Molecular insights into the activation mechanism of GPR156 in maintaining auditory function

Abstract

The class C orphan G-protein-coupled receptor (GPCR) GPR156, which lacks the large extracellular region, plays a pivotal role in auditory function through G_i2/3. Here, we firstly demonstrate that GPR156 with high constitutive activity is essential for maintaining auditory function, and further reveal the structural basis of the sustained role of GPR156. We present the cryo-EM structures of human apo GPR156 and the GPR156-G_i3 complex, unveiling a small extracellular region formed by extracellular loop 2 (ECL2) and the N-terminus. The GPR156 dimer in both apo state and G_i3 protein-coupled state adopt a transmembrane (TM)5/6-TM5/6 interface, indicating the high constitutive activity of GPR156 in the apo state. Furthermore, C-terminus in G-bound subunit of GPR156 plays a dual role in promoting G protein binding within G-bound subunit while preventing the G-free subunit from binding to additional G protein. Together, these results explain how GPR156 constitutive activity is maintained through dimerization and provide a mechanistic insight into the sustained role of GPR156 in maintaining auditory function.

Fig. 1. Knockdown of GPR156 causes hearing loss and hair cell loss in adult mice.

a The experimental design diagram. AAV dose: 6 × 10¹⁰ GC /ear. b Representative images of the AAV-control virus infecting inner ear HCs in P30 mice from 6 independent experiments. Scale bar, 40 μm. c The ABR results of the GPR156-shRNA-injected ear (n = 9 mice), the contralateral ear (n = 9 mice), WT mice (n = 4 mice) and AAV-control-injected ear (n = 5 mice). All AAVs were injected into the left ear of P30 mice, and ABR experiments were started at P45. **P < 0.01 and ***P < 0.001 were calculated by a two-tailed unpaired t test. (mean ± SEM (bars), WT vs AAV-control: P = 0.26398(4k), P = 0.07594(8k), P = 0.83978(12k), P = 0.44799(16k), P = 0.50538(24k), P = 0.64441(32k). WT vs contralateral ear: P = 0.50064(4k), P = 0.16799(8k), P = 0.88241(12k), P = 0.72864(16k), P = 0.79457(24k), P = 0.50528(32k). GPR156-shRNA injected ear vs contralateral ear: P = 0.00027(4k), P = 0.00014(8k), P = 0.00064(12k), P = 0.00229(16k), P = 0.00559(24k), P = 0.00794(32k). Source data are provided as a Source Data file.). d, e Low and high magnification representative confocal images of Myo7a signaling in P45 GPR156-shRNA-injected cochleae from 6 independent experiments. Apex, Mid, and Base: apical, middle, and basal turn of the cochlea. Scale bar, 200 μm in (d) and 40 μm in (e). f The representative confocal image of Myo7a signaling in the P45 GPR156-shRNA contralateral cochlea from 6 independent experiments. Scale bar, 40 μm. g The number of HCs, OHCs, and IHCs in the P45 GPR156-shRNA-injected ear and contralateral ear per 100 μm. **P < 0.01, ***P < 0.001, and ****P < 0.0001 were calculated by two-tailed unpaired t test. (mean ± SEM (bars), GPR156-shRNA injected ear(10 regions from 4 mice) vs contralateral ear(9 regions from 4 mice): P = 1.6E-07(Apex-OHC), P = 1.7E-06(Apex-IHC), P = 7.6E-10(Apex-HC), P = 8.6E-06(Mid-OHC), P = 7.4E-06(Mid-IHC), P = 1.8E-08(Mid-HC), P = 0.00181(Base-OHC), P = 7.5E-05 (Base-IHC), P = 0.00034(Base-HC). Source data are provided as a Source Data file.). OHC: the outer hair cell. IHC: the inner hair cell. h The ABR results of the GPR156-shRNA-injected ear (n = 9 mice), the contralateral ear (n = 9 mice), WT mice (n = 4 mice) and AAV-control-injected ear (n = 4 mice). All AAVs were injected into the left ear of P60 mice, and ABR experiments were started at P75. ***P < 0.001 and ****P < 0.0001 were calculated by two-tailed unpaired t test. (mean ± SEM (bars), WT vs AAV-control: P = 0.62022(4k), P = 0.61311(8k), P = 0.38670(12k), P = 0.82908(16k), P = 0.70485(24k), P = 0.82310(32k). WT vs contralateral ear: P = 0.10835(4k), P = 0.92272(8k), P = 0.53047(12k), P = 0.16518(16k), P = 0.89175(24k), P = 0.45458(32k). GPR156-shRNA injected ear vs contralateral ear: P = 6E-09(4k), P = 3E-07(8k), P = 3E-07(12k), P = 1E-06(16k), P = 2E-05(24k), P = 0.0001(32k). Source data are provided as a Source Data file.). i, j Low and high magnification representative confocal images of Myo7a signaling in the P75 GPR156-shRNA-injected cochlea from 6 independent experiments. Scale bar, 200 μm in (i) and 40 μm in (j). k The representative confocal image of Myo7a signaling in the P75 GPR156-shRNA contralateral cochlea from 6 independent experiments. Scale bar, 40 μm. l The number of HCs, OHCs, and IHCs in the P75 GPR156-shRNA-injected ear and contralateral ear per 100 μm. **P < 0.01 and ****P < 0.0001 were calculated by a two-tailed unpaired t test. (mean ± SEM (bars), GPR156-shRNA injected ear(10 regions from 4 mice) vs contralateral ear(8 regions from 4 mice): P = 3E-06(Apex-OHC), P = 7E-09(Apex-IHC), P = 6E-8(Apex-HC), P = 6E-05(Mid-OHC), P = 5E-08(Mid-IHC), P = 2E-07(Mid-HC), P = 0.0037(Base-OHC), P = 1E-06(Base-IHC), P = 3E-05(Base-HC). Source data are provided as a Source Data file.). m The representative image of Phalloidin signaling in the GPR156-shRNA-injected cochlea and control group from 5 independent experiments. Scale bar, 10 μm. n, p The representative images of Ctbp2 staining of GPR156-shRNA-injected cochlea in P45 and P75 mice from 5 independent experiments, respectively. Scale bar, 10 μm. o, q The counts of Ctbp2 from the (n) and (p). ***P < 0.001 was calculated by a two-tailed unpaired t test. (mean ± SEM (bars), GPR156-shRNA injected ear(21 cells from 4 mice) vs control(12cells from 4 mice): P = 0.0792(P45). GPR156-shRNA injected ear(18 cells from 4 mice) vs control(11cells from 4 mice): P = 0.0003(P75). Source data are provided as a Source Data file.).

Fig. 2. Cryo-EM structures of human GPR156 in apo state and in complex with G_i3.

a Phylogenetic tree of the 22 class C GPCR family members with different ligand types. GPR156 is highlighted with a red mark. The scale bar indicates the number of substitutions per site. b Summary of the N-terminal lengths of the 22 class C GPCRs. GPR156 and GPRC5A–D are the only five GPCRs of class C whose N-terminal length is less than 60 residues. VFT: Venus Flytrap, CRD: Cysteine-rich Domain, and TMD: Transmembrane Domain. Source data are provided as a Source Data file. c The structural features of class C GPCRs include mGluR or CaSR (purple), GPR158 or GPR179 (green), GB1 subunit (orange), GB2 subunit (yellow), and GPR156 (blue). d, e Cryo-EM maps (left panel) and models (right panel) of human apo GPR156 (d) and GPR156–G_i3 complex (e).

Fig. 3. The unique arrangement of the N-terminus and ECL2.

a Side view (left panel), and a top view (right panel) of the distinct ECL2 and N-terminus conformations. b Sequence alignment logo showing the conservation of C^3.29 among family C GPCRs. c Close-up view of the ECL2 and transmembrane region. In GPR156, C^3.29 is not conservative and is replaced by I120^3.29. d, e Basal activity of WT and three mutant constructs in the N-terminus and ECL2 of GPR156, measured by BRET-based assay (d) (from left to right n  =  6, 6, 6, 6; NΔ23-44 vs WT: P = 0.0170; ECL2Δ192-202 vs WT: P = 0.7947; ECL2Δ192-213 vs WT: P = 0.1190) and NanoBiT-based assay (e) (from left to right n  =  7, 6, 7, 6; NΔ23-44 vs WT: P = 0.1038; ECL2Δ192-202 vs WT: P = 0.8694; ECL2Δ192-213 vs WT: P = 0.1819). Data are presented as the percentage of WT activity and are shown as the mean ± SEM (bars) from at least six independent experiments performed in technical triplicate with individual data points shown (dots). ns (not significant) = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test compared to WT. Supplementary Fig. 7a, b provides the related surface expression level. Supplementary Tables 3 and 4 provide detailed information related to BRET-based assay (d) and NanoBiT-based assay (e). Source data are provided as a Source Data file.

Fig. 4. Transmembrane interface of the apo GPR156 homodimer.

a Detailed interactions of the transmembrane interface in the apo GPR156. The 7TM homodimer interface is formed at two regions (I and II), namely the extracellular and intracellular side. b Detailed interactions on the surface of the region I. The network of electrostatic interactions is shown in Ia, and the affiliated hydrophobic interaction is shown in Ib. c Detailed interactions in the intracellular region II. A hydrophobic contact network is shown in IIa. The van der Waals forces at the homodimer interface are shown in IIb. d Comparison of the dimeric interface of apo GPR156 transmembrane domains with those of apo GABA_B receptor (PDB code: 6VJM) and apo GPR158 (PDB code: 7EWL). e The basal activity of WT and versions with mutations in the dimer interface of GPR156, as measured by BRET-based assay (from left to right n  =  8, 6, 7, 8, 8, 8, 8, 6, 6, 8, 6, 6, 6; D222^5.37A vs WT: P = 2.52817E-06; R279^6.57A vs WT: P = 0.5694; Y280^6.58A vs WT: P = 0.0560; V276^6.54A vs WT: P = 0.0118; V223^5.38A vs WT: P = 0.0006; L237^5.52A vs WT: P = 5.2657E-08; V264^6.42A vs WT: P = 0.0015; V268^6.46A vs WT: P = 0.0309; M261^6.39A vs WT: P = 3.1286E-07; Y241^5.56A vs WT: P = 0.0014; L234^5.49A vs WT: P = 6.6258E-06). Data are presented as a percentage of WT activity and are shown as the mean ± SEM (bars) from at least six independent experiments performed in technical triplicate with individual data points shown (dots). ns (not significant) = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and  ****P < 0.0001 by two-tailed unpaired t test compared to WT. Supplementary Fig. 7c provides the related surface expression level, and Supplementary Table 3 provides detailed information. Source data are provided as a Source Data file. f The key residues in the ionic lock motif (3.50 and 6.35) are aligned among members of class C GPCRs. g Close-up view of the conserved ionic lock motif showing the different conformations between GPR156 and other members of the class C subfamily in the apo state, including GABA_B receptor (PDB code: 6VJM) and GPR158 (PDB code: 7EWL).

Fig. 5. Structural details and G_i-associated transitions of the TMD in GPR156.

a Side views of the superposed structures of the GPR156_apo and GABA_B2(G) (PDB code: 7EB2) aligned by the TMD of GABA_B2(G). b Magnified views of the detailed interactions within the TMD of apo GPR156 (top panel), GABA_B2(G) (left panel, PDB code: 7EB2), and GABA_B2(inactive) (right panel, PDB code: 6WIV). c Sequence alignment logo showing the conservation of Y^3.44 or F^3.44 among family C GPCRs (left panel). And magnified views of the critical residue F^3.44 in the apo state of GPR156 and different states of GABA_B2 (right panel). d The basal activity of WT and mutant versions as measured by BRET-based assay (from left to right n  =  12, 6, 11, 6, 7, 6; K141^3.50E vs WT: P = 3.9825E-11; R144^3.53E vs WT: P = 8.9060E-12; S84^2.35A vs WT: P = 0.0086; N88^2.39A vs WT: P = 0.9080; F135^3.44W vs WT: P = 1.5807E-07). Data are presented as the percentage of WT activity and are shown as the mean ± SEM (bars) from at least six independent experiments performed in technical triplicate with individual data points shown (dots). ns (not significant) = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t-test compared to WT. Supplementary Fig. 7d provides the related surface expression level. Supplementary Table 3 provides detailed information. Source data are provided as a Source Data file. e Side, extracellular, and intracellular views of the superposed structures of apo GPR156 dimer and G_i-bound GRP156 dimer. f Upon G protein coupling, the dimeric TMD of five class C GPCRs (including mGlu3_free–mGlu2_G (Cα of V699; PDB code: 8JD3), mGlu4_free–mGlu2_G (Cα of V699; PDB code: 8JD5), mGlu2_free–mGlu2_G (Cα of V769; PDB code: 7MTS), CaSR_free–CaSR_G (Cα of M771; PDB code: 8WPU), and GABA_B1(free)–GABA_B2(G) (Cα of C614; PDB code: 7EB2)) undergo conformational rearrangements, except for GPR156_free–GPR156_G. Source data are provided as a Source Data file. g, h Comparison of the GPR156 G-bound subunit and GPR156 G-free subunit (g), and the GPR156 G-bound subunit and apo GPR156 subunit (h). The RMSD levels were calculated.

Fig. 6. GPR156–G_i coupling and the special role of the C-terminus.

a Close-up view of the GPR156-G_i3 complex at the cytoplasmic end, showing a long C-terminal tail (320–338aa). b, c Detailed interactions of the C-terminal tail (320–330aa) of GPR156_G with the TMD of GPR156_G and Gα_i (b), and detailed interactions of the C-terminal tail (331–338aa) of GPR156_G with the TMD of GPR156_free (c). d Close-up views of the seven key residues in the GPR156_G and GPR156_free subunits show conformational changes upon G protein coupling. e Schematic representation of the seven key residues’ contacts between the GPR156_G and GPR156_free subunits and the C-terminal tail of GPR156_G and Gα_i. Residues from the GPR156_G subunit are shown on the left, and those of the GPR156_free subunit are shown on the right. The C-terminal tail of GPR156_G–GPR156_free is in red, the C-terminal tail of GPR156_G–GPR156_free is in orange, the C-terminal tail of GPR156_G–Gα_i is in yellow, and both C-terminal tails of GPR156_G–GPR156_G and GPR156_G–Gα_i are in green boxes. f The basal activity of WT and the three mutant constructs of the C-terminal tail from GPR156, as measured by NanoBiT-based assay (from left to right n  =  7, 7, 7, 7; CΔ331-338 vs WT: P = 2.4064E-05; CΔ320-330 vs WT: P = 2.1818E-07; CΔ320-338 vs WT: P = 9.9574E-08). Data are presented as a percentage of WT activity and are shown as the mean ± SEM (bars) from at least six independent experiments performed in technical triplicate with individual data points shown (dots). *P< 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test compared to WT. Supplementary Fig. 7e provides the related surface expression level, and Supplementary Table 4 provides detailed information. Source data are provided as a Source Data file. g, h The basal activity of WT and mutant versions of GPR156 (including mutant constructs of the C-terminal tail) (g) (from left to right n  =  9, 7, 6, 7, 6, 6, 6, 9, 7, 6, 8, 6; C∆331-338 vs WT: P = 7.4009E-09; C∆320-330 vs WT: P = 5.5541E-08; C∆320-338 vs WT: P = 4.6108E-09,; Q323^C-termA vs WT: P = 4.2212E-09; T324^C-termA vs WT: P = 0.0017; I325^C-termA vs WT: P = 0.0470; M328^C-termA vs WT: P = 0.0002; K330^C-termA vs WT: P = 3.4903E-07; Y331^C-termA vs WT: P = 1.8316E-07; F332^C-termA vs WT: P = 0.0001; K337^C-termE vs WT: P = 9.1879E-08) and mutant constructs of the seven key residues (h) (from left to right n  =  9, 8, 6, 6, 7, 7, 6, 6, 7; R78^ICL1E vs WT: P = 3.5544E-10; M82^ICL1A vs WT: P = 2.1033E-07; F149^3.58A vs WT: P = 3.9032E-11; R152^ICL2E vs WT: P = 1.4385E-08; R157^ICL2E vs WT: P = 7.0588E-08; H248^5.63A vs WT: P = 3.5652E-07; F318^7.58A vs WT: P = 9.5730E-06; F318^7.58W vs WT: P = 3.2740E-07) as measured by BRET-based assay. Data are presented as a percentage of WT activity and are shown as the mean ± SEM (bars) from at least six independent experiments performed in technical triplicate with individual data points shown (dots). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test compared to WT. Supplementary Fig. 7f, g provides the related surface expression level, and Supplementary Table 3 provides detailed information. Source data are provided as a Source Data file. i Schematic diagrams summarizing the conformational changes of the GPR156 homodimers upon G protein coupling.