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.

Fig. 1

Schematic representation of the GABA_B receptor. a GABA_B forms an obligatory heterodimer made of the two subunits GABA_B1 (GB1, blue) and GABA_B2 (GB2, grey). GABA binds to the extracellular domain (ECD) of GB1, while the GB2 heptahelical domain (7TM) is responsible for G-protein activation. b GABA_B has the tendency to form stable higher-order hetero-oligomers that are likely organized through interactions between the GB1 subunits, while GB2 is likely not directly involved in these contacts. c Recently reported loss-of-function genetic mutations in GB2 7TM in human diseases. Most of these mutations affect residues in GB2^TM6 (Gly693, yellow; Ser695, red; Ile705, orange; Ala707, cyan). These mutations produce a constitutively active receptor, except the mutation of Gly693 that has not been studied in functional assays

Fig. 2

Cysteine cross-linking identifies TM5 and TM6 at the 7TM heterodimer interface. a Schematic representation of the GB1^Ctr and GB2^Ctr constructs used in the study. To easily distinguish GB1-GB2 and GB1-GB1 cross-linking in SDS-PAGE experiments, the molecular weight of the two subunits was modified. The SNAP-tagged full-length GB1 was truncated in the C-terminal region downstream of the coil-coiled region. Halo-tagged full-length GB2 was enlarged by adding a GFP tag at the C-terminal end of the subunit. To prevent the endogenous Cys producing unwanted disulphide bridges, the two indicated Cys residues in GB2^TM4 were changed to alanine. b 3D model of the 7TM of GB1 (blue) and GB2 (grey). All cysteine substitutions are highlighted by a yellow ball (α carbon), and those that cross-linked well in TM5 and TM6 (see panel c) by a red ball. c Cross-linking of the indicated cell surface SNAP-GB1 subunits labelled with fluorescent SNAP substrates, after treatment (+) or without treatment (−) with CuP. After SDS-PAGE in non-reducing conditions, GB1 monomers and GB1-GB2 dimers were detected via the fluorophore covalently attached to the receptors. MW, molecular weight. Data are representative of a typical experiment performed three times. d Change of GB1-GB2 dimer rate induced by CuP treatment for the “Control” heterodimer (GB1^Ctr co-expressed with GB2^Ctr) and every indicated mutant (both GB1 and GB2 subunits having a Cys residue in the same position). Positions with a significant change were highlighted in red. Data are mean ± SD from at least three independent experiments (n = 3–6). Unpaired t test with Welch’s correction with ****P < 0.0001 and ***P < 0.001, the other data being not significant. e Dimerization interface based on the results of the cross-linking experiments in the absence of ligand. TMs that can cross-link between GB1 and GB2 are highlighted in red

Fig. 3

The interface of the heterodimer is switched from TM5 to TM6 during activation. a, b The cell surface SNAP-GB1 containing the indicated single cysteine substitution was cross-linked with the indicated GB2 cysteine mutant. The results were obtained for the symmetric interface TM5 and TM6, after pre-incubation with the agonist GABA or the competitive antagonist CGP54626 and with CuP. The percentage of GB1-GB2 heterodimers (in red) and GB1-GB1 homodimers (in blue) relative to the total amount of GB1 subunit was quantified by imaging the fluorescent blots. c Change of GB1-GB2 dimer rate induced by the agonist and determined by GB1-GB2 dimer quantification before and after GABA treatment. Data are mean ± SD from at least three independent experiments (n = 3–5). Unpaired t test with Welch’s correction with ****P < 0.0001, ***P < 0.001 and **P < 0.01, the other data being not significant. GABA and CGP54626 were used at 100 μM. d Model highlighting the TMs involved in the dimerization of GB1-GB2 heterodimers in the inactive state (TM5, yellow) and in the active state (TM6, red)

Fig. 4

Disulfide cross-linking confirms the GB1-GB2 TM6 active interface and the resting interface. a Inositol phosphate (IP) production in cells that co-express the mutants GB1^6.59 and GB2^6.59 after treatment with or without CuP, and stimulation with GABA. Results are mean ± SD from three independent experiments performed in triplicates. b In both the control receptor (after stimulation with GABA) and the co-expressed mutants GB1^6.59 and GB2^6.59, IP production is proportional to the amount of SNAP-tagged GB1 at the cell surface, as measured by fluorescence after labelling with SNAP-Red substrate and then treatment with CuP. GABA was used at 100 μM. Data are mean ± SD from a typical experiment performed three times. c Treatment with the indicated competitive antagonist does not reverse the constitutive activity after GB1-GB2 TM6s cross-linking. GABA and CGP54626 were used at 1 and 10 μM, respectively. Data are mean ± SEM from a typical experiment performed three times. Unpaired t test with Welch’s correction with *P < 0.1, ns, not significant. d–f Stabilizing the inactive GB1-GB2 interface (d) by co-expressing the indicated mutants that cross-link well upon CuP treatment (e), impairs IP accumulation induced by GABA (f). Data are mean ± SD from a typical experiment performed three times

Fig. 5

Rearrangement of the transmembrane domain interface during GABA_B heterodimer activation 3D model of the GB1-GB2 7TM heterodimer (a) and mGluR2 7TM homodimer (b) in the resting and active orientations. Based on these models, the amplitude of the relative reorientation of two 7TMs in the dimer might be smaller in the GABA_B receptor than in mGluR2

Fig. 6

Interactions between GB1 7TMs in GABA_B oligomers during activation. a, b Schematic representation of a GABA_B oligomer in lateral (a) and top view (b). c, d Blots showing cross-linking of cell surface SNAP-GB1 subunits containing a single cysteine substitution in TM1, TM4, TM5, TM6 or TM7, with GB2^Ctr after pre-incubation or not with GABA (agonist) and with CuP, as indicated. The percentage of GB1-GB1 homodimers (in red) relative to the total amount of GB1 subunit was quantified from the fluorescent images. Data are mean ± SD from three independent experiments. Paired t test with Welch’s correction with ****P < 0.0001, ***P < 0.001 and **P < 0.01, or not significant (ns). e, f Model for the structural organization of the GABA_B 7TMs in higher-order oligomers in the inactive and active state. Interfaces at the GB1 subunits are highlighted

Fig. 7

High-molecular-weight complexes confirm the two interfaces between GB1s in oligomers. a Quantification of the oligomers obtained after cross-linking of the double cysteine substitution in different TMs of the GB1 subunit, after pre-incubation or not with GABA and with CuP, as indicated. The percentage of oligomers (in purple) relative to the total amount of GB1 subunit was quantified from the fluorescent blots. The pictograms indicate the possible cross-linking of three GB1 subunits that could form the oligomer band of the corresponding blot. These schemes are from snapshots of the GABA_B oligomer 3D model when morphing are performed between the inactive and active states (see Figs. 9a, b). GABA was used at 100 μM. Data are mean ± SD of at least three individual experiments (n = 3–5). Paired t test with Welch’s correction with ***P < 0.001, **P < 0.01 and *P < 0.1, the other data being not significant (ns). b Quantification of the oligomers (% of total GB1) obtained for the indicated pairs of cysteines in panel (a), after cross-linking in presence of CuP and GABA. c Model of the 7TM of GABA_B oligomers highlighting the two distinct and possible interfaces between the GB1 subunits during activation

Fig. 8

A disease-causing mutation stabilizes the active interface of the dimer and oligomer. a, b Quantification of the GB1-GB2 cross-linking for the GB1^6.56 and GB2^6.56 cysteine mutants containing or not the genetic mutation S695I^6.42 in the GB2 subunit, in the indicated conditions and as described in Fig. 3. Both cysteine mutation and the genetic mutation have been introduced in the rat GB1^Ctr and GB2^Ctr. c, d Quantification of the GB1-GB1 cross-linking for the GB1^5.42 single cysteine mutant co-expressed with GB2^Ctr containing or not the genetic mutation S695I^6.42. GABA and CGP54626 were used at 100 μM. Data are mean ± SD from at least three independent experiments (n = 3–5). Unpaired t test with Welch’s correction with ****P < 0.0001, or not significant (ns)

Fig. 9

Agonist-induced rearrangement of the 7TMs in the GABA_B oligomer during activation. a, b 3D model of the 7TM oligomer in the inactive and active orientations. The dashed line highlights a minimal functional receptor made of GB1 and GB2 (heterodimer B). Heterodimer A is proposed to assemble the tetramer with B, and C to form oligomer with the tetramer A-B. In this model, stabilization of the tetramer interactions is made by the symmetric interfaces with GB1^TM6 in the resting state, and with GB1^TM1−TM7 in the active state. Stabilization of the oligomer would be through the symmetric interfaces with GB1^TM4 in the resting state and with GB1^TM4−TM5 in the active state. TM4, TM5 and TM6 of GB1 and GB2 are in yellow, green and red, respectively. c Model of the active oligomer coupled to four Gαβγ proteins based on the structure of the complex between the active β₂-adrenergic receptor and the G protein previously reported