The Construction And Function Detection Of MGluR2Mutants Used For Drug Screening And Mglur5Mutants Utilized For The Study On The Mechanism Of Desensitization

Metabotropic glutamate receptors(mGluRs) belong to class C GPCRs, and thedisorder of their function is able to induce a number of nervous system diseases, such asAlzheimer s disease, anxiety, dejection, schizophrenia. mGluRs exist as dimers in vivo,and evety monomer consists of VFT(Venus flytrap module), HD(heptahelical domain),Cys-rich domain(CRD) and the intracellular C-terminal. The activity and signaltransduction of mGluRs is modulated by ligands and allosteric modulators. The structureof high operation and the important physical function of mGluRs make them the crititaltarget of drugs that treat nervous system diseases. In addition to being regulated byexogenous ligands, the activity and transduction of mGluRs is also modulated by therelevant intracellular proteins.The low toxin and high genus specificity of Positive Allosteric Modulator(PAM)make it the hot spot for recent drug exploitation. However, the research of the PAMneed to added tow kind of compounds, agonists and PAMs, binging about a large quantityof work and a high signal-to-noise rate, which contribute to the difficulty of the researchprocess. Meanwhile, the VFT deleted mGluRs can be activated by PAMs alone andscreening drugs with this module will undoubtedly diminish the quantity of word andsignal-to-noise rate.For diffirent recepors couple diffirent signal pathways, the traditional drug screeningmethod is combined with the low application. Herein, the VFT deleted and SNAP/CLIPtag inserted mutants of mGluR2are constructed to serve as the Alternative models. Thedeletion of VFT avoids the extra work and high signal-to-noise rate that brought about bythe addition agonists in the process of PAM screening and the utilization of SNAP/CLIPbased TR-FRET averts the low applicaion induced by the combination of varies signalpathways. In this study, a series of alternative cell models were constructed, including theslicing of VFT or CRD, the insertion of SNAP/CLIP into the N-terminal or e2-loop, andthe replacement of C1KKXX/C2KKXX of mGluR2. As the mutants may impact theexpression, especially the membrane expression, the expression of the mutants wasdetected and the VFT deleted mutant was confirmed to have function.In addition, it was confirmed that the mutant, losing VFT with CRD retained, shows a higher signal sthenththan that losing both VFT and CRD.According to reports, the interaction among mGluR1，Gαq and GRK2is needed forthe GRK2mediated mGluR1desensitization. Here the mutants of GRK2RH, PH, D110Awere constructed to demontrate that the expression of RH domain alone is efficient tomediate mGluR5desensitization, which illustrate that it is the RH domain of GRK2that isresponsible for mGluR5desensitization. Meanwhile, D110A, losing the ability to bind tomGluR5, fails to mediate mGluR5desensitizaion, which illuminates the direct interactionbetween GRK2and Gαq is essential for GRK2mediated mGluR5desensitization.Additionally, it was proved by GRK2mutant D527A that the reciprocity between PH ofGRK2and mGluR5is not essential for GRK2mediated mGluR5desensitization.Furthermore, it was demonstrated by i2-loop mGluR5mutants K667A, K678A, K697A,KAA, AAA that the interaction between the i2-loop of mGluR5and GRK2is notnecessary for GRK2mediated mGluR5desensitization, which intimates, in contrast tomGluR1, the GRK2mediated mGluR5do not necessitate the direct interaction betweenthe i2-loop and GRK2.