代谢型谷氨酸受体组成性活性突变体的构建和功能检测-生物化学与分子生物学专业论文.docx

华中科 技 大学 华 中 科 技 大 学 硕 士 学 位 论 文 II II 关键词：代谢性谷氨酸受体，半胱氨酸富集区，组成性活性，反向变构剂，药物筛 选，G 蛋白偶联受体 III III Abstract L-glutamic acid is an important excitatory synaptic neurotransmitter in the mammalian central nervous system，which exerts biological effects by binding to the glutamate receptor. There are two types of glutamate receptors in mammalian neural cells, ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The mGluRs are family C G-protein-coupled receptors that participate in the modulation of synaptic transmission and neuronal excitability throughout the central nervous system, which is widely involved in learning, memory and neurodegenerative diseases, such as a variety of physiological 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 mutants will be benificial to the study of GPCR activation and regulation mechanism and drug screening. This study is based on the viewpiont that the cysteine rich domain (CRD) of the mGluRs is directly linked with the activation of the mGluRs. And any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. Moreover, a specific crosslinking of the CRDs with intersubunit sidulfide bridges leads to fully constitutively active receptors. In this study we predict the distribution of disulfide bridges in other subtypes mGluRs by bioinformatic, introduction of mutation points in other subtypes mGluRs CRD and get 14 mGluR mutants, as a result, we found that the 14 mGluR mutants are able to expressed correctly. Meanwhile, ELISA results show that with the exception of mGluR1 C524A, the other 13 mGluR mutants can be expressed in the cell membrane. The IP3 experiments revealed that the IP3 accumulation between the absence of ligand (Glutamate) b