Rearrangement Of The Transmembrane Domain Interfaces Associated With The Activation Of A GPCR Hetero-Oligomer

G protein-coupled receptors?GPCR?are major players in cell communication.They all contain a seven transmembrane domain?7TM?responsible for G protein activation.Although they can activate G proteins as monomers,they also form dimeric and even oligomeric complexes.GPCR complexes likely play roles in signal integration through their associated receptors.However,the molecular and structural basis for allosteric interaction in these complexes remain elusive.As a model,we used the?-amino-butyric acid receptor type B?GABA_BR?,since the GABA_BR functional unit is the first mandatory heterodimer of two homologous subunits GABA_B1?GB1?and GABA_B2?GB2?.GABA_B receptors have the propensity to form stable hetero-oligomers organized through interactions between the GB1 subunits in vivo.Allosteric interactions between the 7TM of GB1 and GB2 within heterodimer,and heterodimeric units within such oligomers have been identified.As an important inhibitory neurotransmitter receptor in the central nervous system,GABA_BR represents a promising target in drug development for the treatment of multiple neurological diseases such as Rett syndrom and Epileptic encephalitis.Despite the known structure of the active and inactive heterodimeric extracellular domain,little is known about 7TM structure.To Clarify the structural bases of the allosteric interaction between GABA_B subunits,we studied the dynamic conformation changes of GABA_BR transmembrane domain in heterodimer and oligomer during activation.In our study,we used cysteine cross-linking associated with biochemical appoaches to identify the transmembrane domain interfaces in dimer and oligomer.The use of the SNAP-and Halo-tag techonologies associated with cell-impermeant SNAP-and Halo-tag fluorescent substrates allowed us to specifically analyze surface receptors,including their conformational state during activation.Our study brings important information on the structural dynamics of a GPCR oligomer at the plasma membrane of living cells:?i?We demonstrate that a major concerted movement between the transmembrane domains in GABA_BR heterodimer during activation.While TM5 is the main dimer interface of the inactive state,the TM6 is involved in the active interface.By locking of GB1-GB2 TM6 interface,we observed a robust constitutive activity,suggesting that this major rearrangement is required for the activation by GABA and orthosteric ligands.?ii?Two main interfaces are formed between the GB1 subunits in the GABA_BR oligomers,one being TM4/5 and the other TM1/6/7 interface.In the inactive state,one GB1 subunit forms two different interfaces with two other GB1 subunits,one mediated by TM4 and the other by TM6.During activation,two novel interfaces are formed,one being TM4/TM5 by rotation of TM4 and the other TM1/7 by rotation of TM6.?iii?We propose a 3D model of well-organized oligomers both in the inactive and active state,based on our experimental results.In this model,we propose that the minimal organization within this oligomer are tetramers organized in arrays.The GABA_B tetramer has a rhomboid shape structure with GB1 subunits that form rows whereas the GB2subunits are in side branches.?iv?A disease-causing naturally occurring mutation stabilizes the active interface of both the heterodimer and oligomer.This is a complete characterization of the transmembrane interface of a well recognized GPCR oligomer,that will guide studies of other cell surface proteins.The orientation of GB1 TM6 in the oligomer interfaces is shown here to be critical for the activation process.This provides information for our understanding of the cooperativity phenomena within GPCR dimers and oligomers.TM6 is the major site of genetic mutations of GABA_B in human diseases,our study will therefore likely have therapeutic consequences.