Rearrangement of the transmembrane domain interfaces associated with the activation of a GPCR hetero-oligomer

Abstract

G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABA B receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABA B1 -GABA B2 , as well as GABA B1 -GABA B1 interactions. Our data are consistent with an oligomer made of a row of GABA B1 . We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers.

Cryo-EM structures of inactive and Gi-coupled GABAB heterodimer

“…2b, Fig. 3d), consistent with previous cross-linking studies 40 . This interface is also observed in the agonist-bound structure of the mGlu5 homodimer 33 , suggesting the TM6/TM6 interface is likely a hallmark of activation of class C GPCR.…”

“…2, Extended Data Fig. 7a, b), propagating through the stalk domains to the TM domains, which in turn reorients the TM interface between two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state, in perfect agreement with previous crosslinking data for GABA B receptor and mGlu homodimers 40,46 . Therefore, the overall domain rearrangements upon activation of GABA B receptor resemble the published cryo-EM structures of mGlu5 33 , suggesting this reorientation of TM domains is possibly the hallmark of class C GPCR activation.…”

The GABAB Receptor—Structure, Ligand Binding and Drug Development

“…Binding studies of isolated GABA B2 subunits and studies manipulating the receptor composition showed that the GABA B2 7TM domain is mainly responsible for recruiting G-proteins, in addition to hosting an allosteric binding site [23][24][25]. There is also evidence from biochemical and biophysical studies that the GABA B1 7TM participates in formation of GABA B -R oligomers by interacting with other GABA B1 7TMs in the inactive state [26][27][28]. The receptor was found to be in equilibrium between heterodimers and oligomers by applying SNAP-tag technology [27].…”

“…Changes in the 7TM subunit interaction interface upon agonist activation of the GABA B -R have been quantified by cysteine cross-linking studies [26], and is also described in the in the newly released papers describing cryo-EM structures [6,7]. The 7TM domain interface interactions in the inactive state is formed by TM3 (GABA B1 )-TM5 (GABA B2 ) and TM5 (GABA B1 )-TM3 (GABA B2 ) interactions and that the interactions between the protomers are facilitated through ionic interactions stabilized by aromatic residues [6,9,26]. The distance between the backbones of TM5 in each monomer is 8 Å and thereby much shorter than the distance between the homodimers in the inactive mGlu5, which is 21 Å [6,44].…”

Capturing Peptide–GPCR Interactions and Their Dynamics

“…Systems investigated via disulfide trapping include ligand-bound class B GPCRs [ 93 , 94 , 95 ], chemokine receptors [ 96 , 97 ], the M3 muscarinic acetylcholine receptor [ 98 , 99 , 100 ], and other receptors [ 101 , 102 ]. Disulfide trapping has been used also to study GPCR dimerization [ 103 , 104 , 105 , 106 ] and to characterize interaction interfaces between GPCRs and G protein or arrestin [ 107 ]. Unfortunately, disulfide trapping does not always yield clear results, probably because of the requirement of nonreducing conditions during analysis of samples [ 93 , 95 , 96 , 98 ].…”