Study On The Mechanism Of GLP-1R Nuclear Localization In Rat Aortic Vascular Smooth Muscle Cells

Glucagon-like peptide 1 receptor(GLP-1R),which is widely present in pancreatic islets,central and peripheral nervous systems,gastrointestinal tract,heart and vasculature,belongs to family B of the G-protein-coupled receptor(GPCR).Like other G protein-coupled receptors,classical GLP-1R at the plasma membrane experiences desensitization,internalization,and recycling back to the plasma membrane under the control of Glucagon-like peptide 1(GLP-1)in islet ?-cells and promotes insulin release,delays gastric emptying,and suppresses appetite,so it is used as a treatment target for type 2 diabetes.GLP-1R expressed in vascular smooth muscle cells and can provide cardiovascular protection under the action of agonists.However,the subcellular localization of GLP-1R in the vascular system has not yet been determined.This subcellular localization and nuclear import mechanism of GLP-1R in rat aortic vascular smooth muscle cells was focused in the Master's thesis.The main research contents include the following parts:Part 1: The subcellular localization of GLP-1R in rat aorta media,liver,kidney,pancreas and RASMC cells,Hep G2 cells,Skov3 cells,and MIN6 cells were observed through immunofluorescence and immunohistochemistry experiments.The results showed that GLP-1R waslocalized in the nucleus of arterial media and RASMCs or enriched in cytoplasm and cell membrane in other tissues and cells.In order to analyze the nuclear import mechanism of GLP-1R,c NLS Mapper was first used to predict the putative nuclear localization signal(NLS)of GLP-1R.The results showed that GLP-1R may have three putative NLSs,two of which were located in the transmembrane domain of GLP-1R,and the third NLS was located in the C-terminus of GLP-1R.According to the structure of GLP-1R,it was truncated into N-terminus(1-145aa),transmembrane region(146-408aa),and C-terminus(409-463aa),that were fused with Green fluorescence protein(GFP)respectively to express in the RASMCs.The results showed that only GFP-GLP-1R(409-463aa)was localized in the nucleus.When NLS(412-442aa)was deleted in the full-length GLP-1R and all the basic amino acids in this segment were mutated,and fused with GFP,to express in RASMCs respectively.It is shown that nuclear import of GFP-GLP-1R(?412-442)and GFP-NLSmut GLP-1R were inhibited and significantly enriched in the cytoplasm.Therefore,(412-442aa)was identified as the NLS of GLP-1R,which is necessary for its nuclear import.In addition,the classical pathway importin?/? inhibitor Bimax2 also inhibited the nuclear import of GLP-1R.The interaction between GLP-1R and importin? was confirmed GST-Pull down experiments in vitro,which disappeared after NLS mutation.These results indicated that GLP-1R enters the nucleus through its NLS(412-442aa)interaction with importin?/?classical pathway.Part 2: In order to investigate the effect of phosphorylation on nuclear import of GLP-1R,5 pairs of Ser at the C-terminus were gradually truncated and Ser416 in NLS was mutated in the full-length GLP-1R.Immunofluorescence showed that 5 pairs of Ser at the C-terminus had no effect on nuclear localization of GLP-1R,but Ser416 was mutated to Asp(GLP-1R S416 D,mimicking phosphorylation),GLP-1R is significantly distributed in the cytoplasm and nuclear import was inhibited;it is mutated to Ala(GLP-1R S416 A,mimicking dephosphorylation),GLP-1R can still perform normal nuclear import.It is shown that the phosphorylation of Ser416 in NLS regulates nuclear import of GLP-1R.GST-Pull down experiment revealed that GLP-1R S416 D no longer interacted with importin? in vitro.The phosphorylation of GLP-1R Ser416 affected the nuclear import of the protein by affecting the interaction between NLS and importin?.Nuclear import of endogenous GLP-1R was observed to be inhibited by Phorbol myristate acetate(PMA),a protein kinase C(PKC)activator.But GFP-GLP-1R S416 A was still localized in the nucleus induced by PMA,indicating that PKC or its downstream kinases inhibits nuclear import of GLP-1R by phosphorylating Ser416 in NLS.Finally,GLP-1R was remained in the cytoplasm by mutating the basic amino acid in the NLS(GFP-NLSmut GLP-1R)and mimicking phosphorylation(GFP-GLP-1R S416D)to study the effects of increasedGLP-1R in the cytoplasm of RASMC cells on the function of cells by MTT,cell colony-formation assays,and western blot.The results showed that the proliferation of RASMC cells was promoted significantly.In summary,in RASMCs,GLP-1R entered the nucleus through the interaction between the NLS and importin alpha/beta.Phosphorylated by protein kinase such as PKC,GLP-1R entered the cytoplasm from the RASMC nucleus and promoted cell proliferation.These findings may provide clues for further understanding the function of GLP-1R in the cardiovascular system.