Structural And Functional Research Of Prostaglandin E Receptor Subtype3

Prostaglandin E receptor （EP） is the receptor of prostaglandin E2（PGE2）,including four subtypes, namely prostaglandin receptor EP1, EP2, EP3and EP4,which all belong to the family of G-protein coupled receptor （GPCR）. PGE2has themost diversed biological activity and is the most widely distributed prostaglandins invivo. PGE₂is involved in regulating a variety of physiological and pathologicalfunctions through its receptors, such as body temperature regulation, inflammatoryresponse and so on. Above the four PGE2receptor, EP3receptor is the most specialone. A variety of transcriptional variants of EP3receptor, which can be formed byalternative splicing of the C-terminal intracellular domain, have been found in theformer researches. Different transcriptional variants have different types ofG-protein-coupled properties and signal transduction pathways. Recent studiesshowed that many prostaglandin receptors can form heterodimers with other receptorsor subtypes to achieve various functions. For example, thromboxane A2receptor （TP）and prostaglandin I receptor （IP） can form heterodimer, and the two subtypes of EP1can also heterodimerize with each other. The C terminus of the receptor was thoughtto be involved in this phenomenon. Also, EP3receptor has been reported to beassociated with severe inflammation, tumor invasion and metastasis, but there are alot of controversy and contradictions about these pathological features which may berelated to heterodimerization of EP3receptor isoforms or the heterodimerization ofEP3receptor and other prostaglandins receptors. In addition, the C-terminus of EPreceptors can mediate the internalization and desensitation of GPCR, and thusregulate the activity of receptors. Therefore, the researches on the structure of EP3receptor, especially the C-terminal structure, are of great significance for theunderstanding of the heterodimer phenomenon, as well as physiological and pathological functions of heterodimers.In this thesis, on one hand, we researched on the structure of EP3subtype withthe longest C-terminus （EP3f C-terminus） by NMR. We constructed high expressingplasmids and baterial strains, expressed the EP3fC in prokaryotic cells, denaturized,purified, refolded the identical protein and tried a variety of optimization to obtainEP3fC protein. The efficiency of protein expression, purification and refolding wasvery good, and we have also get the results of1D-¹H NMR. Since EP3fC itself tendsto aggregate even in the case of β-ME precipitation aggregation persists, itspolymerization is not entirely dependent on the disulfide bonds. Protein aggregationcan be opened with the presence of high concentrations of DMSO, and the proteinstatus and stability were good, and a narrow one-dimensional hydrogen spectrallines was obtained.Then high sensitivity, two-dimensional NMR analysis of itsstructure was obtained, and had laid a good foundation of the future use of¹⁵Nlabeled protein and three-dimensional NMR experiments.One the other hand, in this thesis, the method of homologous modeling wasutilized to obtain structural model of the extracellular domain, trans-membranedomain and intracellular loop, namely the model without the C terminus of EP3f. Theresults showed that the quality of this model was relatively high, and a cavity could beobserved with the size of300, which was the potent ligand binding site of EP3f.Then the flexible molecular docking was conducted between the ligand PGE₂and thenewly established model, and the analysis of this joint’s results revealed that the twoamino acids that mainly participated in forming the hydrogen bonds with PGE₂wereMet101and Lys287, and that Arg297, which was reported to be significant to thebinding to the ligand, was precisely at the bottom of this pocket. The establishment ofthis model will contribute to better knowledge of the interaction between EP3receptors and ligands, and has laid a foundation to the future experiments such aspoint mutation for the identification of the interaction sites.Moreover, Co-Immunoprecipitation （Co-IP） and Fluorescent Quantitative PCR（RT PCR） were also conducted to study the homo-or hetero-dimerization between EP3’s different subtypes, and its influence to the gene expression level of MMP-9，mPGES-1，PKC and PKCζ. The results of CO-IP showed that EP3f has interactionbetween itself as well as all other subtypes, and this interaction does not depend onthe addition of ligands. Among all these interactions, the one with EP3b, which hasthe shortest C terminus, has the strongest interaction, indicating that the length of EP3receptor C terminus might affect the polymerization. The results of RT PCR showedthat EP3receptor has significant inhibiting effect on the expression of MMP-9, whilein some other researches it might enhance the MMP-9expression in other tumor cellslines. Thus, we think that EP3’s regulation to the expression of MMP-9iscell-dependent, and that other EP receptors in MCF-7（such as the EP4receptor whichhas reversed effect） might be able to inhibit the function of EP3receptor by thepolymerization with it.To sum up, in this thesis, the structure of EP3f receptor was studied, as well asthe gene expression regulating effects of the hetero-dimerization of EP3receptors.These researches has laid a foundation for the further understanding of EP3receptors‘molucular structures and their relationships to the functions. In the future,we will continue focussing on the molecualr structure of different subtypes of EP3receptors using3D-NMR. At the same time, we will also work on the interactionmechanism of the interactions between receptors of each subtypes and that betweeneach transcripts variants, and their relevance to some deseases. Also, the interactionbetween ligands and receptors, ligand mediated internalization and desensitation willalso be paid efforts to.