Structural And Functional Studies Of Metabotropic Glutamate Receptor 3

G-protein coupled receptor(GPCR)super family is the largest membrane protein receptor family in human beings,which can sense a variety of extracellular signaling molecules and regulate intracellular activities.Glutamate is the major excitatory neurotransmitter in the brain and is responsible for excitatory transmission between neuron cells.Metabotropic glutamate receptor(mGluR)belongs to class C family GPCR and responsible for the regulation of glutamate.There are eight members in this family and can detect levels of glutamate concentration from the tens of nanomolar to the tens of millimolar.The mGluR3 receptor is of much interest as it is the one responsible for the detection of very low concentrations of glutamate.mGluR3 is also considered as a potent therapeutic target for both psychiatric disorders and neurodegenerative diseases such as schizophrenia,anxiety,depression,pain and addiction.However,due to the high homology of mGluR3 and mGluR2,the development of selective ligand for mGluR3 is a great challenge,and no FDA-approved drug targeting mGluR3 has been marketed so far.In addition,the structures of the full-length mGluR3 have not been solved,and the understanding of the activation mechanism and the allosteric ligand induced regulation mechanism of mGluR3 is relatively limited.In this dissertation,we studied the structure and function of mGluR3 by cryo-EM and mutation assay.Firstly,we studied the activation mechanism of mGluR3 by cryo-EM and functional assay.We solved the structures of LY341495 bound mGluR3 in the inactive state and LY2794193 bound mGluR3 in the active state with the resolution of 4.2 ? and 3.7 ?,respectively.The interaction between M-methoxyphenyl on LY2794193 and residues of orthosteric binding pocket enhances the affinity of the ligand,and the interaction with D279 is the molecular basis of the ligand’s selectivity to the receptor subtype.Upon agonist binding to mGluR3,the extracellular VFTD(Venus flytrap domains)was closed and CRD(cystine rich domains)was twisted that caused the TMD(transmembrane domains)interface rearranged from the TM5 in the inactive state to TM6 in the active state,which was conserved in class C family GPCR.Our functional data suggest that the interaction between CRD and ECL2 on TMD is critical for receptor activation and transduction.Then we focused on the allosteric ligand induced regulation mechanism of mGluR3.We solved structure of LY341495 and VU0650786 bound mGluR3 in the full inactive state with resolution of 3.7 A by adding NAM VU0650786 to inactive mGluR3 sample.NAM binds to the central region of TMD,and the allosteric binding pocket of NAM are formed by residues from TM3,TM5,TM6 and TM7.Comparison of these three different state structures of mGluR3,we found that NAM induced TMD rotation in opposite direction compare with agonist mediated TMD rotation.Upon NAM bound to mGluR3,the TMD interface rearranged from TM5 of the inactive state to TM3/TM4 of full inactive state and the distance between TMD of two subunits reduced from 21.9 ? to 11.6 ?,which caused the receptor more difficult to rotate to TM6 interface of active state,thus inhibiting activity of the receptors.For the purpose of mGluR3-G protein complex structural study,we obtained the agonist-mGluR3-Gai1β1γ2-scFv16 complex sample after preliminary condition optimization.The negative staining EM results showed that complex particles has formed,but the current complex sample was unstable and in bad homogeneity.It requires more efforts on optimizing the stability of the complex for high cryo-EM sample preparation.In this dissertation,we successfully solved three different state structures of mGluR3 by cryo-EM,and the activation mechanism and allosteric modulation mechanism of mGluR3 were revealed through structural analysis and functional assay.In addition,the binding mode of orthosteric ligand and NAM also provided a structural basis for the development of selective orthosteric and allosteric ligands for mGluR3.