Experimental Study On Heterodimerization Of Orexin Type 1 Receptor And Cholecystokinin Type 1 Receptor

In mammals,the central nervous system is the main distribution location of Orexin type 1 receptors(OX1R),with the positive feedback way to increase food intake of the body,thus to enhance the role of appetite.Cholecystokinin 1 receptor,(CCK1R)are mainly distributed in the central nervous system and digestive system in a negative feedback manner to regulate feeding behavior in mammals.OX1 R and CCK1 R belong to the members of the super family of G protein coupled receptor.The research of monomer has been relatively mature in appetite regulation,but dimerization have not been reported.The aim of experient is to prove whether OX1 R and CCK1 R could from heterologous dimers and how two dimers affect cell signal transduction.Further analysis help us to elaborate the dimerization of OX1 R and CCK1 R for appetite regulation mechanism,so as to provide a theoretical basis for clinical treatment of appetite disease(such as obesity,diabetes).Firstly,five recombinant plasmids(CCK1R-EGFP,CCK1R-Rluc,CCK1R-Myc,OX1R-YFP,CCK1R-CFP)using molecular cloning.HEK293 T cells expression OX1R-EGFP and CCK1R-Myc were observed under confocal laser scanning microscope using immunofluorescence co-localization,determining the expression of OX1 R and CCK1 R on cell membrane.Using BRET(biological fluorescence resonance energy transfer),FRET(fluorescence resonance energy transfer technology)and co-immunoprecipitation(CO-IP),the heterodimer of OX1 R and CCK1 R were detected at time,space and protein levels.Using by dual luciferase assay,serum response element(SRE),nuclear factor of activated T cells(NFAT),cAMP response element(CRE),serum response factor(SRF)were detected in HEK293 T cells transfecting after stimulating OX1 R,CCK1R and OX1R/CCK1 R.The results show that,five recombinant plasmids(CCK1R-EGFP,CCK1R-Rluc,CCK1R-Myc,OX1R-YFP,CCK1R-CFP)were successfully constructed.OX1 R and CCK1 R were proved to co-express at the cell member using laser confocal scanning microscope.BRET,FRET andCO-IP further proved the heterologous dimerization of OX1 R and CCK1 R.TheBRET signal was significantly enhanced and showed the ability of OX1 R and CCK1 R to dimerize.FRET proved that the two dimerization in space and CO-IP has proved that dimerization at the proved the protein level.In stably transfected cell lines HEK293T-OX1 R,HEK293T-CCK1 R and HEK293T-OX1R/CCK1 R,the action of CRE,SRE,SRF or NFAT were detected after CRE-luc,SRE-luc,NFAT-luc or SRF-luc was increase of NFAT and SRF in cells expressing OX1 R and CCK1 R with OX1 R or CCK1 R single cell.However,the expression of SRE decreased.The results indicated that dimerization of OX1 R and CCK1 R reduced the interaction of two receptor with the G?i subtypes and increased the interaction of two receptor with G?q andG?12/13 subtypes.This study demonstrated that OX1 R and CCK1 R can form a heterologous dimerization in HEK293 T cells transient transfectingOX1 R and CCK1 R.After stimulating by Orexin A and CCK(26-33),OX1R/CCK1 R changed the original single signal transduction pathway.Therefore,the research on OX1 R and CCK1 R of heterologous dimerization provides a new experimental basis for in-depth study of the OX1 R and CCK1 R and appetite,and provides a new idea for clinical treatment of diseases caused by the appetite regulation,and provide new drug targets for new drug research and development.