Eukaryotic Expression And Pharmacological Characterization Of Porcine Orexin 2 Receptors And Mutants

Orexins,involves orexin A(OXA)and orexin B(OXB),are neuropeptides which are mainly synthesized from the hypothalamic and lateral hypothalamic central neurons.Orexins act through the activation of orexin 1 receptor(OX1R)and orexin 2 receptor(OX2R).The two hypothalamic neuropeptides orexin A and orexin B are important homeostatic mediators in central control of food intake,maintenance of sleep/wake states,energy metabolism and neuroendocrine homeostasis.Up to now,lots of studies on orexins and orexin receptor focused on OX2R of human,and there are few studies on porcine orexin.In order to further investigate the effects of DOX2R on the energy regulation and the intracellular signal transduction of ligand receptors,and provide basis for breeding optimization.Four mutants were selected based on the relationship between gene polymorphism and signal transduction in the existing literature:P10S,P11T,V308I and T4011.The study used the established plasmid pcDNA3.1(+)-myc/pOX2R WT as the template to construct four mutants by single site-directed mutagenesis.The eukaryotic expression vectors of WT and mutants were transiently transfected into HEK293T cell(human embryonic kidney)to prove the receptor expression by western blot in vitro.Total cyclic adenosine monophosphate(cAMP)expression in wild type and mutants stimulated under different concentrations of ligands were detected by dual luciferase reporter gene assay.The phosphorylated extracellular regulated protein kinases 1/2(ERK1/2),p38 and phosphorylated cAMP-response element binding protein(CREB)of WT and mutant pOX2R in the HEK293T cells under the stimulation of high concentrations of ligands were evaluated.By using the above methods to study the effects of POX2R on the energy regulation,the results were as follows:1.Using the established eukaryotic expression vector pOX2R wild-type pcDNA3.1(+)-myc/pOX2R-WT as a template,four amino acids were mutated successfully.The four constructed mutants were named as pcDNA3.1(+)-myc/pOX2R-P10S,pcDNA3.1(+)-myc/pOX2R-P11T pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I2.The constructed mutant eukaryotic expression vector plasmids and wild-type plasmid were transfected into HEK293T cells.The total protein was extracted at 24 h after transfection and verified by Western blot.The results showed that the wild type and four mutants of pOX2R were expressed correctly.3.To detect the intracellular basal cAMF3 level,the pOX2R wild type and four mutants with the firefly and renilla luciferase reporter plasmid pGL4.29[luc2P/CRE/Hygro]and pRL-TK at a ratio of 2:10:1 separately were co-transfected into HEK293T cells.The results showed that the intracellular basal cAMP levels of the four mutants were not significantly different from the wild type.Cells expressing WT and four mutants of pOX2R were stimulated with different concentrations(10-6M-10-10 M)of endogenous agonists OXA and OXB,respectively.The results showed dose-dependent response in pOX2R WT or mutant.The responses of mutants P10S,P11T and T401I were significantly reduced compared to wild type under the same concentration of agonist OXA.Mutant P11T was significantly reduced in sensitivity to wild-type stimulation under the same concentration of agonist OXB.These results indicated that mutations in amino acids 10,11 and 401 can affect intracellular PKA signaling pathways.4.The pOX2R wild type and mutant were separately transfected into HEK293T cells,and the cells were stimulated with 10-6 M OXB and 10-7 M OXA respectively.Total protein was extracted and the protein expression levels of p-CREB,p-ERK1/2 and p-p38 were evaluated by Western blot.The results showed that the expression levels of p-CREB in the four mutants were significantly lower than that in the wild type ligands OXA and OXB.Compared to the effect of orexin A stimulated wild type receptor,the p-ERK1/2 protein expression level of the mutant V308I was significantly reduced,and meanwhile the p38 phosphorylation levels of mutants P10S,P11T and V308I were also significantly reduced,under the induction of ligand OXB,the intracellular p-ERK/2 level of mutant T401I was significantly decreased,and the phosphorylation p38 level of mutant P11T was significantly decreased.These results showed that mutants at these four sites have an impact on the PKC and MAPK pathways.