| Melanocortin receptor-4 (MC4R) is one of the family members of melanocortin receptors (MCRs), belongs to the superfamily of G protein-coupled receptors (GPCRs).MC4R is widely exist in the central nervous system of animals, the main physiological function of MC4R is to control the appetite and weight, so as to maintain body weight homeostasis. The cAMP/PKA signaling pathway is the classic signaling pathway of MC4R. After the agonist or antagonist combined with MC4R, change the levels of intracellular cAMP, and then change the state of protein kinase A (PKA), result in a corresponding physiological effect.Currently, the research on the signaling pathways of MC4R and mutants of human, swine and other animals had been reported, and the high-throughput drug screening model which took people MC4Ras the target was established successfully. But the study of the MC4R and mutants of chick only focused on the relationship between gene polymorphism and economic character. The researches on the signaling pathways of the MC4R and mutants of chick were few.Therefore, this study on the basis of eukaryotic expression vectors of MC4R WT and four mutants of chick were constructed successfully, the cAMP/PKA signaling pathway of MC4R WT and four mutants of chick in cells was researched by using the agonists (a-MSH, β-MSH, NDP-MSH) and antagonist (SHU9119) of MC4R. And based on the signaling pathway of MC4R, a cell screening model which took chicken MC4R as the target was established by reporter gene method. This study laid the foundation for extracting the agonist and antagonist of cMC4R. The results were as follows:1. The eukaryotic expression vectors of MC4R WT and four mutants (Q18H, G21R, S76L and L299P), the reporter gene plasmid pGL4.29[luc2p/CRE/Hygro] and pRL-TK vector were co-trans fected into HEK293T cells. Then we detected the basal activities of MC4R and mutants by dual reporter gene assay in the absence of ligand. The results showed Ql 8H had significantly increased basal signaling compared with WT cMC4R (P<0.01), L299P had significantly decreased basal signaling compared with WT cMC4R (P<0.01) whereas G21R and S76L had normal constitutive activity.2. The eukaryotic expression vectors of MC4R WT and four mutants, the reporter gene plasmid pGL4.29[luc2p/CRE/Hygro] and pRL-TK vector were co-transfected into HEK293T cells. And the signaling properties of the WT and mutants had been researched with three agonists as the ligand. The results showed that the rank order in terms of potency for these three agonists is:NDP-MSH> a-MSH=β-MSH for cMC4R WT and mutants. For cMC4R L299P, with decrease in proportional impairments in three agonists stimulated cAMP production.3. The eukaryotic expression vectors of MC4R WT, the reporter gene plasmid pGL4.29 and pRL-TK vector were co-transfected into HEK293T cells. And we detected the properties of the MC4R WT with three agonists and antagonist as the ligand. The results showed that no matter which agonist interact with cMC4R WT, high and middle concentration of antagonist (SHU9119) were able to inhibit the activity of agonist significantly, but the inhibitory action of low concentration of SHU9119 was not obvious.4. The eukaryotic expression plasmid cMC4R-pcDNA3.1(+)-myc and a reporter gene plasmid pGL4.29[luc2p/CRE/Hygro] were co-transfected with the ratio of 1:5 into CHO-K1 cell line. After G418 (800μg/ml) and Hygromycin B (400μg/ml) being selected,5 monoclonal (A2, F7, F8, F9, H10) were obtained by limiting dilution method, while took A2 as the positive cell lines which the relative expression of firefly luciferase was highest; the cMC4R gene (996bp), luciferase report gene (1653bp) were detected in the A2 cell lines by RT-PCR; a table cell line was established for cMC4R antagonist screening, the Z’-factor was 0.82 on the condition the final concentration of NDP-MSH was 10-9mol/L, the incubation time was 8h and the final concentration of DMSO was less than 2%. |
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