The Construction And Function Detection Of MGluR Constitutive Activity Mutants (CAM)

L-glutamic acid is an important excitatory synaptic neurotransmitter in the mammaliancentral nervous system，which exerts biological effects by binding to the glutamatereceptor. There are two types of glutamate receptors in mammalian neural cells, ionotropicglutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The mGluRsare family C G-protein-coupled receptors that participate in the modulation of synaptictransmission and neuronal excitability throughout the central nervous system, which iswidely involved in learning, memory and neurodegenerative diseases, such as a variety ofphysiological and pathological processes. mGluRs are regulated very precisely in vivo,excessive activation or inhibition will cause a variety of neurological disorders. Therefore,to investigat the constitutive activity of mGluR and get the constitutive activity mutantswill be benificial to the study of GPCR activation and regulation mechanism and drugscreening.This study is based on the viewpiont that the cysteine rich domain (CRD) of themGluRs is directly linked with the activation of the mGluRs. And any of the four disulfidebridges of the CRD are required for the allosteric coupling between the VFT and the7TMdomains. Moreover, a specific crosslinking of the CRDs with intersubunit sidulfide bridgesleads to fully constitutively active receptors. In this study we predict the distribution ofdisulfide bridges in other subtypes mGluRs by bioinformatic, introduction of mutationpoints in other subtypes mGluRs CRD and get14mGluR mutants, as a result, we foundthat the14mGluR mutants are able to expressed correctly. Meanwhile, ELISA results showthat with the exception of mGluR1C524A, the other13mGluR mutants can be expressedin the cell membrane. The IP3experiments revealed that the IP3accumulation betweenthe absence of ligand (Glutamate) binding10mGluR mutants and the activation of wild-type mGluR is without distinction.The results of this study show that the CRD of mGluR4, mGluR5, mGluR6, mGluR7,mGluR8and mGluR2has a similar structure and activation mechanism, the CRD ofmGluR1is very close to the other subtypes in the sequence, but the structure and functionare really difference. The mGluR4C502A, mGluR4H523C, mGluR5C512A, mGluR5 T533C, mGluR6C512A, mGluR6H533C, mGluR7C523A, mGluR7T544C, mGluR8C516A, mGluR8H537C display constitutive activity. This study has made the contribute toinvestigate the activation mechanism of the different subtypes of mGluRs, while helping tobuild the drug screening model and screen negative allosteric modulators.