Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

The heterodimeric human GABAB receptor was assembled using baculovirus-infected mammalian cells. Each subunit was truncated at the carboxyl terminal end according to the domain boundary of the intracellular coiled-coil[17] to eliminate flexible regions (Supplementary Fig. 1). Upon extraction and purification with detergent (Extended Data Fig. 1a,b; Supplementary Fig. 2), the complex bound radioactive [3H]GABA with a dissociation constant comparable to the GABA affinity reported for native receptors[3,4,7] (Extended Data Fig. 1c). Functional analysis incorporating a chimeric Gαqi5 protein and inositol phosphate (IP) assay[18] in mammalian cells showed that agonist baclofen activated the C-terminally truncated and full-length receptor with similar potency and efficacy, indicating that the deleted regions in the GABAB1b and GABAB2 cytoplasmic tails are not required for ligand-mediated G protein activation (Extended Data Fig. 1d).

Extended Data Fig. 1 |

Purification and functional analysis of human GABAB receptor.

a, Superose 6 size exclusion chromatography profile of detergent-purified GABAB1b(1–802)-GABAB2(1–819) complex.

b, SDS PAGE gel of size exclusion peak fraction from (a) under reducing conditions. For gel source data, see Supplementary Fig. 2.

c, Dose-dependent [3H]GABA binding to purified GABAB1b(1–802)-GABAB2(1–819) complex, reaching maximum molar ratios of GABA-to-receptor binding at 0.98 ± 0.03 mol/mol. Data points represent the mean of triplicate measurements of a typical experiment. The experiment was repeated four times with similar results. Data were subjected to non-linear regression fitting, and the kinetic constants are reported as mean ± s.e.m. of the fit.

d, Functional analysis comparison of full-length and truncated WT GABAB receptor. Dose-dependent baclofen-stimulated receptor response in cells transiently expressing Gαqi5 (abbreviated as Gqi) with full-length GABAB heterodimer or the C-terminally truncated GABAB1b(1–802)-GABAB2(1–819) complex. Cells transfected with Gαqi5 alone were used as negative control. Relative agonist-stimulated activity was measured by IP1 accumulation and expressed as percentage of maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent average ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. Cell surface expression level was 106% for the GABAB1b(1–802)-GABAB2(1–819) complex in comparison with the full-length WT/WT heterodimer.

We determined the structure of the heterodimeric GABAB receptor by cryo-EM to an overall resolution of 3.3 Å (Extended Data Fig. 2a–e; Supplementary Table 1). The global density map displayed directional anisotropy due to linker flexibility (Extended Data Fig. 2f). Performing local refinement separately on the extracellular (ECD) and TM domains yielded 3.1 Å and 3.4 Å resolution reconstructions, respectively (Extended Data Figs. 2g,h). A composite map combining the ECD and TM reconstructions was used for model building and refinement (Extended Data Fig. 3). By applying three-dimensional variability analysis of the data, we found that the receptor is in dynamic motion, and its functional state corresponds to a continuum of conformations along multiple dimensions (Supplementary Video 1–4).

Extended Data Fig. 2 |

Cryo-EM imaging of human GABAB receptor.

a, Workflow of cryo-EM data processing.

b, A representative motion-corrected cryo-electron micrograph of GABAB receptor.

c, Reference-free 2D class averages highlighting clear density for TM helices.

d, Global density map colored according to local resolution in full and clipped views perpendicular to the plane of the membrane.

e, Global FSC curve (purple) corrected by high-resolution noise substitution. The overall resolution as determined by an FSC cut-off value of 0.143 (blue line) is 3.3 Å.

f, 3D-FSC curves measuring directional resolution anisotropy. Plots show global half-map FSC (red solid line), together with the spread of directional resolution values within ± 1 standard deviation of the mean (area encompassed by green dash lines), and a histogram of 100 such directional resolution values sampled evenly over the 3D-FSC threshold value of 0.143 (blue, right axis). The sphericity value reported by 3D-FSC is 0.958 out of 1.

g,h, Separate FSC curves for the locally refined reconstructions of ECD (g) and TM (h) domains. Blue line marks the resolution corresponding to an FSC value of 0.143 (ECD: 3.1 Å; TM: 3.4 Å). The inset shows the mask used for each local refinement.

Extended Data Fig. 3 |

Structural model of GABAB receptor fit within the cryo-EM map.

a-d, Cryo-EM density map and refined model are shown for the LB1 interface helices (H-B and H-C) in the extracellular VFT (a), the linker between VFT and TM domain (b), all seven TM helices of GABAB1b subunit (c) the ten modeled transmembrane cholesterols (d) and all seven TM helices of GABAB2 subunit (e). The density map is a composite of the locally refined reconstructions for the ECD and TM domains. The N- and C-terminal residues of each helix are labeled.

The heterodimeric GABAB receptor is assembled by GABAB1b and GABAB2 subunits interacting side-by-side while facing opposite directions (Fig. 1a,b; Extended Data Fig. 4). Both the VFT and TM components of the two subunits are related by pseudo two-fold axes. Extracellular and intracellular loops (ECLs and ICLs) that interconnect adjacent helices within each TM are visible in the density map, except for ICL2. The cytoplasmic tail including the coiled-coil domain is disordered, possibly due to its flexible attachment to the TM domain.

Fig. 1 |

Cryo-EM structure of human GABAB receptor.

a, Ribbon representation of GABAB receptor structure composed of GABAB1b (blue) and GABAB2 (green) subunits. Ca2+ (green): sphere. Phospholipids (PE 38:5; PC 38:2; yellow): space-filling models. N-linked glycans (NAG, gray) and cholesterols (CLR, pink): ball-and-stick models. TM helices 1 through 7 are marked for each subunit.

b, Cryo-EM density map of GABAB receptor composed of local reconstructions for extracellular (3.1 Å) and TM (3.4 Å) domains, in an orthogonal view from (a).

c, Linker domains of GABAB1b and GABAB2, showing the main-chain and side-chain hydrogen-bonding patterns between linker region and ECL2.

Extended Data Fig. 4 |

Architecture of GABAB receptor.

a, GABAB receptor structure in four views related by 90°-rotations about an axis perpendicular to the membrane. GABAB1b (blue) and GABAB2 (green) subunits are rendered as cartoon, while Ca2+ (green) is shown as a sphere. Phospholipids (PE 38:5 and PC 38:2; yellow) are presented as space-filling models. The observed N-linked glycans (NAG, gray) and cholesterols (CLR, pink) are in ball-and-stick models. TM helices 1 through 7, along with N- and C-termini, are marked for each subunit.

b, Cryo-EM density map of GABAB receptor, in the same orientation and color scheme as (a). The map is composed of local reconstructions for the ECD and TM domains, which were independently refined to 3.1 Å and 3.4 Å, respectively.

c, Linker and TM domain of GABAB receptor viewed from the extracellular and intracellular sides.

The elongated peptide linker joining the VFT to the TM is buttressed through its interaction with a β-hairpin formed by ECL2 (Fig. 1c). ECL2 twists across the linker, forming a united mechanical junction to transmit the conformational changes in the VFT to the TM and vice versa. Additionally, the ECD and TM domains of the receptor spontaneously flex back and forth about the linker, exhibiting the region’s intrinsic flexibility (Supplementary Video 1).

Since no ligand was added during protein purification, we expected the receptor to be in an apo form and inactive conformation. To our surprise, we observed multiple ligands bound to the receptor. GABAB1b contains a Ca2+ at the interdomain cleft of VFT. In addition, an endogenous phospholipid is bound within the TM pocket of each subunit, with a phosphatidylethanolamine (PE 38:5) in GABAB1b, and a phosphatidylcholine (PC 38:2) in GABAB2. Finally, ten cholesterol or cholesteryl hemisuccinate molecules, which we modeled as cholesterol (Methods), are distributed around the exterior of the TM complex, including two that interface both subunits.

Inactive conformation of GABAB receptor

The cryo-EM structure of GABAB receptor occupies an inactive conformation based on its similarity to the known crystal structures of GABAB VFT in the apo and antagonist-bound states[16]. First, the VFT module, composed of LB1 and LB2 domains, adopts an open interdomain conformation in both subunits (Extended Data Fig. 5a,b). Second, while an N-terminal LB1-LB1 dimer interface is present in all functional states, the distinct lack of a heterodimer interface between the membrane-proximal LB2 domains is shared by the near full-length cryo-EM structure and inactive-state VFT structures (Extended Data Fig. 5c,d). In contrast, a hallmark of the active-state VFT structures is a novel heterodimer interface between LB2 domains, which results from agonist-induced GABAB1b closure[16].

Extended Data Fig. 5 |

Heterodimer conformation and interface features of the GABAB receptor.

a,b, Cryo-EM structure of near full-length GABAB receptor (cyan) superimposed with crystal structure of its extracellular VFT module in the inactive-state (PDB code: 4MQE; purple) (a) or active state (PDB code: 4MS3; red) (b). The middle panel shows the heterodimeric receptor structures superimposed based on the LB1 domain of GABAB1b subunit. The two side panels show superposition of individual GABAB1b and GABAB2 subunits based on their respective LB1 domains. In (b), Green line denotes the axis of rotation that relates the LB2 domains of near full-length and VFT structures of GABAB1b (rotation χ = 28°, screw translation τχ = 0.6 Å), or near full-length and VFT structures of GABAB2 (rotation χ = 7°, screw translation τχ = 0.3 Å).

c,d, Extracellular LB2 domains viewed from the C-terminal end. Superposition of near full-length (cyan) and extracellular VFT structures (inactive state: purple (c); active state; red (d)) was based on the LB1 domain of GABAB1b subunit. Within each heterodimeric complex, the C-termini of the LB2 domains in GABAB1b and GABAB2 subunits are shown as spheres, and the distance between the two C-termini is marked by a dotted line.

e, Cryo-EM structure of full-length mGlu5 in the inactive (PDB code: 6N52) and active (PDB code: 6N51) conformations[20].

f, Molecular surface of GABAB1b-GABAB2 complex showing the plane of heterodimer interface for extracellular and TM domains. Structural elements involved in heterodimer formation are highlighted in cartoon (ectodomain: H-B and H-C helices; TM domain: TM5 and TM3 helices).

g, GABAB TM domain viewed from the extracellular side comparing the locations of core (I, II, IIIa, IIIb) vs. peripheral cholesterol-mediated heterodimer contacts from different layers. Heterodimer contacts mediated by two cholesterols (CLR6, CLR3) are displayed in panels at the bottom.

Using conformational variability analysis, we observed that the LB2 domains of both subunits fluctuate by approaching and withdrawing from the central vertical axis of the heterodimer, yet never make contact as in active-state VFT crystal structures (Supplementary Video 2,3). The motion exhibited by the LB2 domains and their associated linkers suggests that the inactive functional state corresponds to an ensemble of conformations, where the separation between the membrane proximal regions falls within a small range around the coordinates of the current structure.

TM heterodimer interface in the inactive state

We identified a novel heterodimer interface between the TM5 and TM3 helices of both subunits, that embodies the signature of the GABAB inactive conformation (Fig. 2a). A TM5-TM5 contact has previously been detected through crosslinking of the GABAB receptor[19], but TM dimer interfaces of any kind are yet to be found in other inactive class C GPCRs, including the recent inactive metabotropic glutamate receptor mGlu5 structure[20] (Extended Data Fig. 5e).

Fig. 2 |

Transmembrane heterodimer interface of GABAB receptor.

a, TM heterodimer interface formed by TM5 and TM3 of both subunits. Three layers (I, II, III) of interfacial contacts are identified by dotted circles. Direct heterodimer contacts within each layer are displayed in panels on the right.

b,c, Functional analysis of the ′intersubunit latch′. Basal activity (b) and dose-dependent baclofen-stimulated receptor response (c) in cells transiently expressing the Gαqi5 chimera protein (abbreviated as Gqi) with different combinations of wild-type (WT) and mutant GABAB subunits (GABAB1b-E673R, abbreviated as E673R; GABAB2-H579E, abbreviated as H579E). IP1 accumulation was measured in the presence and absence of 20 μM CGP54626 (abbreviated as INV). Cells transfected with empty pcDNA3.1 vector, Gαqi5 alone, or WT GABAB subunits in the absence of Gαqi5 were used as controls. Relative activity is expressed as percentage of maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent average ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. ****P<0.0001, one-way ANOVA with Bonferroni’s post hoc test was used to calculate statistical differences in basal activity (b). Cell surface expression level was 90% for the E673R/WT and 76% for WT/H579E mutants in comparison with the WT/WT heterodimer.

Positioned at 30° from the extracellular dimer interface, the TM dimer interactions bury approximately 740 Å2 of surface area and exhibit high shape complementarity (Extended Data Fig. 5f). The pair of TM5 helices scissor at their central residues before contacting the transverse TM3 of opposing subunits at their intracellular ends (Fig. 2a). TM5 extracellular ends display conformational variance wherein they approach and withdraw but fail to make contact (Supplementary Video 4). All direct heterodimer interactions occur near the cytoplasmic membrane surface and can be divided into three core layers (I-III) along the helical path of TM5 (Fig. 2a).

The surface layer I caps the extracellular end of the TM heterodimer interface. It is comprised of hydrophobic contacts between four leucine residues of both subunits. The middle layer II lies directly beneath layer I, and consists of three phenylalanine residues packing against one another, as well as their neighboring leucine residues. Both layers solely incorporate TM5 residues.

Layer III, consisting of sections IIIa and IIIb, completes the interface at the intracellular end. IIIa possesses a network of salt-bridges that tether the cytoplasmic ends of GABAB1b and GABAB2 TM domains. This critical interaction is mediated by a quartet of charged residues from TM3 and TM5 helices (GABAB1b: His5723.55 and Glu6735.60; GABAB2: His5793.55 and Glu6775.60) (Supplementary Table 2), a feature we refer to as the ′intersubunit latch′ for securing the TM orientation of the two subunits in the inactive conformation. One of the ′intersubunit latch′ residues shares nonpolar contacts with a lysine of GABAB2 TM5, establishing the accessory site IIIb.

In addition, the central layers of direct heterodimer contacts are flanked on each side by a cholesterol molecule. One mediates the interaction between TM5 helices of both subunits (CLR3), while the other bridges TM3 of GABAB1b and TM5 of GABAB2 (CLR6) (Extended Data Fig. 5g).

The ′intersubunit latch′

To determine the importance of the ′intersubunit latch′ in controlling the inactive state of GABAB receptor, we examined the effect of single charge-repelling mutations (GABAB1b-E673R; GABAB2-H579E) within the motif. Wild-type GABAB receptor exhibited basal activity in the absence of agonist (Fig. 2b), as previously reported[14]. Both mutants substantially increased basal activity when compared to wild-type, suggesting that each mutation promotes agonist-independent constitutive activity (Fig. 2b). The basal activity displayed by these mutants reached approximately 80–90% of the maximal agonist-dependent wild-type response (Fig. 2b,c). Application of the agonist baclofen raised receptor activity further to the highest wild-type level (Fig. 2c). Neither mutation altered the agonist potency. After treating each construct with the inverse agonist CGP54626, the basal activity of mutants still remained higher than that of wild-type, providing further evidence that the ′intersubunit latch′ mutations serve to shift the conformational equilibrium of GABAB receptor towards an active state. Taken together, our mutational data indicate that the ′intersubunit latch′ is fundamental to maintaining the inactive state of the receptor.

Endogenous ligands bound to GABAB1b VFT

We identified a novel potential Ca2+-binding site in the vicinity of the orthosteric ligand-binding cleft on the LB2 surface of GABAB1b (Fig. 3a; Extended Data Fig. 6a,b). The metal ion density has remarkable peak height in the cryo-EM density map (10σ) and is surrounded by residues chemically favorable for Ca2+ coordination. The Ca2+ is anchored by the carboxylate groups of three acidic residues (Asp281, Glu309, and Glu423) as well as the backbone carbonyl atoms of two additional residues (Gly277 and Tyr279) (Fig. 3b). The Ca2+-oxygen bond distances are between 3.0 Å and 4.6 Å, suggesting that the ion is in a hydrated state. The Ca2+ location in GABAB1b is different from any of the multiple sites found in calcium-sensing (CaS) receptor[21], another class C GPCR (Fig. 3c).

Fig. 3 |

Ca2+ binding in GABAB1b.

a, Ribbon representation of GABAB1b subunit showing the location of Ca2+-binding site at the interdomain crevice of VFT.

b, Specific interactions between GABAB1b and Ca2+. Mesh represents the cryo-EM density map contoured at 7.5 σ surrounding Ca2+.

c, CaS receptor ECD crystal structure (PDB code: 5K5S) highlighting its bound Ca2+ (black spheres, numbered 1–4 and 1′−4′ in the two protomers) and the corresponding location of the Ca2+-binding site in GABAB1b (green sphere).

Extended Data Fig. 6 |

Extracellular ligand binding in GABAB1b.

a,b, Molecular surface (a) and ribbon representation (b) of GABAB1b subunit showing the location of Ca2+-binding site and an unmodeled density at the interdomain crevice of VFT.

c,d, Functional analysis of the impact of endogenous Ca2+. Basal activity (c) and dose-dependent baclofen-stimulated receptor response (d) in cells transiently expressing the Gαqi5 (abbreviated as Gqi) with different combinations of WT and mutant GABAB receptor subunits (GABAB1b-E309K, abbreviated as E309K; GABAB1b-E423R, abbreviated as E423R). IP1 accumulation of WT/WT heterodimer was measured in the presence and absence of 2.5 mM EGTA. Cells transfected with Gαqi5 alone were used as negative control. Relative activity in both graphs is expressed as percentage of maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent average ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. **P=0.0016, ***P=0.0002, ****P<0.0001, one-way ANOVA with Bonferroni’s post hoc test was used to calculate statistical differences in basal activity (c). Cell surface expression level was 107% for the E309K/WT and 87% for E423R/WT mutants in comparison with the WT/WT heterodimer.

e, Fitting of GABA into the extra density (contoured at 7.0 σ) at the orthosteric ligand-binding site and its potential interaction with GABAB1b.

f, Concentration of endogenous GABA in the supernatant and lysate of HEK 293 GnTI− cells after recombinant expression of GABAB receptor, as well as cell culture media and lysis buffer controls, as detected by mass spectrometry.

Using inductively coupled plasma mass spectroscopy, we detected the presence of Ca and Cu above background level in purified GABAB receptor. The amounts of other metal elements were negligible (Supplementary Table 3). The molar ratios of Ca and Cu relative to the receptor protein (0.43:1 and 0.51:1) suggest partial occupancy of the ion-binding sites. An unmodeled density within the interdomain cleft, coordinated by tryptophan and histidine, may serve as a potential Cu2+-binding site but lacks sufficient signal to be labeled confidently.

In exploring the functional role of the bound ion, we found that the Ca2+ chelator EGTA substantially reduced GABAB receptor basal activity, and that mutating specific coordinating residues (GABAB1b-E309K and GABAB1b-E423R) had similar, although less drastic, effects (Extended Data Fig. 6c,d). Consistent with previous findings, these data suggest that Ca2+ may act as a positive allosteric modulator of GABAB receptor[22,23]. Our structure implies that Ca2+ stabilizes residues adjacent to the critical agonist-binding residue Trp278, thereby indirectly reinforcing its conformation (Fig. 3b).

We also found density in the orthosteric agonist-binding site of GABAB1b, and its shape suggests a GABA-like endogenous ligand (Extended Data Fig. 6a,b). GABA is a potential candidate since it fits the density and was detected in the lysate of cells used to express the receptor (Extended Data Fig. 6e,f). This GABA-like endogenous ligand bound in an inactive receptor conformation may reflect a pre-activation state; however, further investigation is required.

Discovery of endogenous phospholipid ligands

Our structure revealed endogenous phospholipids within the TM domains of both GABAB subunits. Using CaS receptor as control in mass spectrometry, we identified two phospholipids specifically bound to GABAB receptor, PE 38:5 and PC 38:2 (Fig. 4a–d). Both lipids consist of two long-chain fatty acyl moieties of 18 and 20 carbons. We further assigned PE 38:5 to GABAB1b and PC 38:2 to GABAB2 based on the size difference between phosphoethanolamine and phosphocholine head groups of the two lipids (Extended Data Fig. 7a,b). The lipid density in the GABAB2 TM domain has a bulkier head group that can better accommodate the larger choline moiety of PC 38:2.

Fig. 4 |

Identification of endogenous phospholipid ligands of GABAB receptor.

a,c, LC-MS/MS analysis of phospholipids bound to GABAB receptor (n=1). LC traces showing the abundance of PC 38:2 (a) and PE 38:5 (c) lipids in GABAB preparation relative to a CaS receptor control.

b,d, High-resolution MS spectra of the peaks in (a, c) (black) matched with standard spectra of PC 38:2 (b) and PE 38:5 (d) in red.

e,f, Molecular surface of GABAB1b (e) and GABAB2 (f) TM domain showing the binding pocket for PE 38:5 and PC38:2, respectively.

g-j, Three views of the specific interactions between phospholipid and each GABAB subunit. Contacts are shown between the phospholipid head group and residues from GABAB1b (g) and GABAB2 (h), as well as phospholipid fatty acyl chains and GABAB1b (i) and GABAB2 (j).

Extended Data Fig. 7 |

Endogenous phospholipid-binding sites of GABAB receptor.

a,b, Ribbon representation of GABAB1b (a) and GABAB2 (b) TM domain highlighting the cryo-EM density for phospholipids contoured at 4.0 σ. Phospholipids are rendered in ball-and-stick representation.

c,d, Electrostatic potential surface of the lipid-binding pocket in GABAB1b (c) and GABAB2 (d). The phospholipids are shown in sphere models. Charged residues that directly contact the phosphate group of each lipid are marked.

e-h, Comparison of phospholipids bound to GABAB subunits with ligands bound to class A GPCRs rhodopsin[27] (PDB code: 1F88) and S1P1 receptor[24] (PDB code: 3V2Y) (e), class B GPCR corticotropin-releasing factor receptor 1[57] (CRF1; PDB code: 4K5Y) (f), class C GPCRs mGlu1[29] (PDB code: 4OR2) and mGlu5[28] (PDB code: 4OO9) (g), and class F GPCR smoothened[58] (PDB code: 4JKV) (h). In each panel, the Cα trace of GABAB1b linker and TM domain is shown in two orthogonal views in gray, and the superimposed GABAB ligands PE 38:5 and PC 38:2 are in stick models in blue and green, respectively. Various GPCRs were overlapped onto the TM domain of GABAB1b to bring their bound ligands into superposition

i,j, Schematic diagram of the specific contacts between GABAB1b and PE 38:5 (i), and between GABAB2 and PC 38:2 (j). Selected contacts between residues and phospholipids are highlighted; hydrogen bonds, red dotted lines; hydrophobic contacts, black wiggled lines; polar interactions, green curved lines; pi-stacking interactions, orange box wave. Red zigzags indicate contacting atoms belong to main chain.

Lipid-interacting residues that are conserved in the two subunits are highlighted in bold and include: (1) head group (GABAB1b: His643 and Arg5493.32; GABAB2: His647 and Arg5563.32), (2) 20-carbon fatty acyl chain (GABAB1b: Phe5573.40, Tyr6575.44, and Ala7036.54; GABAB2: Tyr5643.40, Tyr6615.44, and Ala7076.54), (3) 18-carbon fatty acyl chain (GABAB1b: Ile7247.36; GABAB2: Ile7287.36).

Mirroring the amphipathicity of phospholipids, the lipid-binding pocket of each GABAB subunit retains a hydrophilic trunk and two lipophilic branches for binding the lipid polar head and nonpolar tails, respectively. The trunk consists of a negatively charged patch covering the amine moiety, and a positively charged area surrounding the phosphate (Extended Fig. 7c,d). The lipid-binding pockets are notably deep, extending from the extracellular membrane surface to the center of the TM domain (Fig. 4e,f). Each lipid occupies nearly the entire range of ligand-binding positions in class A, B, C and F GPCRs (Extended Data Fig. 7e–h).

Each GABAB subunit makes extensive contacts with the bound lipid, utilizing a majority of the TM helices, including TM2, 3, 5, 6, and 7 (Extended Data Fig. 7i,j). Approximately 2,400 Å2 of surface area is buried by either lipid-subunit pair. In addition, ECL2 directly contacts PE 38:5 in GABAB1b, while the linker and all three ECLs interact with PC 38:2 in GABAB2.

Key elements of the lipid-receptor interactions are conserved in GABAB1b and GABAB2 (Fig. 4g–j; Extended Data Figs. 7i,j; Supplementary Fig. 3). First, the hydrophilic head of each lipid is anchored through interactions with conserved histidine and arginine residues (GABAB1b: His643 of ECL2 and Arg5493.32; GABAB2: His647 of ECL2 and Arg5563.32) (Fig. 4g,h). GABAB2 also incorporates Arg714 of ECL3, rendering the lipid-binding pocket more electropositive than that of GABAB1b (Fig. 4h).

Second, the 20-carbon fatty acyl chain of both lipids follows a perpendicular turn to pass between two aromatic residues (GABAB1b: Phe5573.40 and Tyr6575.44; GABAB2: Tyr5643.40 and Tyr6615.44) (Fig. 4i,j). A cis double bond in each fatty acyl chain forms π interactions with the aromatic side chains. The bend is further buttressed by extensive nonpolar contacts with the aliphatic part of a conserved lysine (GABAB1b-Lys6605.47; GABAB2-Lys6645.47) lying parallel to the chain. Finally, the 18-carbon fatty acyl chain of both lipids is relatively straight, extending toward the cytoplasm in a binding pocket lined by small aliphatic residues on TM2, 3, and 7 (Fig. 4i,j).

The GABAB receptor TM domains are covered by the linker and ECLs, which form a lid over the lipid-biding pocket. Phospholipids may access the lipid-binding pocket of designated subunits laterally through gaps between TM5 and 6 (Extended Data Fig. 8a–d). One of the fatty acyl tails of each phospholipid even protrudes through this opening. The lipid-binding sphingosine 1-phosphate (S1P1) receptor possess a similar gap between TM1 and TM7[24] (Extended Data Fig. 8e,f). The endogenous lipids of other GPCRs can be readily replaced[24], but the size and engagement of the endogenous lipids bound to GABAB receptor suggest that they are critical for maintaining receptor integrity and stability.

Extended Data Fig. 8 |

Endogenous phospholipid interactions with GABAB receptor.

a,c,e, Orthogonal views of potential access channel in GABAB1b, GABAB2, and S1P1 in molecular surface representation, along with phospholipids PE 38:5 and PC 38:2, as well as sphingolipid mimic ML056, respectively, in space-filling representation. Side view (left) shows opening between helices TM5 and TM6 in GABAB1b (a) and GABAB2 (c), and between TM1 and TM7 in S1P1 (e), while top view (right) highlights blocked entrance to lipid-binding pocket from the extracellular side. In all cases, ECL1 and ECL3 (orange), ECL2 (pale brown), as well as the linker of GABAB receptor subunits (purple) and N-terminal helix of S1P1 (purple) are distinguished by color.

b,d,f, The same information presented in (a, c, e) but with ribbon model used for GABAB1b (b), GABAB2 (d), and S1P1 (f). Lipids are in stick model.

g,h, Functional effect of a GABAB2 lipid-binding site mutation. Basal activity (g) and dose-dependent baclofen-stimulated receptor response (h) in cells transiently expressing Gαqi5 (abbreviated as Gqi) with WT GABAB receptor or WT GABAB1b and mutant GABAB2-R714A (abbreviated as R714A) heterodimer. Cells transfected with Gαqi5 alone were used as negative control. Relative activity in both graphs was measured by IP1 accumulation, and expressed as percentage of maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent average ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. **P=0.0016, one-way ANOVA with Bonferroni’s post hoc test was used to calculate statistical difference in basal activity (g). Cell surface expression level was 77% for the WT/R714A mutant in comparison with the WT/WT heterodimer.

To explore the physiological relevance of the endogenous phospholipids, we mutated residues that hydrogen bonded with the phosphate head group. We identified R714A in GABAB2 ECL3, which displayed a small gain of function despite reduced cell surface expression (Extended Data Fig. 8g,h). This mutation is expected to enhance the movement of PC 38:2 within GABAB2 by eliminating a critical interaction with the lid. Our results suggest that PC 38:2 may act as a negative allosteric modulator of GABAB receptor by stabilizing the inactive conformation of GABAB2.

Comparison of individual subunits with other GPCRs

The GABAB1b and GABAB2 subunits have highly similar VFT and TM components but differ in their relative orientation (Extended Data Fig. 9a,b). Each subunit differs from all other class C GPCRs in possessing an extended peptide linker between the VFT and TM domain instead of a cysteine-rich domain. (Fig. 1c).

Extended Data Fig. 9 |

Comparison of GABAB TM domain with other GPCRs.

a, Superposition of GABAB1b and GABAB2 subunits based on their VFT modules. Purple line denotes the axis of rotation that relates the linker and TM domains of GABAB1b and GABAB2 (rotation χ = 23°, screw translation τχ = 0.01 Å).

b, Superposition of the linker and TM domains of GABAB1b and GABAB2 subunits.

c, Superposition of the linker and TM domains of each GABAB subunit with class C GPCR mGlu5[20] (PDB code: 6N52) in three different views, with arrows revealing inward extracellular shifts in TM5 and 7, as well as an inward intracellular shift in TM3, in mGlu5 compared to either GABAB subunit.

d-f, Superposition of the TM helices of each GABAB subunit with the class A GPCR rhodopsin[27] (PDB code: 1F88) (d), class B GPCR CRF1[57] (PDB code: 4K5Y) (e), and class F GPCR smoothened[58] (PDB code: 4JKV) (f). Arrows indicate large shifts in TM helix positions between GABAB subunits and other GPCRs, such as outward extracellular movements in class A (TM2 and 6), class B (TM1, 2, and 7), and class F (TM2, 4, and 5). There are also inward intracellular shifts in TM7 in all comparisons, and outward intracellular shifts in TM5 in class A, B and F.

GABAB subunits also display distinct helix positions in the seven-helix bundles among inactive GPCRs (Extended Data Fig. 9c–f). However, these differences are minor compared to the dramatic movement of TM6 in class A and B GPCRs when activated upon G protein coupling[25,26]. This corroborates our conclusion that we have captured an inactive conformation of the GABAB receptor.

GABAB receptor presents unique variations of conserved TM motifs (Extended Data Fig. 10a,b). In most class A GPCRs such as rhodopsin[27], the ′ionic lock′ tethers TM3 and TM6 to stabilize the inactive state of an individual TM domain[27] (Extended Data Fig. 10c). The ′ionic locks′ of both GABAB subunits consist of an aspartate from ICL3 and a lysine in TM3 (3.50) (Extended Data Fig. 10a,b). Although only the Lys/Asp pair in GABAB1b are within hydrogen bond distance, their backbone Cα-Cα separations (9.3–9.4 Å) are comparable to that of rhodopsin[27] (8.7 Å) and inactive mGlu receptors[28,29] (10.9–11.2 Å), indicating that the ′ionic lock′ is intact within both GABAB subunits (Extended Data Fig. 10a–e).

Extended Data Fig. 10 |

Conserved motifs in GABAB, rhodopsin and mGlu receptors.

a,b, The ′ionic lock′ and FxPKxx motifs in GABAB1b (a) and GABAB2 (b) subunits. The ′ionic locks′ consist of Asp684 of ICL3 and Lys5673.50 in GABAB1b and Asp688 of ICL3 and Lys5743.50 in GABAB2. The FxPKxx motifs include the conserved Lys7397.51 of GABAB1b and Lys7437.51 of GABAB2, which interact with the ′ionic locks′ through Asn2.39 (GABAB1b-N5132.39; GABAB2-N5202.39) and a serine (GABAB1b-S508; GABAB2-S515) in ICL1.

c, The ′ionic lock′ and NPxxY motifs in class A rhodopsin[27].

d,e, The ′ionic lock′ and FxPKxY motifs in class C receptors mGlu1[29] (d) and mGlu5[28] (e), which are in close proximity as in GABAB subunits.

Key residues of the motifs are displayed in stick models. Hydrogen bonds are indicated by black dotted lines. Interactions between participating residues of the ′ionic lock′ are denoted by red dotted lines with distances labeled. Distances between the Cα atoms of the ′ionic lock′ residues are marked by brown dotted lines.

The ′ionic lock′ in each GABAB subunit resides in close proximity to an FxPKxx sequence in TM7, which is the counterpart of the NPxxY(x)5,6F motif in class A GPCRs[25]. Specifically, the conserved Lys7.51 participates in a network of hydrophilic contacts with the ′ionic lock′ through Asn2.39 and a serine in ICL1 (Extended Data Fig. 10a, b; Supplementary Fig. 3). These interactions unite the ′ionic lock′ and FxPKxx motif into a larger and integral system for maintaining the inactive TM conformation of individual GABAB subunits.

Conclusion

The combination of our previous VFT structures and current cryo-EM data supports the occurrence of three critical events during GABAB receptor activation: (1) agonist-induced VFT closure of GABAB1b, (2) association of membrane-proximal LB2 domains, and (3) dissociation of the ′intersubunit latch′ and the ensuing rearrangement of the TM heterodimer interface. This hypothesis is consistent with our finding that an inverse agonist bound to the extracellular domain can inhibit the constitutive activity stemming from the spontaneous closure of GABAB1b VFT, but not that resulting from direct ‘downstream’ disruption of the ′intersubunit latch′.

The GABAB receptor structure also yields surprising findings regarding its endogenous ligand composition. We suspect that the phospholipids discovered well inside each TM cavity are necessary structural components, as they are pre-bound within each subunit and their interactions with the receptor are extensive. These endogenous lipids may be unique to GABAB receptor since the lipid-binding residues are not conserved among class C GPCRs (Supplementary Fig. 3). Pre-occupation of the TM pocket suggests that GABAB allosteric modulators may bind to yet unknown sites, with the heterodimer interface being a potential location. An active structure of GABAB receptor would confirm whether the phospholipids are integral receptor components or functional modifiers.

Methods

Protein expression and purification

Human GABAB1b (UniProt code: Q9UBS5–2) and GABAB2 (UniProt code: O75899) subunits were each cloned into a modified pEG BacMam vector[30] for co-expression in baculovirus-infected mammalian cells. GABAB1a and GABAB1b are two major isoforms of GABAB1, and have identical pharmacological profiles[7]. Different C-terminal truncations of each receptor subunit were tested for heterodimeric receptor assembly. The optimal GABAB1b expression construct consisted of residues 1–802 [GABAB1b(1–802)], while GABAB2 construct included residues 1–819 [GABAB2(1–819)]. This allowed the heterodimeric construct to transport to the cell membrane as it retained the intracellular coiled-coil region present in the intracellular tails of each subunit[31,32]. Signal peptides for GABAB1b and GABAB2 occupied residues 1–29 and 1–41, respectively. A Flag tag was engineered at the C terminus of each subunit to facilitate affinity purification.

Human embryonic kidney (HEK) 293 GnTI− cells[33] were grown in suspension culture at 37°C in 8% CO2 using 293 freestyle media (Life Technology, Carlsbad, USA). The cells were co-infected with recombinant baculoviruses carrying the GABAB1b(1–802) and GABAB2(1–819) genes at 37°C. To enhance expression level, 10 mM sodium butyrate was added 18 hours post infection, and the cells were incubated for an additional 72 hours at 30°C before harvest.

Cell membrane was isolated by differential centrifugation method. The cells were lysed using an EmulsiFlex-C3 high pressure homogenizer (Avestin, Ottawa, Canada) in a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol and a cocktail of protease inhibitors (Roche, Basel, Switzerland). Cell debris was removed by centrifugation of the lysed cell suspension at 10,000 rpm. The cell membrane was then pelleted by ultracentrifugation at 45,000 rpm.

GABAB receptor was extracted from the cell membrane with 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% lauryl maltose neopentyl glycol (LMNG) (Anatrace, Maumee, USA), and 0.2% cholesteryl hemisuccinate (CHS) (MilliporeSigma, Burlington, USA) at 4°C overnight. After the insoluble matter was removed by centrifugation, the supernatant was applied to an anti-Flag M2 antibody affinity column. The column was washed stepwise with decreasing concentrations of detergent, from 0.1% to 0.002% LMNG. The heterodimeric GABAB1b(1–802)-GABAB2(1–819) complex was then eluted with 50 mM HEPES, pH 7.5, 50 mM NaCl, 0.002% LMNG, 0.0004% CHS, and 0.2 mg/ml Flag peptide.

The receptor was further purified by Mono Q (GE Healthcare, Chicago, USA) ion exchange chromatography using a linear salt gradient from 50 mM to 1 M NaCl in 50 mM HEPES, pH 7.5, 0.002% LMNG, and 0.0004% CHS. Finally, the assembled GABAB receptor was subjected to Superose 6 (GE Healthcare, Chicago, USA) size exclusion chromatography in 50 mM HEPES, pH 7.5, 50 mM NaCl, 0.002% LMNG, and 0.0004% CHS.

HEK 293 GnTI− cells were purchased from and authenticated by American Type Culture Collection (ATCC No. CRL-3022). Cell morphology was examined for each passage of cells. The cells were certified by ATCC to be free of mycoplasma contamination, but they were not tested again during culturing.

Cryo-EM specimen preparation and data acquisition

Specimens were composed of vitrified GABAB protein samples occupying UltraAuFoil R 0.6/1, 300 mesh holey Au/Au grids (Quantifoil Micro Tools, Jena, DEU). The surfaces of the grids were rendered hydrophilic by glow-discharging using H2 and O2 for 25 seconds at 10 watts with a Solarus 950 plasma cleaner system (Gatan, Cranberry, USA). For vitrification, 3ul of purified GABAB receptor at a concentration of approximately 0.3 mg/ml was applied to each grid, blotted for 4 seconds at a blot force of 3 inside a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, USA), and plunge-frozen in a liquid propane:ethane mixture (63:37, v/v) cooled with liquid nitrogen.

Data collection was performed on a Titan Krios transmission electron microscope (Thermo Fisher Scientific, Waltham, USA) equipped with a K2 Summit direct electron detection camera (Gatan, Cranberry, USA) in counting mode and a post-column GIF Quantum energy filter (Gatan, Cranberry, USA) in zero-energy-loss mode with a slit width of 20 eV. Micrographs were accrued at a calibrated pixel size of 1.06 Å and with nominal defocus range of −0.5 to −2 μm. Each micrograph consisted of 60 frames collected over a 12-second exposure at a dose rate of ~8 e−/pixel/second for a total dose of ~85 e−/Å2. A total of 3,435 micrographs were acquired as dose-fractionated image stacks.

Cryo-EM image processing

Image processing began with frame alignment and dose-weighting of the image stacks using the CPU-based implementation of MotionCor2[34] in Relion 3.0[35]. Estimation of contrast transfer function (CTF) for each non-dose weighted micrograph was determined by Gctf[36] v1.06. After visual inspection of the micrographs and their power spectra, 3,334 were selected for further processing.

Approximately 3,000 particles were manually picked in Relion 3.0[35], and extracted from a 4x binned dataset with a pixel size of 4.24 Å. This data produced an initial set of two-dimensional (2D) classes that were used as templates to select 1,048,241 particles automatically, all of which were subsequently imported into cryoSPARC v2[37] for extensive 2D classification. After elimination of unfit classes, a total of 312,840 particles from the high-quality 2D classes were combined to produce an ab initio 3D reference in cryoSPARC v2[37]. Based on the ab initio model, particles were re-extracted at full scale from the unbinned dataset with a pixel size of 1.06 Å in Relion 3.0[35], and re-introduced into cryoSPARC v2[37] for 3D refinement. Homogeneous refinement of the ab initio 3D model against the unbinned set of particles yielded a density map with nominal resolution of 3.6 Å according to the Fourier shell correlation (FSC) = 0.143 gold standard criterion[38]. Heterogenous refinement of multiple models obtained before and after homogeneous refinement allowed removal of additional poor-quality particles and reduced the particle count to 233,737. Non-uniform refinement then improved the resolution to 3.5 Å. At this point, CTF refinement and Bayesian polishing were conducted in Relion 3.0[35], followed by an additional round of non-uniform refinement in cryoSPARC v2[37], further improving the resolution to 3.3 Å.

Although the TM domains of GABAB receptor exhibit pseudo two-fold symmetry, bulky carbohydrate densities that are visible only in the ECD of GABAB1b subunit resulted in sufficient low-resolution asymmetry to prevent particle misalignment. These include partial carbohydrate densities attached to Asn323 and Asn365 of GABAB1b that do not have counterparts in GABAB2.

The global density map exhibited directional anisotropy[39] that is caused by inter-domain movement about a flexible linker. To eliminate the adverse effect of such movement on map quality, we performed local refinement of the ECD and TM domains of GABAB receptor independently. A mask was created covering each region, and the non-uniform refinement algorithm was used as implemented in cryoSPARC v2[37]. The resulting reconstructions for the individual ECD and TM domains reached 3.1 Å and 3.4 Å resolution, respectively. A composite map was generated in UCSF Chimera[40] by taking the maximum values pointwise from the two locally refined maps after alignment to the global reconstruction (vop maximum command in UCSF Chimera[40]). This composite map was used for subsequent model building and refinement.

Resolutions of cryo-EM reconstructions were determined using a cutoff value of 0.143 in gold standard half-map Fourier shell correlation (FSC) curves[38].

Three-dimensional (3D) variability analysis[41] was conducted in cryoSPARC v2[37] using the 233,737 particles from global non-uniform refinement as input. Calculations were performed for the entire receptor, the ECDs, and the TM domains, respectively. In each case, multiple modes of variability were solved, and represented as eigenvectors along which conformational changes occur. To visualize the transformation of density, five reconstructions were calculated along each eigenvector, with a filter resolution of 4.5 Å. A movie that combines these reconstructions as frames was generated in Chimera[40] for each dimension of motion.

Model building and refinement

Model building was carried out in COOT[42]. The crystal structure of human GABAB1b VFT-GABAB2 VFT complex in the apo form (PDB code: 4MQE) was used as the initial model for extracellular domain of the receptor. The VFT modules of GABAB1b and GABAB2 were separately placed into density as rigid bodies. Individual residues were then adjusted to optimize the fit. The linker and TM domain of each subunit was traced de novo based on the density. The final model contained residues 48–368, 377–576 and 588–747 of GABAB1b, and 54–294, 302–376, 385–584, and 595–749 of GABAB2.

In addition to the polypeptide chains, we built models for a Ca2+ in the extracellular domain of GABAB1b, as well as one phospholipid (PE 38:5 in GABAB1b; PC 38:2 in GABAB2) within the TM domain of each subunit. Density for carbohydrate was observed at three N-linked glycosylation sites on GABAB1b (Asn365, Asn385 and Asn397), and one site on GABAB2 (Asn404). An N-glucosamine residue was modeled at each of these glycosylation sites. Continuous density was also identified for ten cholesterol or CHS molecules surrounding the TM domains of both GABAB subunits. Cholesterols were modelled to optimize the fit with density, however, these densities may belong to CHS molecules with disordered parts. Density for an endogenous ligand was found at the interdomain cleft of GABAB1b VFT. Although this density could be fit with GABA, it was not modelled because the origin and identity of the ligand remained ambiguous without confirmation by an independent method.

The entire structure was refined by the real-space refinement algorithm, and validated with the comprehensive validation application as implemented in Phenix[43]. Ramachandran statistics was calculated using MolProbity[44]. The refined model also has an overall EMRinger[45] score of 2.7, while the extracellular and TM domains have scores of 3.4 and 1.8, respectively. The final model revealed that VFT and TM components are related to their counterparts in the other subunit by 177° and 179°-rotations about the vertical axis, respectively.

Pairwise structural alignment was performed using LSQMAN[46]. Figures were generated using Pymol Molecular Graphics System Version 2.3 (Schrödinger), UCSF Chimera[40] and UCSF ChimeraX[47]. Software installation support was provided by SBGrid[48].

Scintillation proximity assay

Binding of [3H]GABA (60 Ci/mmol; American Radiolabeled Chemicals, Inc., St. Louis, USA) to GABAB receptor was measured with the scintillation proximity assay (SPA)[49,50]. Purified GABAB1b(1–802)-GABAB2(1–819) complex (100 ng) was extensively dialyzed against the purification buffer to remove any residual endogenous ligand. The dialyzed protein was then immobilized to yttrium silicate (YSi) protein A SPA beads (62.5 μg) (PerkinElmer, Waltham, USA) using anti-Flag M2 antibody (12.5 pg) (Sigma-Aldrich, Inc., St. Louis, USA), and incubated at 4°C for 30 minutes in the same buffer as used for the final step of protein purification (50 mM Hepes, pH 7.5, 50 mM NaCl, 0.002% LMNG, 0.004% CHS). Increasing concentrations (ranging from 0.2–25 μM) of [3H]GABA (2.5 Ci/mmol final specific radioactivity) were added to the protein/antibody/SPA-bead mixture and the samples were allowed to reach equilibrium at 4°C for 16 hours. Reaction performed with an antibody/SPA-bead mixture in the absence of GABAB receptor was used to determine the non-proximity signal originating from non-specific interaction between the radioligand and SPA beads.

All samples were counted in a Microbeta™ counter (PerkinElmer, Waltham, USA) in counts per minute (cpm) in the SPA mode. The efficiency of detection was calculated with a standard curve of known [3H]GABA concentrations, and this was used to transform cpm into pmol. Specific binding was determined by subtracting the non-proximity signal (non-specific binding) from the total binding signal and was plotted as a function of free radioligand concentration. Nonlinear regression fitting of the data was performed in SigmaPlot 13.0 to obtain the dissociation constant (Kd) and the molar ratio of GABA-to-receptor binding.

Identification of phospholipid ligands and GABA

Identification of bound endogenous lipids was conducted utilizing published protocol[51] with modifications. Briefly, intact GABAB and control CaS receptor were digested with trypsin overnight at 37°C. Digested proteins were dried and extracted with 1 mL of ice-cold methanol:water (9:1, v/v). The supernatants were dried and re-suspended with methanol:toluene (9:1, v/v) to equivalent concentration of 2 μM. For LC-MS/MS analysis[52], the lipid extracts were separated on a Waters Acquity UPLC CSH C18 column (100 × 2.1 mm; 1.7 μm) coupled to an Acquity UPLC CSH C18 VanGuard precolumn (5 × 2.1 mm; 1.7 μm). The column was maintained at 65°C at a flow rate of 0.6 mL/min. The mobile phases consisted of A: acetonitrile:water (60:40, v/v) with ammonium formate (10 mM) and formic acid (0.1%), as well as B: 2-propanol:acetonitrile (90:10, v/v) with ammonium formate (10 mM) and formic acid (0.1%). The 15 min separation was conducted under the following gradient: 0 min 15% B; 0–2 min 30% B; 2–2.5 min 48% B; 2.5–11 min 82% B; 11–12 min 99% B; 12–15 min 15% B. Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, USA) was operated in electrospray ionization (ESI) in positive mode with the following parameters: mass range 100–1500 m/z; spray voltage +3.6 kV; sheath gas (nitrogen) flow rate 60 units; auxiliary gas (nitrogen) flow rate 25 units, capillary temperature 320°C, full scan MS1 mass resolving power 60,000, data-dependent MS/MS acquisition (dd-MS/MS) 4 scans per cycle, dd-MS/MS mass resolving power 15,000. The mass features that were differentially higher in GABAB receptor were subjected to targeted MS/MS in re-injections to acquire tandem mass spectra. Thermo Xcalibur 4.0.27.19 was used for data acquisition. Data processing and identification were performed in MS-DIAL v3.40. Identification was conducted by matching accurate mass, tandem mass spectra, and chromatographic retention time with built-in lipid library LipidBlast[53]. The identified endogenous lipids bound to GABAB receptor include phosphatidylcholine (PC) 38:2 [PC(18:1_20:1); International Chemical Identifier (InChI) Key: QLEJPADMSQQACL-WWUFLCHTSA-N], and phosphatidylethanolamine (PE) 38:5 [PE(18:1_20:4); InChIKey: VFUVYNGTMNUBMF-ZRVIQYDLSA-N]. Although many isoforms exist for these lipids, both phospholipids share two long-chain fatty acyl moieties of 18 and 20 carbons based on mass spectrometry fragmentation pattern and biological relevance.

Identification of GABA was conducted in targeted LC-MS/MS. Briefly, cell supernatant, cell lysate, together with culture media and lysis buffer controls were dried from 1 mL and extracted with 1 mL of ice-cold methanol:water (9:1, v/v). The supernatants were dried and re-suspended with 200 μL of acetonitrile:water (8:2, v/v). The extracts were separated on a Waters Acquity UPLC BEH Amide column (150 × 2.1 mm; 1.7 μm) coupled with an Acquity UPLC BEH Amide VanGuard precolumn (5 × 2.1 mm; 1.7 μm). The column was maintained at 45 °C at a flow rate of 0.4 mL/min. Mobile phase A was water with ammonium formate (10 mM) and formic acid (0.125%) while B was acetonitrile:water (95:5, v/v) with ammonium formate (10 mM) and formic acid (0.125%). Separation was conducted using the gradient: 0–2 min 100% B; 2–7.7 min 70%B; 7.7–9.5 min 40% B; 9.5–12.5 min 30% B; 10.25–12.75 100% B, 12.75–16.75 100% B. Q Exactive HF mass spectrometer was operated in the same parameters as above. GABA standard was injected along with the samples to confirm its spectrum and retention time. The responses of GABA in samples were normalized to that of GABA standard with known concentration.

Cell surface expression

Full-length human GABAB1b and GABAB2 were each cloned into a pcDNA3.1(+) vector (Life Technologies, Carlsbad, USA), with a Flag tag inserted after the signal peptide of GABAB1b, and an HA tag after the signal peptide of GABAB2. Similar constructs were also generated for the C-terminally truncated GABAB1b(1–802) and GABAB2(1–819). Single mutants of full-length GABAB1b (E673R, E309K, and E423R) and GABAB2 (H579E, R714A) were constructed using the QuikChange mutagenesis system (Agilent Technologies, Santa Clara, USA).

The cell surface expression levels of wild-type (WT) and mutant GABAB receptor were measured following previously published protocol[17]. Briefly, HEK293 T/17 cells (ATCC) were cultured in monolayer in DMEM/F12 media (Life Technology, Carlsbad, USA) supplemented with 10% FBS at 37°C in the presence of 5% CO2. The cells were co-transfected with GABAB1b and GABAB2 plasmids using Lipofectamine 3000 (Life Technologies, Carlsbad, USA). Each GABAB1b and GABAB2 mutant was paired with its wild-type partner. Since GABAB1b is retained inside the cells unless it is chaperoned by GABAB2, we used the surface expression level of GABAB1b on intact cells to measure the amount of assembled heterodimeric GABAB receptor on the cell surface. The amount of surface GABAB1b protein detected for each pair of constructs was normalized with the cell count in each experiment. The cell surface expression level of each mutant is calculated as a percentage of the wild-type receptor.

After blocking with 1% BSA, the cells were incubated with mouse anti-Flag M1 antibody (MilliporeSigma, Burlington, USA) as the primary antibody to measure GABAB1b expression, followed by donkey anti-mouse IRDye 800-labeled antibody (Li-Cor Biosciences, Lincoln, USA) as the secondary antibody. Fluorescent signals were measured with an Odyssey Infrared Imager (Li-Cor Biosciences, Lincoln, USA). Each experiment was performed in triplicates.

HEK 293 T/17 cells were purchased from and authenticated by American Type Culture Collection (ATCC No. CRL-11268). Cell morphology was examined for each passage of cells. The cells were certified by ATCC to be free of mycoplasma contamination, but they were not tested again during culturing.

Inositol phosphate measurement

HEK293 T/17 cells were co-transfected with plasmids encoding GABAB1b, GABAB2, and Gαqi5. The Gαqi5 chimera was constructed by replacing the five C-terminal amino acids of murine Gαq with those of murine Gαi[18]. Exchanging the C-terminal end of Gαq with that of Gi/o permits it to couple with GABAB receptor and allows the functional activity of the receptor to be tracked through phospholipase C (PLC). Control experiments were conducted using cells transfected with an empty pcDNA3.1(+) vector, Gαqi5 alone, or wild-type GABAB1b and GABAB2 in the absence of Gαqi5.

Inositol phosphate (IP) accumulation was quantified with the homogenous time-resolved fluorescence (HTRF) IP-one Tb kit (Cisbio Bioassays, Codolet, FRA), which measures the accumulation of inositol 1-monophosphate (IP1), a metabolite of inositol 1,4,5-triphosphate (IP3). One day post transfection, the cells were stimulated with increasing concentrations of baclofen for one hour at 37°C. The stimulated cells were lysed, and the native IP1 which had been produced was incubated with a d2 fluorophore-labeled IP1 analog (acceptor) to compete for binding to an Eu Cryptate-coupled anti-IP1 monoclonal antibody (donor). The fluorescence data was collected at 620 and 665 nm with a PHERAstar FS plate reader (BMG LABTECH, Cary, USA) after laser excitation at 320 nm. The fluorescence resonance energy transfer (FRET) signal was calculated as the fluorescence ratio (665 nm/620 nm) and is inversely proportional to the concentration of native IP1 produced following GABAB activation through a chimeric Gαqi5 G protein. The agonist-induced receptor response of each mutant was calculated as a percentage of the maximum activity of wild-type receptor relative to the activity observed for Gαqi5 alone. Basal activity was determined in the absence of baclofen stimulation and calculated similarly as the agonist-dependent receptor response. Data analysis was performed using the non-linear regression algorithms in Prism (GraphPad Software, San Diego, USA). Data points represent average ± s.e.m. of multiple experiments, each consisting of quadruplicate measurements.

Application of a known antagonist, CGP54626, reduced agonist potency as expected. In addition, the compound lowered the basal activity of GABAB receptor, indicating that it has inverse agonist activity as previously reported[54]. Therefore, we refer to the compound as an inverse agonist.

Inductively coupled plasma mass spectrometry

Purified GABAB receptor (200 μl, 11.1 μg/μL or 57.7 μM) was collected in metal-free tubes and digested overnight with the addition of 1 mL of concentrated nitric acid (HNO3) (Fisher, Hampton, USA; Optima grade). The digested protein was then diluted to a total volume of 10 ml with 8.7 ml of deionized water supplemented with 500 μg/L of gold (Au) and 100 μL of an internal standard solution containing 500 μg/L each of gallium (Ga) and rhodium (Rh) in 2% HNO3. The protein purification buffer (200 μl) containing 10 mM HEPES, pH 7.5, 50 mM NaCl, 0.002% LMNG, and 0.0004% CHS, was similarly mock digested and diluted for analysis.

Inductively coupled plasma mass spectrometry (ICP-MS) was conducted using a NexION 350S ICP-MS instrument (Perkin Elmer, Waltham, USA) equipped with dynamic reaction cell (DRC) feature and a SC-4 DX FAST Autosampler (Elemental Scientific, Omaha, USA). The DRC-ICP-MS experimental method was developed based on previously published procedures[55,56] and a laboratory protocol for multi-element DCR-ICP-MS from the Centers for Disease Control (CDC) (https://www.cdc.gov/nchs/data/nhanes/nhanes_13_14/UM_UMS_UTAS_UTASS_H_MET.pdf). The concentrations of magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn) and strontium (Sr) in the digested protein and buffer samples were measured. Data points represent average ± c.v. of eight measurements within two experiments, where c.v. corresponds to coefficient of variance.

One multi-element calibration standard was prepared from concentrated single-element stocks, and used for instrument calibration. The calibration standard was diluted to various concentrations using a solution containing 10% HNO3 and 500 μg/L of Au to cover the expected concentration range of each analyte in the protein sample: 0.01, 0.02, 0.05, 0.1, 0.2 μg/L for Co and Sr; 0.05, 0.1, 0.25, 0.5, 1.0 μg/L for Mn and Ni; 0.25, 0.5, 1.25, 2.5, 5.0 μg/L for Mg and Cu; 0.5, 1.0, 2.5, 5.0, 10.0 μg/L for Fe and Zn; and 2.5, 5.0, 12.5, 25, 50 μg/L for Ca.

The instrument was also calibrated against a set of blank solutions, including commercially available quality controls containing digested hair samples from Public Health Expertise and Reference Center, Quebec (INSPQ, Quebec, Canada), and a water sample containing a broad range of metals from National Institute of Technology (NIST, Gaithersburg, USA).

Special attention was given to correction for matrix-induced interferences. Matrix suppression was compensated by the selection of suitable internal standards, which were matched to masses and, if possible, to ionization properties of the analytes. The internal standards were added to each calibration standard and quality control sample to the same final concentrations as that in the protein sample and buffer (5 μg/L each of Ga and Rh). The elements Mg, Ca, Mn, Fe, Co, Ni, Cu, and Zn were corrected with Ga, while Sr was corrected with Rh. Polyatomic interferences were suppressed with the instrument’s DRC technology feature, utilizing ammonia as a second gas for Mn and Fe, while Mg, Ca, Sr, Co, Ni, Cu, and Zn were measured in standard mode without a second gas.

MALDI mass spectrometry

Purified GABAB1b-GABAB2 complex (0.3 mg/ml, 1 μl) was mixed with 1 μl sinapinic acid matrix solution (Bruker Daltonics, Billerica, USA) containing 10 mg of sinapinic acid in 1 ml of 2.5% trifluoroacetic acid (MilliporeSigma, Burlington, USA) and 50% acetonitrile (MilliporeSigma, Burlington, USA). The protein-matrix suspension (2 μl) was added to the ground steel MALDI target plate and dried at room temperature. Mass spectra were collected using a UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonic, Billerica, USA) operated with FlexControl software in linear positive mode, i.e. using a mass range of 30,000 to 120,000 daltons. The instrument was externally calibrated with Proteins MALDI-MS calibration kit (MilliporeSigma, Burlington, USA). Each individual mass spectrum was analyzed and adjusted for smoothness and baseline using FlexAnalysis software 3.0 (Bruker Daltonics, Billerica, USA). The molecular mass of the heterodimeric GABAB1b-GABAB2 complex was determined to be 192,647.967 daltons.