Class B1 GPCR activation by an intracellular agonist

Expression and purification of human PTH1R

The plasmid encoding human PTH1R (GenBank identifier: U17418.1; residues 27–491) was constructed and purified as previously reported17. The construct was expressed in HEK293 GnTI (N-acetylglucosaminyltransferase I-negative) cells (American Type Culture Collection, CRL-3022) using the BacMam system (Thermo Fisher Scientific), and the cells were grown and maintained in FreeStyle 293 medium (Gibco) at 37 °C, with 8% CO2 under humidified conditions. Note that the cells were infected by the baculovirus generated in Sf9 cells (Life Technologies), supplemented with 10 mM sodium butyrate to boost protein expression after 18 h and cultivated in suspension at 30 °C for another 48 h. The cultured cells were collected by centrifugation (5,000g, 10 min, 4 °C) and disrupted by sonication in a hypotonic lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 20% glycerol). Cell debris was removed by centrifugation (10,000g, 10 min, 4 °C). The membrane fraction was collected by ultracentrifugation at 180,000g for 1 h, and solubilized in a solubilization buffer (20 mM Tris-HCl, pH 9.0, 200 mM NaCl, 1% LMNG, 0.1% CHS, 20% glycerol and 100 μM PCO371) for 2 h at 4 °C. The solubilized receptors were separated from the insoluble material by ultracentrifugation at 180,000g for 20 min and incubated with Ni-NTA resin (Qiagen) for 30 min. Detergent micelles were replaced by washing with 20 column volumes of wash buffer (20 mM Tris-HCl, pH 9.0, 500 mM NaCl, 0.03% GDN, 10% glycerol, 100 μM PCO371 and 30 mM imidazole). The receptor was eluted in elution buffer (20 mM Tris-HCl, pH 9.0, 500 mM NaCl, 0.01% GDN, 10% glycerol, 100 μM PCO371 and 300 mM imidazole). The eluate was treated with TEV protease to cleave the GFP-His10 tag and dialysed against dialysis buffer (20 mM Tris-HCl, pH 9.0, 500 mM NaCl, 100 μM PCO371 and 10% glycerol). The cleaved GFP-His10 tags and the TEV proteases were removed with Ni-NTA resin. The receptor was purified by size-exclusion chromatography on a Superdex 200 10/300 Increase column, equilibrated in SEC buffer (20 mM Tris-HCl, pH 9.0, 150 mM NaCl, 0.01% GDN and 100 μM PCO371). The peak fractions were collected and concentrated to about 5 mg ml–1.

Expression and purification of the mini-Gs heterotrimer

The plasmid encoding mini-Gs was constructed and purified as previously reported34. Mini-Gs was expressed in Escherichia coli (BL21) cells. The cells were cultured in LB medium supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25 °C. After 20 h, the cells were disrupted by ultrasonication in hypotonic buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 μM GDP), and the mini-Gs protein was purified by Ni-NTA affinity chromatography and then subjected to size-exclusion chromatography on a HiLoad Superdex75 16/600 column.

His6-tag-fused rat Gβ1 and bovine Gγ2 were also constructed, expressed and purified as previously reported17. Cell cultures were grown to a cell density of 4 × 106 cells per ml in Sf900 II medium (Gibco) for 60 h at 27 °C. The cells were collected by centrifugation and lysed in hypotonic buffer. The Gβ1–Gγ2 heterodimer was purified by Ni-NTA affinity chromatography and then subjected to size-exclusion chromatography on a HiLoad Superdex75 16/600 column.

The purified mini-Gs and Gβ1–Gγ2 were mixed and incubated overnight on ice. The mixture was concentrated and loaded onto a Superdex 75 10/300 Increase size-exclusion column and equilibrated in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 μM GDP). Peak fractions containing the mini-Gs heterotrimer were pooled and concentrated to 5 mg ml–1.

Expression and purification of Nb35

The plasmid encoding Nb35 was prepared as previously reported17,35. The protein was expressed in the periplasm of E.coli C41 (Rosetta) cells cultured in LB medium supplemented with 1 mM IPTG for 20 h at 25 °C. After 20 h, the cells were collected and disrupted by ultrasonication in hypotonic buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 2 mM MgCl2), and the Nb35 protein was purified by Ni-NTA affinity chromatography and then subjected to size-exclusion chromatography on a HiLoad Superdex75 16/600 column. Peak fractions were pooled and concentrated to 3 mg ml–1.

Formation and purification of the PCO371–PTH1R–mini-GSβ1γ2–Nb35 complex

PCO371 was synthesized at Chugai Pharmaceutical, and its purity and stability was confirmed by liquid chromatography–mass spectrometry. The purified PCO371-bound PTH1R proteins were mixed with a 1.2-fold molar excess of mini-Gsβ1γ2 and a 1.5-fold molar excess of Nb35, and the mixture was incubated on ice for overnight in pH 9.0. The sample was purified using Ni-NTA affinity resin and loaded onto a Superdex 200 10/300 Increase size-exclusion column, equilibrated in buffer (20 mM Tris-HCl, pH 9.0, 150 mM NaCl, 0.01% GDN and 100 μM PCO371) to separate the complex from contaminants. Peak fractions of the PCO371–PTH1R–mini-Gs heterotrimer–Nb35 complex were pooled and concentrated to 7 mg ml–1.

EM data collection and processing

The purified complex was applied onto a freshly glow-discharged Quantifoil holey carbon grid (R1.2/1.3, Au, 300 mesh) using a Vitrobot Mark IV at 4 °C in 100% humidity. The prepared grids were transferred to a Titan Krios G4 microscope (Thermo Fisher Scientific), operated at an accelerating voltage of 300 kV with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in nanoprobe EFTEM mode. Images were collected at a nominal magnification of 105 K, corresponding to a calibrated pixel size of 0.83 Å per pixel (The University of Tokyo, Japan). The dataset was acquired using the Serial EM (v.3.7.4) software, with a defocus range of −0.8 to −1.6 μm. Each image was dose-fractionated to 72 frames at a dose rate of 7.5 e pixel−1 s−1 to accumulate a total dose of 54.578 e Å−2. The 6,333 dose-fractionated movies were subjected to beam-induced motion correction using RELION-3 (ref. 36), and the contrast transfer function and the defocus parameters were estimated using CTFFIND4 (ref. 37). The 4,880,293 particles were initially picked from the 6,333 micrographs using the Laplacian-of-Gaussian picking function in RELION-3 and extracted in 3.63 Å per pixel. These particles were subjected to several rounds of 2D and 3D classifications. The best class contained 109,422 particles, which were then re-extracted with a pixel size of 1.11 Å per pixel and subjected to 3D refinement. The homogenous subset was subjected to per-particle defocus refinement, beam-tilt refinement, Bayesian polishing and 3D refinement. The final 3D refinement and post-processing produced maps with global resolution at 2.9 Å according to the Fourier shell correlation = 0.143 criterion. The processing strategy is described in Extended Data Fig. 2.

Model building and refinement

The initial template for the PTH1R–Gs–Nb35 complex was derived from the PTH–PTH1R–Gs–Nb35 structure (PDB identifier: 7VVL), followed by extensive remodelling using COOT-0.9.3 EL38. The models of PCO371–PTH1R–Gs were manually readjusted using COOT, refined using phenix.real_space_refine (v.1.14-3260)39 REFMAC540 and Servalcat41 against the working maps, and validated in MolProbity42.

Molecular dynamics simulation

The system included the PTH1R, PCO371, 1-phosphoryl-2-oleoylphosphatidylcholine (POPC), TIP3P water and 150 mM NaCl. The initial model of PTH1R containing amino acids 27–491 was created using MODELLER (v.10.1)43, with the cryo-EM structure of PTH1R in complex with PCO371 as the template. The missing hydrogen atoms were built with the program VMD (v.1.9.3)44. The protein was embedded into the POPC membrane using the MemProtMD pipeline45. The net charge of the simulation system was neutralized through the addition of 150 mM NaCl. The simulation system was 96 × 96 × 180 Å3 and contained 123,567 atoms. The molecular topologies and parameters from the Charmm36 force field46 were used for the protein, lipid and water molecules. The molecular topology and parameters for PCO371 were prepared using the CHARMM-GUI ligand reader and modeller47,48.

Molecular dynamics simulations were performed with the program NAMD (v.2.13). The simulation systems were energy minimized for 1,000 steps with fixed positions of the non-hydrogen atoms. After minimization, another 1,000 steps of energy minimization were performed with 10 kcal mol−1 restraints for the non-hydrogen atoms, except for the lipid molecules within 5.0 Å of the proteins. Next, equilibrations were performed for 0.1 ns under NVT conditions, with 10 kcal mol−1 Å−2 restraints for the heavy atoms of the proteins. Finally, equilibration was performed for 2.0 ns under NPT conditions, with the 1.0 kcal mol−1 Å−2 restraints for all Cα atoms of the proteins. The production runs were performed for 200 ns without restraints while maintaining a constant temperature at 310 K using Langevin dynamics and constant pressure at 1 atm using a Nosé–Hoover Langevin piston49,50. The long-range electrostatic interactions were calculated using the particle mesh Ewald method50. The simulations were independently performed three times. The simulation results were analysed and visualized using mdtraj (v.1.9.8)51, seaborn (zenodo.org/record/54844) and CUEMOL (v.2.2.3.443) (www.cuemol.org).

Glo-sensor cAMP accumulation assay

PTH1R-induced Gs activation was measured using the GloSensor cAMP accumulation assay. We first constructed a plasmid wherein the human full-length PTH1R gene was N-terminally fused to the Flag epitope tag with the preceding haemagglutinin-derived signal sequence. HEK293A cells were seeded in a 6-cm culture dish at a concentration of 2 × 105 cells per ml (4 ml per well in DMEM (Nissui Pharmaceutical) supplemented with 10% FBS (Sigma, F7524, lot 0001641439) and penicillin–streptomycin–glutamine (complete DMEM)) 1 day before transfection. The transfection solution was prepared by combining 10 μl (per well hereafter) of 1 mg ml–1 polyethylenimine MAX (Polysciences) solution and a plasmid mixture consisting of 1,000 ng Glo-22F cAMP biosensor (human codon-optimized and gene-synthesized)-encoding pCAGGS plasmids and 400 ng Flag–PTH1R plasmids in 400 µl of Opti-MEM (ThermoFisher Scientific). After incubation for 24 h, transfected cells were collected using 0.53 mM EDTA-containing Dulbecco’s PBS (D-PBS), centrifuged and suspended in 2 ml of HBSS containing 0.01% BSA (fatty-acid-free grade; SERVA) and 5 mM HEPES (pH 7.4) (assay buffer). The cell suspension was dispensed into a white 96-well plate at a volume of 40 μl per well and loaded with 10 μl of 10 mM d-luciferin potassium solution (FujiFilm Wako Pure Chemical) diluted in assay buffer. After 2 h of incubation at room temperature, the plate was measured for baseline luminescence (SpectraMax L equipped with 2PMT, Molecular Devices; SoftMax Pro (v.7.03), Molecular Devices) and 20 μl of 6× ligand diluted in the assay buffer or the assay buffer alone (vehicle) was manually added. The plate was read for 20 min with an interval of 30 s at room temperature. The luminescence counts over 8–10 min after ligand addition were averaged and normalized to the initial counts, and the fold changes in the signals over the vehicle treatment were further normalized to forskolin (10 µM) and plotted for the cAMP accumulation response. Using the Prism 9 software (GraphPad), the response were fitted to all data using the nonlinear regression. The variable slope (four parameter) in the Prism 9 tool with a constraint of the Hill slope of absolute value less than 2 was used. pEC50 and Emax values were obtained from the nonlinear regression curve of the averaged data. For multiple comparison analysis, one-way or two-way ANOVA and followed by Dunnett’s test was used.

NanoBiT β-arrestin recruitment assay

β-arrestin recruitment to PTH1R was measured using the NanoBiT β-arrestin-recruitment assay52 with minor modifications. In brief, plasmid transfection was performed in a 6-cm culture dish with a mixture of 200 ng N-terminal large BiT-fused β-arrestin 1 (Lg-β-arrestin 1) or Lg-β-arrestin 2 and 1,000 ng C-terminal small BiT-fused PTH1R (PTH1R-Sm) plasmids. After 24 h of incubation, the transfected cells were collected using 0.53 mM EDTA-containing D-PBS, centrifuged at 190g for 5 min and suspended in 4 ml of the assay buffer described for the GloSensor assay. The cell suspension was dispensed into a white 96-well plate at a volume of 80 μl per well (hereafter 96-well plate) and loaded with 20 μl of 50 μM coelenterazine (Carbosynth) diluted in the assay buffer. After 2 h of incubation at room temperature, the plate was measured for baseline luminescence (SpectraMax L equipped with 2PMT, Molecular Devices; SoftMax Pro (v.7.03), Molecular Devices) and 20 μl of 6× ligand diluted in the assay buffer or vehicle was manually added. The plate was read for 15 min with a 40 s interval at room temperature. The luminescence counts from 13 to 15 min after ligand addition were averaged and normalized to the initial counts.  the response were fitted to all data using the same procedure as described for the GloSensor assay.

Flow cytometry analysis

Cell surface expression of PTH1R was measured using a previously described flow cytometry method17. In brief, HEK293A cells were seeded in a 6-well culture plate at a concentration of 2 × 105 cells per ml 1 day before transfection. Transfection was performed using the same procedure as described for the GloSensor assay. One day after transfection, the cells were collected by adding 100 μl of 0.53 mM EDTA-containing D-PBS, then 100 μl of 5 mM HEPES (pH 7.4) containing HBSS. The cell suspension was transferred into a 96-well V-bottom plate and fluorescently labelled with an anti-Flag epitope (DYKDDDDK) tag monoclonal antibody (Clone 1E6, FujiFilm Wako Pure Chemicals; 10 μg ml–1 diluted in 2% goat serum and 2 mM EDTA-containing D-PBS (blocking buffer) and a goat anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Fluor 488 (1:200) (ThermoFisher Scientific, 10 μg ml–1 diluted in the blocking buffer). After washing with D-PBS, the cells were resuspended in 100 μl of 2 mM EDTA-containing D-PBS and filtered through a 40-μm filter. The fluorescence intensity of single cells was quantified using a flow cytometer (EC800 equipped with a 488 nm laser, Sony). The fluorescent signal derived from Alexa Fluor 488 was recorded in a FL1 channel, and the flow cytometry data were analysed using the FlowJo 10 software (FlowJo). Live cells were gated with a forward scatter (FS-Peak-Lin) cutoff at the 390 setting, with a gain value of 1.7. Values of mean fluorescence intensity from approximately 20,000 cells per sample were used for analysis.

Live-cell imaging by confocal microscopy

Transfection was performed using a mixture of 500 ng mVenus–β-arrestin 2 and 200 ng Flag–PTH1R plasmids (per well in a 6-cm dish). After incubation for 1 day, the transfected cells were collected and reseeded on a 35-mm, collagen-coated glass bottom dish (Matsunami). After 1 day, medium was changed to DMEM without phenol-red and FBS (starvation buffer). After 1 h of incubation, the cells were incubated with Alexa-647-labelled (1:2,000) Flag-M1 antibody for 1 h, washed once in the starvation buffer and set on the confocal microscope. Live-cell imaging was performed using LSM880 with Airyscan (Zeiss) equipped with a ×100/1.46 alpha-Plan-Apochromat oil-immersion lens and ImmersolTM 518F/37 °C (444970-9010-000, Zeiss). During live-cell imaging, the dish was mounted in a chamber (STXG-WSKMX-SET, TOKAI HIT) to maintain the incubation conditions at 37 °C and 5% CO2. We took dual-colour time-lapse images with the following settings: time interval of 5 min; total time of 35 min. The 200 µl ligand solution in 0.01% BSA-HBSS was added between time points 1 and 2. Acquired serial images were Airyscan processed using Zeiss ZEN 2.3 SP1 FP3 (black, 64 bit) (v.14.0.21.201). Co-localization analysis was performed using Fiji (v.2.0.0-rc-69/1.52p).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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