutant c/d/gMC5R-F254A and pMC5R-F339A were detected only.3.The wild-types and mutants eukaryotic expression vectors of MC5R in four species were transfected into HEK293T,and total proteins were extracted.The intracellular phosphorylation level of ERK1/2 and the phosphorylation level after ligand stimulation were detected by Western Blot.The results showed that the basic pERK1/2 level of c/gMC5R-F254A/H257A decreased,the basic pERK1/2 level of pMC5R-F339A/H342A increased significantly,and the three mutants of dMC5R showed no significant changes.Under the activation of agonist NDP-a-MSH,the pERK1/2 level of cMC5R-F254A increased,and the pERK1/2 level of pMC5R-F254A decreased,while the pERK1/2 level of other receptors and mutants showed no significant change.Under the activation of the antagonist AgRP,there was no significant changes in the pERK1/2 level of c/d/gMC5R-H257A and pMC5R,cMC5R-F254A and dMC5R-F254A showed increased pERK1/2 level,and the other receptors and mutants showed decreased pERK1/2 level.