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Molecular basis for the selective G protein signaling of somatostatin receptors

Abstract

G protein-coupled receptors (GPCRs) modulate every aspect of physiological functions mainly through activating heterotrimeric G proteins. A majority of GPCRs promiscuously couple to multiple G protein subtypes. Here we validate that in addition to the well-known Gi/o pathway, somatostatin receptor 2 and 5 (SSTR2 and SSTR5) couple to the Gq/11 pathway and show that smaller ligands preferentially activate the Gi/o pathway. We further determined cryo-electron microscopy structures of the SSTR2‒Go and SSTR2‒Gq complexes bound to octreotide and SST-14. Structural and functional analysis revealed that G protein selectivity of SSTRs is not only determined by structural elements in the receptor–G protein interface, but also by the conformation of the agonist-binding pocket. Accordingly, smaller ligands fail to stabilize a broader agonist-binding pocket of SSTRs that is required for efficient Gq/11 coupling but not Gi/o coupling. Our studies facilitate the design of drugs with selective G protein signaling to improve therapeutic efficacy.

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Fig. 1: Ligands in smaller size preferentially signal through the Gi/o pathway of SSTR2 and SSTR5.
Fig. 2: Cryo-EM structures of the SSTR2–Go and SSTR2–Gq complexes bound to octreotide or SST-14.
Fig. 3: Molecular basis of octreotide and SST-14 recognition of SSTR2.
Fig. 4: Comparison of structures of octreotide-bound SSTR2–Go and SSTR2–Gq complexes.
Fig. 5: Molecular basis of the selective G protein signaling.

Data availability

The atomic structures have been deposited at the PDB under accessions 7Y24, 7Y26 and 7Y27. The electron microscopy maps have been deposited at the Electron Microscopy Data Bank under the accessions EMD-33585, EMD-33586 and EMD-33587. Source data are provided with this paper.

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Acknowledgements

We thank S. Rajagopal for his suggestion on interpretation of bias signaling. We thank staff at Shuimu BioSciences for their help with cryo-EM data collection. All electron microscopy images were collected at Shuimu BioSciences. This work was supported by the Chinese Ministry of Science and Technology, Beijing Municipal Science & Technology Commission (Z201100005320012, to S.Z.) and Tsinghua University.

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Contributions

S.C. and X.T. purified the protein complex, collected cryo-EM data and performed cryo-EM data processing and model building and performed all functional assays with the supervision by S.Z. S.Z. and S.C. wrote the manuscripts.

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Correspondence to Sanduo Zheng.

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Nature Chemical Biology thanks Asuka Inoue and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 SSTR2 and SSTR5 activate the Gq/11 pathway.

a, Primary sequences of SST-14, CST-17, octreotide and seglitide, and chemical structure of L-054,264. b-d, Dose-response curves of cAMP inhibition in HEK293 cells expressing SSTR1 (b), SSTR3 (c), or SSTR4 (d) upon stimulation with octreotide and SST-14. e, NanoBiT mini-Gq recruitment assays demonstrated that mini-Gq is recruited to the SST-14 activated SSTR2 and SSTR5. Measurements were performed with three independently biological replicates. f-h, Dose-response curves of IP1 production in cells expressing SSTR1 (f), SSTR3 (g) or SSTR4 (h) treated with SST-14 or octreotide. i-j, Effects of a specific Gq/11 inhibitor YM-254890 on activation of Gq/11(i) and Gi/o (j) by SSTR2. Each data point of dose-response curves is shown as mean ± SEM from three independent experiments. All IP1 data are normalized as percentage of maximum response of SSTR2 treated with SST-14.

Source data

Extended Data Fig. 2 Smaller ligands lead to the selective Gi/o coupling of SSTR2 and SSTR5.

a-b, Summary of the maximum effects (Emax), potencies (pEC50) and bias factors of different ligands at SSTR2 (a) and SSTR5 (b) from the cAMP inhibition assay and IP1 assay. Emax represents maximum effects of ligands. pEC50 is the logarithm of the concentration of ligands that produces 50% of Emax. Bias factor is calculated by comparing the Gi/o pathway to the Gq/11 pathway with SST-14 as a reference ligand. Positive value of bias factor indicates Gi/o-biased. n, number of independent repeats. ND, not determined because of no response. c-d, BRET assay for monitoring Go (c) and Gq (d) activation by SSTR2 stimulated by different ligands. All data represent mean ± SEM from three independent repeats. e, Summary of the maximum effects (Emax), potencies (pEC50) and bias factors of different ligands at SSTR2 from the BRET assay. ND, not determined because of low response and poor fit.

Source data

Extended Data Fig. 3 Cryo-EM data collection and processing of the octreotide-bound SSTR2-mini-Gαo/Gβ1γ2/scFv16 complex.

a, Size-exclusion chromatography profile. b, SDS-PAGE of the purified complex. c, Representative micrograph from 2349 micrographs. Scale bar represents 50 nm. d, Representative 2D classes. e, Cryo-EM data workflow. f, Gold standard FSC curves.

Source data

Extended Data Fig. 4 Cryo-EM data collection and processing of the octreotide- and SST-14-bound SSTR2-mini-Gαq/Gβ1γ2/scFv16 complex.

a, Size-exclusion chromatography profile. b, SDS-PAGE of the purified complex. c-d, Representative micrograph from 6480 micrographs (c) and 2D classes (d) of the octreotide-bound SSTR2-mini-Gq/Gβ1γ2/scFv16 complex. Scale bar represents 50 nm. e, Representative micrograph of the SST-14-bound SSTR2-mini-Gq/Gβ1γ2/scFv16 complex from 4881 micrographs. Scale bar represents 50 nm. f-g, Cryo-EM data workflow (f) and gold standard FSC curves (g) of the octreotide-bound SSTR2-mini-Gq/Gβ1γ2/scFv16 complex. h-i, Cryo-EM data workflow (h) and gold standard FSC curves (i) of the SST-14-bound SSTR2-mini-Gαq/Gβ1γ2/scFv16 complex.

Source data

Extended Data Fig. 5 Characterization of ligand binding of SSTR2.

a, Solution structures of octreotide alone in two different states. b, Crystal structure of octreotide alone in two different states. c-d, NanoBiT assay shows that SSTR2 equally activates Gi1 and Go when stimulated by octreotide (c) or SST-14 (d). Each data point is presented as mean +/− SEM from three independent experiments. e, Sequence alignment of SSTR subtypes from Homo sapiens. Secondary structures are shown above the alignment. Residues that are important for ligand binding and G protein activation of SSTR2 based on the structure and the NanoBiT Gi1 dissociation assay are denoted with orange circles below the alignment.

Source data

Extended Data Fig. 6 Comparison of structures of CCKAR and NK1R bound to different G protein subtypes.

a, Structural overlay of the CCKAR-Gi and CCKAR-Gq complexes from two orthogonal views. b, Structural overlay of the NK1R-miniGs and NK1R-miniGsq complexes from two orthogonal views.

Extended Data Fig. 7 Interaction interfaces between SSTR2 and the C-terminal helix of Gα.

a, Structural superposition of the octreotide-bound SSTR2-Go and SSTR2-Gq complex with the receptor aligned. b, Detailed interactions between TM3, TM5 and TM6 of SSTR2 and the extreme C terminus of Gαo. c, Detailed interactions between TM3, TM5 and TM6 of SSTR2 and the extreme C terminus of Gαq. d-e, Dose-response curves of cAMP inhibition (c) and IP1 production (d) in cells expressing SSTR2 or N186ECL2G mutant upon stimulation by SST-14. Each data point is presented as mean +/− SEM from three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–5 and Supplementary Note.

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Chen, S., Teng, X. & Zheng, S. Molecular basis for the selective G protein signaling of somatostatin receptors. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01130-3

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