Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Nature Structural & Molecular Biology volume 12, pages 814–821 (2005)Cite this article

Abstract

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structure of unliganded hGHR ECD.
Figure 2: Rotation of subunits results in constitutive hGHR activation in the absence of hGH in stable BaF/3 populations.
Figure 3: Rotation of subunits results in constitutive GHR activation in the absence of hGH in CHO-K1 cells transiently transfected with hGHR.
Figure 4: Evidence for constitutive receptor dimers by coimmunoprecipitation.
Figure 5: Evidence for constitutive GHR dimerization by FRET.
Figure 6: Evidence for constitutive GHR dimerization by BRET.
Figure 7: A schematic model of GHR activation.

Similar content being viewed by others

Accession codes

Accessions

BINDPlus

Protein Data Bank

References

  1. Fuh, G. et al. Rational design of potent antagonists to the human growth hormone receptor. Science 256, 1677–1680 (1992).

    Article  CAS  Google Scholar 

  2. Rowlinson, S.W. et al. Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signaling. J. Biol. Chem. 273, 5307–5314 (1998).

    Article  CAS  Google Scholar 

  3. Jiang, J. et al. A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: impact on GH signaling and GHR proteolysis. Mol. Endocrinol. 18, 2981–2996 (2004).

    Article  CAS  Google Scholar 

  4. Wan, Y., McDevitt, A., Shen, B., Smythe, M.L. & Waters, M.J. Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization. J. Biol. Chem. 279, 44775–44784 (2004).

    Article  CAS  Google Scholar 

  5. Ross, R.J. et al. Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer. J. Clin. Endocrinol. Metab. 86, 1716–1723 (2001).

    CAS  PubMed  Google Scholar 

  6. Harding, P.A. et al. Growth hormone (GH) and a GH antagonist promote GH receptor dimerization and internalization. J. Biol. Chem. 271, 6708–6712 (1996).

    Article  CAS  Google Scholar 

  7. Constantinescu, S.N., Huang, L.J., Nam, H. & Lodish, H.F. The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif. Mol. Cell 7, 377–385 (2001).

    Article  CAS  Google Scholar 

  8. Livnah, O. et al. Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283, 987–990 (1999).

    Article  CAS  Google Scholar 

  9. Seubert, N. et al. Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. Mol. Cell 12, 1239–1250 (2003).

    Article  CAS  Google Scholar 

  10. McKinstry, W.J. et al. Crystallization and preliminary X-ray diffraction analysis of the unliganded human growth hormone receptor. Acta Crystallogr. D Biol. Crystallogr. 60, 2380–2382 (2004).

    Article  Google Scholar 

  11. de Vos, A.M., Ultsch, M. & Kossiakoff, A.A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306–312 (1992).

    Article  CAS  Google Scholar 

  12. Clackson, T., Ultsch, M.H., Wells, J.A. & de Vos, A.M. Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity. J. Mol. Biol. 277, 1111–1128 (1998).

    Article  CAS  Google Scholar 

  13. Ottemann, K.M., Xiao, W., Shin, Y.K. & Koshland, D.E., Jr. A piston model for transmembrane signaling of the aspartate receptor. Science 285, 1751–1754 (1999).

    Article  CAS  Google Scholar 

  14. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    Article  CAS  Google Scholar 

  15. Rost, B., Yachdav, G. & Liu, J. The PredictProtein server. Nucleic Acids Res. 32, W321–W326 (2004).

    Article  CAS  Google Scholar 

  16. Gent, J., van Kerkhof, P., Roza, M., Bu, G. & Strous, G.J. Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. Proc. Natl. Acad. Sci. USA 99, 9858–9863 (2002).

    Article  CAS  Google Scholar 

  17. Baumgartner, J.W., Wells, C.A., Chen, C.M. & Waters, M.J. The role of the WSXWS equivalent motif in growth hormone receptor function. J. Biol. Chem. 269, 29094–29101 (1994).

    CAS  PubMed  Google Scholar 

  18. Dinerstein, H. et al. The proline-rich region of the GH receptor is essential for JAK2 phosphorylation, activation of cell proliferation, and gene transcription. Mol. Endocrinol. 9, 1701–1707 (1995).

    CAS  PubMed  Google Scholar 

  19. Eidne, K.A., Kroeger, K.M. & Hanyaloglu, A.C. Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol. Metab. 13, 415–421 (2002).

    Article  CAS  Google Scholar 

  20. Damjanovich, S. et al. Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: a fluorescence resonance energy transfer study. Proc. Natl. Acad. Sci. USA 94, 13134–13139 (1997).

    Article  CAS  Google Scholar 

  21. Couturier, C. & Jockers, R. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J. Biol. Chem. 278, 26604–26611 (2003).

    Article  CAS  Google Scholar 

  22. Issad, T., Boute, N. & Pernet, K. A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer. Biochem. Pharmacol. 64, 813–817 (2002).

    Article  CAS  Google Scholar 

  23. Waters, M.J. & Friesen, H.G. Purification and partial characterization of a nonprimate growth hormone receptor. J. Biol. Chem. 254, 6815–6825 (1979).

    CAS  PubMed  Google Scholar 

  24. Gurezka, R., Laage, R., Brosig, B. & Langosch, D. A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments. J. Biol. Chem. 274, 9265–9270 (1999).

    Article  CAS  Google Scholar 

  25. Hanyaloglu, A.C., Seeber, R.M., Kohout, T.A., Lefkowitz, R.J. & Eidne, K.A. Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2. J. Biol. Chem. 277, 50422–50430 (2002).

    Article  CAS  Google Scholar 

  26. Kubatzky, K.F. et al. Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor. Curr. Biol. 11, 110–115 (2001).

    Article  CAS  Google Scholar 

  27. Bernat, B., Pal, G., Sun, M. & Kossiakoff, A.A. Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization. Proc. Natl. Acad. Sci. USA 100, 952–957 (2003).

    Article  CAS  Google Scholar 

  28. Zhang, Y., Jiang, J., Kopchick, J.J. & Frank, S.J. Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization. J. Biol. Chem. 274, 33072–33084 (1999).

    Article  CAS  Google Scholar 

  29. van Kerkhof, P., Smeets, M. & Strous, G.J. The ubiquitin-proteasome pathway regulates the availability of the GH receptor. Endocrinology 143, 1243–1252 (2002).

    Article  CAS  Google Scholar 

  30. Schneider, H. et al. Homodimerization of erythropoietin receptor by a bivalent monoclonal antibody triggers cell proliferation and differentiation of erythroid precursors. Blood 89, 473–482 (1997).

    CAS  PubMed  Google Scholar 

  31. Waters, M. The growth hormone receptor. in Handbook of Physiology—Section 7: The Endocrine System., Vol. 5 (ed. Goodman, M.) 445–480 (Oxford University Press, New York, 1999).

    Google Scholar 

  32. Chen, C., Brinkworth, R. & Waters, M.J. The role of receptor dimerization domain residues in growth hormone signaling. J. Biol. Chem. 272, 5133–5140 (1997).

    Article  CAS  Google Scholar 

  33. Gent, J., Van Den Eijnden, M., Van Kerkhof, P. & Strous, G.J. Dimerization and signal transduction of the growth hormone receptor. Mol. Endocrinol. 17, 967–975 (2003).

    Article  CAS  Google Scholar 

  34. Blott, S. et al. Molecular dissection of a quantitative trait locus: a phenylalanine-to-tyrosine substitution in the transmembrane domain of the bovine growth hormone receptor is associated with a major effect on milk yield and composition. Genetics 163, 253–266 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Angers, S., Salahpour, A. & Bouvier, M. Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells. Life Sci. 68, 2243–2250 (2001).

    Article  CAS  Google Scholar 

  36. Kroeger, K.M., Hanyaloglu, A.C., Seeber, R.M., Miles, L.E. & Eidne, K.A. Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J. Biol. Chem. 276, 12736–12743 (2001).

    Article  CAS  Google Scholar 

  37. Piper, R.C., Hess, L.J. & James, D.E. Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am. J. Physiol. 260, C570–C580 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by National Health and Medical Research Council (NHMRC; Australia) project grants to M.J.W. and K.A.E. We thank H. Tong and other staff at BioCARS for their help at the Advanced Photon Source (supported by the US Department of Energy, Basic Energy Sciences, Office of Energy Research). The crystallographic work, and use of the BioCARS sector, was supported by the Australian Synchrotron Research Program, funded by the Commonwealth of Australia under the Major National Research Facilities Program. We thank E. Holliday and E. Lim for their skilled assistance. W.J.M. is a NHMRC Industry Fellow and M.W.P. is an NHMRC Senior Principal Research Fellow and M.J.W. and K.A.E. are NHMRC Principal Research Fellows.

Author information

Author notes

  1. Richard J Brown, Julian J Adams and Rebecca A Pelekanos: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia

    Richard J Brown, Rebecca A Pelekanos, Yu Wan, Kathryn Palethorpe, Thea A Monks & Michael J Waters

  2. Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia

    Julian J Adams, William J McKinstry & Michael W Parker

  3. The University of Western Australia, Western Australian Institute for Medical Research, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia

    Ruth M Seeber & Karin A Eidne

Authors
  1. Richard J Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julian J Adams
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rebecca A Pelekanos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William J McKinstry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kathryn Palethorpe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ruth M Seeber
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thea A Monks
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Karin A Eidne
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michael W Parker
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Michael J Waters
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael J Waters.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brown, R., Adams, J., Pelekanos, R. et al. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nat Struct Mol Biol 12, 814–821 (2005). https://doi.org/10.1038/nsmb977

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb977

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing