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Structure of class B GPCR corticotropin-releasing factor receptor 1

Nature volume 499, pages 438–443 (2013)Cite this article

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Abstract

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

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Figure 1: Schematic representation of the CRF1R structure.
Figure 2: Overall structure.
Figure 3: Comparison of the antagonist-bound structures of CRF1R and D3R.
Figure 4: Conserved sequence motifs.
Figure 5: Antagonist-binding site.

Accession codes

Accessions

Protein Data Bank

Data deposits

Co-ordinates and structure factors have been deposited in the Protein Data Bank under the accession code 4K5Y.

References

  1. Gether, U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev. 21, 90–113 (2000)

    Article  CAS  Google Scholar 

  2. Lagerström, M. C. & Schiöth, H. B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nature Rev. Drug Discov. 7, 339–357 (2008)

    Article  Google Scholar 

  3. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013)

    Article  ADS  CAS  Google Scholar 

  4. Bhavsar, S., Mudaliar, S. & Cherrington, A. Evolution of exenatide as a diabetes therapeutic. Curr. Diabetes Rev. 9, 161–193 (2013)

    PubMed  PubMed Central  Google Scholar 

  5. Berg, C., Neumeyer, K. & Kirkpatrick, P. Teriparatide. Nature Rev. Drug Discov. 2, 257–258 (2003)

    Article  CAS  Google Scholar 

  6. Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)

    Article  ADS  CAS  Google Scholar 

  7. Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013)

    Article  ADS  CAS  Google Scholar 

  8. Congreve, M., Langmead, C. & Marshall, F. H. The use of GPCR structures in drug design. Adv. Pharmacol. 62, 1–36 (2011)

    Article  CAS  Google Scholar 

  9. Pioszak, A. A., Parker, N. R., Suino-Powell, K. & Xu, H. E. Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1. J. Biol. Chem. 283, 32900–32912 (2008)

    Article  CAS  Google Scholar 

  10. Grace, C. R. R. et al. NMR structure and peptide hormone binding site of the first extracellular domain of a type B1 G protein-coupled receptor. Proc. Natl Acad. Sci. USA 101, 12836–12841 (2004)

    Article  ADS  CAS  Google Scholar 

  11. Runge, S., Thøgersen, H., Madsen, K., Lau, J. & Rudolph, R. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. J. Biol. Chem. 283, 11340–11347 (2008)

    Article  CAS  Google Scholar 

  12. Pioszak, A. A. & Xu, H. E. Molecular recognition of parathyroid hormone by its G protein-coupled receptor. Proc. Natl Acad. Sci. USA 105, 5034–5039 (2008)

    Article  ADS  CAS  Google Scholar 

  13. ter Haar, E. et al. Crystal structure of the ectodomain complex of the CGRP receptor, a class-B GPCR, reveals the site of drug antagonism. Structure 18, 1083–1093 (2010)

    Article  CAS  Google Scholar 

  14. Kumar, S., Pioszak, A., Zhang, C., Swaminathan, K. & Xu, H. E. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors. PLoS ONE 6, e19682 (2011)

    Article  ADS  CAS  Google Scholar 

  15. Koth, C. M. et al. Molecular basis for negative regulation of the glucagon receptor. Proc. Natl Acad. Sci. USA 109, 14393–14398 (2012)

    Article  ADS  CAS  Google Scholar 

  16. Perrin, M. H. & Vale, W. W. Corticotropin releasing factor receptors and their ligand family. Ann. NY Acad. Sci. 885, 312–328 (1999)

    Article  ADS  CAS  Google Scholar 

  17. Bale, T. L. & Vale, W. W. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol. 44, 525–557 (2004)

    Article  CAS  Google Scholar 

  18. Hemley, C. F., McCluskey, A. & Keller, P. A. Corticotropin releasing hormone–a GPCR drug target. Curr. Drug Targets 8, 105–115 (2007)

    Article  CAS  Google Scholar 

  19. Chen, Y. L. et al. 2-aryloxy-4-alkylaminopyridines: discovery of novel corticotropin-releasing factor 1 antagonists. J. Med. Chem. 51, 1385–1392 (2008)

    Article  CAS  Google Scholar 

  20. Hoare, S. R. et al. Allosteric ligands for the corticotropin releasing factor type 1 receptor modulate conformational states involved in receptor activation. Mol. Pharmacol. 73, 1371–1380 (2008)

    Article  CAS  Google Scholar 

  21. Zorrilla, E. P. & Koob, G. F. Progress in corticotropin-releasing factor-1 antagonist development. Drug Discov. Today 15, 371–383 (2010)

    Article  CAS  Google Scholar 

  22. Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA 105, 877–882 (2008)

    Article  ADS  CAS  Google Scholar 

  23. Shibata, Y. et al. Thermostabilization of the neurotensin receptor NTS1. J. Mol. Biol. 390, 262–277 (2009)

    Article  CAS  Google Scholar 

  24. Lebon, G., Bennett, K., Jazayeri, A. & Tate, C. G. Thermostabilisation of an agonist-bound conformation of the human adenosine A2A receptor. J. Mol. Biol. 409, 298–310 (2011)

    Article  CAS  Google Scholar 

  25. Piserchio, A., Bisello, A., Rosenblatt, M., Chorev, M. & Mierke, D. F. Characterization of parathyroid hormone/receptor interactions: structure of the first extracellular loop. Biochemistry 39, 8153–8160 (2000)

    Article  CAS  Google Scholar 

  26. Wootten, D., Simms, J., Miller, L. J., Christopoulos, A. & Sexton, P. M. Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations. Proc. Natl Acad. Sci. USA 110, 5211–5216 (2013)

    Article  ADS  CAS  Google Scholar 

  27. Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012)

    Article  CAS  Google Scholar 

  28. Mann, R. J., Al-Sabah, S., De Maturana, R. L., Sinfield, J. K. & Donnelly, D. Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket. Peptides 31, 2289–2293 (2010)

    Article  CAS  Google Scholar 

  29. Chien, E. Y. T. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330, 1091–1095 (2010)

    Article  ADS  CAS  Google Scholar 

  30. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995)

    Article  CAS  Google Scholar 

  31. Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)

    Article  ADS  CAS  Google Scholar 

  32. Schipani, E., Kruse, K. & Jüppner, H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268, 98–100 (1995)

    Article  ADS  CAS  Google Scholar 

  33. Heller, R. S., Kieffer, T. J. & Habener, J. F. Point mutations in the first and third intracellular loops of the glucagon-like peptide-1 receptor alter intracellular signaling. Biochem. Biophys. Res. Commun. 223, 624–632 (1996)

    Article  CAS  Google Scholar 

  34. Hjorth, S. A., Orskov, C. & Schwartz, T. W. Constitutive activity of glucagon receptor mutants. Mol. Endocrinol. 12, 78–86 (1998)

    Article  CAS  Google Scholar 

  35. Vohra, S. et al. Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies. J. R. Soc. Interface 10, 20120846 (2013)

    Article  Google Scholar 

  36. Hoare, S. R. J. et al. Single amino acid residue determinants of non-peptide antagonist binding to the corticotropin-releasing factor1 (CRF1) receptor. Biochem. Pharmacol. 72, 244–255 (2006)

    Article  CAS  Google Scholar 

  37. Gardella, T. J., Luck, M. D., Fan, M. H. & Lee, C. Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands. J. Biol. Chem. 271, 12820–12825 (1996)

    Article  CAS  Google Scholar 

  38. Conner, A. C. et al. A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors. Mol. Pharmacol. 67, 20–31 (2005)

    Article  CAS  Google Scholar 

  39. Chugunov, A. O. et al. Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation. Mol. Pharmacol. 78, 394–401 (2010)

    Article  CAS  Google Scholar 

  40. Ganguli, S. C. et al. Protean effects of a natural peptide agonist of the G protein-coupled secretin receptor demonstrated by receptor mutagenesis. J. Pharmacol. Exp. Ther. 286, 593–598 (1998)

    CAS  PubMed  Google Scholar 

  41. Robertson, N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36–44 (2011)

    Article  CAS  Google Scholar 

  42. Söding, J. Protein homology detection by HMM–HMM comparison. Bioinformatics 21, 951–960 (2005)

    Article  Google Scholar 

  43. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001)

    Article  CAS  Google Scholar 

  44. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

    Article  CAS  Google Scholar 

  45. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic cubic mesophases. Nature Protocols 4, 706–731 (2009)

    Article  CAS  Google Scholar 

  46. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  Google Scholar 

  47. Leslie, A. G. W. & Powell, H. R. Processing diffraction data with Mosflm. Evolv. Methods Macromol. Crystallogr. 245, 41–51 (2007)

    Article  Google Scholar 

  48. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  Google Scholar 

  49. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012)

    Article  ADS  CAS  Google Scholar 

  50. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  52. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  53. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

    Article  CAS  Google Scholar 

  54. Haddadian, E. J. et al. Automated real-space refinement of protein structures using a realistic backbone move set. Biophys. J. 101, 899–909 (2011)

    Article  ADS  CAS  Google Scholar 

  55. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Evans, R. Owen and D. Axford for their support. We are grateful to A. Leslie, R. Read and A. McCoy for advice on data collection and structure determination. We thank R. Henderson, A. Leslie, C. Tate as well as F. Blaney, B. Tehan, R. Miller, F. Deflorian and other colleagues for suggestions and comments.

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Authors and Affiliations

  1. Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK,

    Kaspar Hollenstein, James Kean, Andrea Bortolato, Robert K. Y. Cheng, Andrew S. Doré, Ali Jazayeri, Robert M. Cooke, Malcolm Weir & Fiona H. Marshall

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  1. Kaspar Hollenstein
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Contributions

J.K. carried out the conformational thermostabilization and pharmacological characterization of the constructs and determined the stability of the StaR in a panel of reagents/additives to aid purification and crystallization. A.J. constructed the T4 lysozyme fusion proteins and identified the usefulness of the fusion into intracellular loop 2. K.H. designed and characterized truncation constructs, designed the final T4 lysozyme fusion construct, established procedures for, and carried out expression and purification, established the platform/protocols for and carried out lipidic cubic phase (LCP) crystallization, collected and processed X-ray diffraction data, solved and refined the structure. R.K.Y.C. performed expression and purification, optimized purification and grew crystals in LCP for data collection of the final construct, collected X-ray diffraction data and solved and refined the structure. A.S.D. was involved in construct design, established the platform/protocols for, and carried out LCP crystallization, collected and processed X-ray diffraction data and solved and refined the structure. Computational analysis of the structure and modelling was carried out by A.B. Project management was carried out by A.J., R.M.C., M.W. and F.H.M. The manuscript was prepared by K.H., R.K.Y.C., A.B., J.K., A.J. and F.H.M.

Corresponding author

Correspondence to Fiona H. Marshall.

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Competing interests

The authors are employees and shareholders of Heptares Therapeutics Ltd, a GPCR drug discovery company using structure-based drug design techniques.

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Hollenstein, K., Kean, J., Bortolato, A. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013). https://doi.org/10.1038/nature12357

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