Abstract
Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells1. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade2. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM (ref. 3), 10−2 of its K d value for erythropoietin–EPOR binding site 1 (Kd ≈ 1 nM), and 10−5 of the K d for erythropoietin–EPOR binding site 2 (Kd ≈ 1 μM)4. Overall half-maximal binding (IC50) of cell-surface receptors is produced with ∼0.18 nM erythropoietin, indicating that only ∼6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses5,6 but much less efficiently, requiring concentrations close to their K d values (∼0.1 μM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 Å from two crystal forms, shows that erythropoietin imposes a unique 120° angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Graber, S. E. & Krantz, S. B. Erythropoietin and the control of red blood cell production. Annu. Rev. Med. 29, 51–66 (1978).
Damen, J. E. & Krystal, G. Early events in erythropoietin-induced signaling. Exp. Hematol. 24, 1455–1459 (1996).
Johnson, D. L. et al. Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1. Biochemistry 37, 3699–3710 (1998).
Philo, J. S., Aoki, K. H., Arakawa, T., Narhi, L. O. & Wen, J. Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction. Biochemistry 35, 1681–1691 (1996).
Wrighton, N. C. et al. Small peptides as potent mimetics of the protein hormone erythropoietin. Science 273, 458–464 (1996).
Livnah, O. et al. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 Å. Science 273, 464–471 (1996).
Derby, P., Aoki, K. H., Katta, V. & Rohde, M. F. in Techniques in Protein Chemistry VII (ed. Marshak, D.R.) 109–119 Academic, San Diego, (1996).
Hilton, D. J., Watowich, S. S., Katz, L. & Lodish, H. F. Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor. J. Biol. Chem. 271, 4699–4708 (1996).
Abrahams, J. P. & Leslie, A. G. W. Acta Crystallogr. D 52, 30–42 1996).
Cheetham, J. C. et al. NMR structure of human erythropoietin and a comparison with its receptor bound conformation. Nature Struct. Biol. (in the press).
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).
Sprang, S. R. & Bazan, J. F. Cytokine structural taxonomy and mechanisms of receptor engagement. Curr. Opin. Struct. Biol. 3, 815–827 (1993).
Rozwarski, D. A. et al. Structural comparisons among the short-chain helical cytokines. Structure 2, 159–173 (1994).
Yoshimura, A. et al. Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. J. Biol. Chem. 267, 11619–11625 (1992).
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).
Barbone, F. P. et al. Mutagenesis studies of the human erythropoietin receptor. Establishment of structure–function relationships. J. Biol. Chem. 272, 4985–4992 (1997).
Middleton, S. A. et al. Identification of a critical ligand-binding determinant of the human erythropoietin receptor—evidence for common ligand-binding motifs in the cytokine receptor family. J. Biol. Chem. 271, 14045–14054 (1996).
Grodberg, J., Davis, K. L. & Sykowski, A. J. Alanine scanning mutagenesis of human erythropoietin identifies four amino acids which are critical for biological activity. Eur. J. Biochem. 218, 597–601 (1993).
Wen, D. Y., Boissel, J. P., Showers, M., Ruch, B. C. & Bunn, H. F. Erythropoietin structure–function relationships–identification of functionally important domains. J. Biol. Chem. 269, 22839–22846 (1994).
Matthews, D. J., Topping, R. S., Cass, R. T. & Giebel, L. B. Asequential dimerization mechanism for erythropoietin receptor activation. Proc. Natl Acad. Sci. USA 93, 9471–9476 (1996).
Elliott, S., Lorenzini, T., Chang, D., Barzilay, J. & Delorme, E. Mapping of the active site of recombinant human erythropoietin. Blood 89, 493–502 (1997).
Clackson, T. & Wells, J. A. Ahot spot of binding energy in a hormone-receptor interface. Science 267, 383–386 (1995).
Watowich, S. S. et al. Homodimerization and constitutive activation of the erythropoietin receptor. Proc. Natl Acad. Sci. USA 89, 2140–2144 (1992).
Narhi, L. O. et al. The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022–23026 (1991).
Johnson, D. L. et al. Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli. Prot. Express. Purif. 7, 104–113 (1996).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
Brunger, A. T. X-PLOR, Version 3.1, A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, 1992).
Furey, W. & Swaminathan, S. Phases-95: a program package for processing and analyzing diffraction data from macromolecules. Methods Enzymol. 277, 590–620 (1997).
Acknowledgements
We thank J. Philo, M. McGrath, P. Sprengeler, W. Welch, J. Rupert, L. Narhi, G.Rogers, M. Rohde, S. Jordan, K. Langley, R. Mackman and M. Venuti for valuable discussions. Structure analyses of Form 1 and Form 2 were independently performed at Axys Pharmaceuticals Inc. and Amgen Inc., respectively.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Syed, R., Reid, S., Li, C. et al. Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 395, 511–516 (1998). https://doi.org/10.1038/26773
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/26773
This article is cited by
-
A critical role for erythropoietin on vagus nerve Schwann cells in intestinal motility
BMC Biotechnology (2023)
-
A topological refactoring design strategy yields highly stable granulopoietic proteins
Nature Communications (2022)
-
Erythropoietin-derived peptide treatment reduced neurological deficit and neuropathological changes in a mouse model of tauopathy
Alzheimer's Research & Therapy (2021)
-
Modified aptamers as reagents to characterize recombinant human erythropoietin products
Scientific Reports (2020)
-
Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion
Nature Communications (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.