Structural And Functional Studies Of Metabotropic Glutamate Receptors

Metabotropic glutamate receptors（m Glus）belong to class C G protein-coupled receptors（GPCRs）,which are mostly distributed in the central nervous system and play important roles in synapses.Upon binding to glutamate,one of the major excitatory neurotransmitters,the m Glus are activated to exert their functions in regulating neurotransmitter release and synaptic plasticity.It is well established that the m Glus are involved in many important physiological functions such as learning,memory and emotion.Furthermore,these receptors have been considered as promising drug targets for the treatment of neurodegenerative and neuropsychiatric diseases,including Parkinson’s disease,depressive disorder,anxiety disorder and schizophrenia.According to sequence homology and downstream signal pathway,the m Glus are divided into three subgroups,group I（m Glu1 and m Glu5）,group II（m Glu2 and m Glu3）and group III（m Glu4 and m Glu6-8）.The group I receptors couple to the G_q protein that induces PLC activation,while group II and group III receptors couple to the G_i protein that reduces the accumulation of c AMP.The class C GPCRs contain a large extracellular domain and a canonical seven transmembrane region.Distinct from the other GPCRs,it is mendatory for class C receptors to form homo-or heterodimer to excert their functions.However,due to lack of structural information,how the m Glus recognize downstream signaling transducers such as the G protein and how the subunits in the m Glu dimers cooperate to transduce the signal remain largely unknown.Here,we report the G_i-bound structures of m Glu2 and m Glu4,two representatives of group II and group III m Glus.We extensively screened and optimized the conditions of complex assembly and purification and obtained stable protein samples.Using cryo-electron microscopy（cryo-EM）single-particle analysis,we solved the structures of m Glu2-G_i1 and m Glu4-G_i3 complexes.These two structures reveal a similar asymmetric signal transduction mode of m Glu2 and m Glu4,in which an m Glu dimer coupling to only one heterotrimeric G_i protein.These two m Glus exhibit an unique G_ibinding pattern,with theα5 helix of the G_iprotein binding to a shallow groove formed by three intracellular loops and the intracellular tips of TM3 and TM4 of the receptors.During receptor activation,the two subunits in the m Glu2 and m Glu4 homodimers form an asymmetric dimer interface,which is composed of TM6,TM7 and the extracellular tips of TM1 of the G_i-free subunit and TM6 and the intracellular tip of TM5 in the G_i-bound subunit.Supported by our mutagenesis and functional data,this asymmetric dimer interface was proved crucial for receptor activation.Also,we illustrated the binding mode of m Glu2 with its positive allosteric modulator（PAM）JNJ-40411813and the mechanism of the PAM in modulating the receptor function.Combined with the structures of m Glu2 at inactive and intermediate states,the m Glu2-G_i1 and m Glu4-G_i3 structures provide a full picture of conformational changes during m Glu activation.There is increasing evidence suggesting that the m Glus can form not only homodimers but also heterodimers with distinct physiological and pharmacological profiles.Previous studies indicated the existence of m Glu2-m Glu7 heterodimer in the hippocampus.The m Glu2-m Glu7 heterodimer has distinct pharmacological profiles from the m Glu2 and m Glu7 homodimers,which indicates complexity and diversity of the funcation modulation mechanism of the m Glu family.To explore the assembly and signaling mechanisms of the m Glu heterodimer,we solved the structure of m Glu2-m Glu7 heterodimer at inactive state.We introduced two fusion proteins,FK506 binding protein（FKBP）and rapamycin binding fragment（FRB）,to the C termini of m Glu2 and m Glu7,respectively,to facilitate heterodimerization.We then used tandem affinity chromatography to purify the heterodimer and solved the structure of the m Glu2-m Glu7 heterodimer using single-particle cryo-EM.The TMD assembly pattern of the m Glu2-m Glu7 heterodimer is similar to that of the m Glu7 homodimer,but not the m Glu2 homodimer,implying a dominant role of the m Glu7 subunit in the signaling of the m Glu2-m Glu7 heterodimer.This was further supported by our functional studies,which showed that the m Glu7 subnuit was responsible for G protein coupling.Collectively,these results,for the first time,offer structural evidence for the study of the m Glu heterodimer.In the first study,we solved the structures of G_i-bound m Glu2 and m Glu4.Together with the functional data,these structures provide molecular details of signal transduction of the m Glus.In the second study,we sovled the inactive structure of the m Glu2-m Glu7 heterodimer and performed cell signaling studies,which offer insights into the molecular mechanisms of function modulation and signal transduction of the m Glu heterodimer.These findings deepen our our understanding of the functionality of the m Glus in the central nervous system and extend our knowledge about the signal transduction of class C GPCRs.