Erythropoietin receptor activation by a ligand-induced conformation change

Science

Volumn 283, Issue 5404, 1999, Pages 990-993

  Remy, Ingrid ^a   Wilson, Ian A ^b   Michnick, Stephen W ^a  

^a UNIVERSITÉ DE MONTRÉAL   (Canada)

^b SCRIPPS RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYTOKINE; ERYTHROPOIETIN; ERYTHROPOIETIN RECEPTOR; PHOSPHOTRANSFERASE;

ANIMAL CELL; ARTICLE; AUTOPHOSPHORYLATION; CONFORMATIONAL TRANSITION; CRYSTALLOGRAPHY; DIMERIZATION; FLUORESCENCE; MODEL; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; RECEPTOR OCCUPANCY; STRUCTURE ANALYSIS;

ANIMALS; CHO CELLS; COS CELLS; CRICETINAE; DIMERIZATION; ERYTHROPOIETIN; FLOW CYTOMETRY; FLUORESCEINS; JANUS KINASE 2; LIGANDS; METHOTREXATE; MICROSCOPY, FLUORESCENCE; PEPTIDES, CYCLIC; PROTEIN CONFORMATION; PROTEIN-TYROSINE KINASES; PROTO-ONCOGENE PROTEINS; RECEPTORS, ERYTHROPOIETIN; RECOMBINANT FUSION PROTEINS; TETRAHYDROFOLATE DEHYDROGENASE; TRANSFECTION;

EID: 0033548048     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5404.990     Document Type: Article

References (27)

1. A. D. D'Andrea, G. D. Fasman, H. F. Lodish, Cell 58, 1023 (1989)
2. J. F. Bazan, Proc. Natl. Acad. Sci. U.S.A. 87, 6934 (1990); N. Stahl and G. D. Yancopoulos, Cell 74, 587 (1993).
3. J. F. Bazan, Proc. Natl. Acad. Sci. U.S.A. 87, 6934 (1990); N. Stahl and G. D. Yancopoulos, Cell 74, 587 (1993).
4. C. H. Heldin, Cell 80, 213 (1995).
5. hGH-hGHR: A. M. De Vos, M. Ultsch, A. A. Kossiakoff, Science 255, 306 (1992); Epo-EpoR: R. S. Syed et al., Nature 395, 511 (1998); EMB1-EpoR: O. Livnah et al., Science 273, 464 (1996); EMP33-EpoR: O. Livnah, et al., Nature Struct. Biol. 5, 993 (1998).
6. hGH-hGHR: A. M. De Vos, M. Ultsch, A. A. Kossiakoff, Science 255, 306 (1992); Epo-EpoR: R. S. Syed et al., Nature 395, 511 (1998); EMB1-EpoR: O. Livnah et al., Science 273, 464 (1996); EMP33-EpoR: O. Livnah, et al., Nature Struct. Biol. 5, 993 (1998).
7. hGH-hGHR: A. M. De Vos, M. Ultsch, A. A. Kossiakoff, Science 255, 306 (1992); Epo-EpoR: R. S. Syed et al., Nature 395, 511 (1998); EMB1-EpoR: O. Livnah et al., Science 273, 464 (1996); EMP33-EpoR: O. Livnah, et al., Nature Struct. Biol. 5, 993 (1998).
8. hGH-hGHR: A. M. De Vos, M. Ultsch, A. A. Kossiakoff, Science 255, 306 (1992); Epo-EpoR: R. S. Syed et al., Nature 395, 511 (1998); EMB1-EpoR: O. Livnah et al., Science 273, 464 (1996); EMP33-EpoR: O. Livnah, et al., Nature Struct. Biol. 5, 993 (1998).
9. O. Livnah et al., Science 283, 987 (1999).
10. J. N. Pelletier and S. W. Michnick, Protein Eng. 10, 89 (1997).
11. J. N. Pelletier, F. Campbell-Valois, S. W. Michnick, Proc. Natl. Acad. Sci. U.S.A. 95, 12141 (1998).
12. R. J. Kaufman, J. R. Bertino, R. T. Schimke, J. Biol. Chem. 253, 5852 (1978); D. I. Israel and R. J. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 90, 4290 (1993).
13. R. J. Kaufman, J. R. Bertino, R. T. Schimke, J. Biol. Chem. 253, 5852 (1978); D. I. Israel and R. J. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 90, 4290 (1993).
14. V. Cody, J. R. Luft, E. Ciszak, T. I. Kalman, J. H. Freisheim, Anti-Cancer Drug Des. 7, 483 (1992).
15. 2 and residual error for the Epo curve were 0.98 and 0.045, respectively, and for the EMP1 curve, 0.99 and 0.022, respectively).
16. A. D. D'Andrea et al., Mol. Cell. Biol. 11, 1980 (1991); D. L. Johnson et al., Biochemistry 37, 3699 (1998); N. C. Wrighton et al., Science 273, 458 (1996); N. C. Wrighton et al., Nature Biotechnol. 15, 1261 (1997).
17. A. D. D'Andrea et al., Mol. Cell. Biol. 11, 1980 (1991); D. L. Johnson et al., Biochemistry 37, 3699 (1998); N. C. Wrighton et al., Science 273, 458 (1996); N. C. Wrighton et al., Nature Biotechnol. 15, 1261 (1997).
18. A. D. D'Andrea et al., Mol. Cell. Biol. 11, 1980 (1991); D. L. Johnson et al., Biochemistry 37, 3699 (1998); N. C. Wrighton et al., Science 273, 458 (1996); N. C. Wrighton et al., Nature Biotechnol. 15, 1261 (1997).
19. A. D. D'Andrea et al., Mol. Cell. Biol. 11, 1980 (1991); D. L. Johnson et al., Biochemistry 37, 3699 (1998); N. C. Wrighton et al., Science 273, 458 (1996); N. C. Wrighton et al., Nature Biotechnol. 15, 1261 (1997).
20. I. Remy and S. W. Michnick, data not shown.
21. B. A. Witthuhn et al., Cell 74, 227 (1993).
22. C. Guo, S. K. Dower, D. Holowka, B. Baird, J. Biol. Chem. 270, 27562 (1995); S. Damjanovich et al., Proc. Natl. Acad. Sci. U.S.A. 94. 13134 (1997); T. Gadella Jr. and T. M. Jovin, J. Cell Biol. 129, 1543 (1995).
23. C. Guo, S. K. Dower, D. Holowka, B. Baird, J. Biol. Chem. 270, 27562 (1995); S. Damjanovich et al., Proc. Natl. Acad. Sci. U.S.A. 94. 13134 (1997); T. Gadella Jr. and T. M. Jovin, J. Cell Biol. 129, 1543 (1995).
24. C. Guo, S. K. Dower, D. Holowka, B. Baird, J. Biol. Chem. 270, 27562 (1995); S. Damjanovich et al., Proc. Natl. Acad. Sci. U.S.A. 94. 13134 (1997); T. Gadella Jr. and T. M. Jovin, J. Cell Biol. 129, 1543 (1995).
25. R. J. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 82, 689 (1985).
26. All fusion clones were generated by polymerase chain reaction amplification of the individual genes of interest. The construction of the DHFR F[1,2] and F[3] are described in (7). Oligonucleotides coding for flexible linker peptides were synthesized individually with 5′ and 3′ complementary overhangs corresponding to 5′ or 3′ insertion between EpoR- and DHFR fragment-encoding sequences. Regions of each construct were subcloned into the mammalian expression vector pMT3 (15). Lipofectamine (Life Technologies) was used to stably transfect cells with EpoR-DHFR fragments, and stable colonies were selected on minimum essential medium (Life Technologies) enriched with 10% dialyzed fetal bovine serum (FBS) (Hyclone) (dialyzed to remove nucleotides, rendering cells dependent on exogenous DHFR activity) and in the presence of 2 nM human recombinant Epo (R. W. Johnson Pharmaceutical Research Institute). fMTX (Molecular Probes) was added to each sample at a final concentration of 10 μM and incubated for 22 hours at 37°C without Epo. Cells were then treated with 10 nM Epo or 10 μM EMP1 (RWJ) for 30 min at 37°C. The medium was removed and the cells were washed with PBS and incubated again for 30 min in α-MEM with Epo or EMP1 to allow for efflux of unbound fMTX. Medium was removed and cells were washed with PBS, treated with trypsin, and suspended in 500 μl of cold PBS supplemented with 10% FBS to increase cell viability and kept on ice before cytometric analysis within 20 min.
27. We gratefully acknowledge gifts of pMT3 vector and CHO DUKX-B11 cell line and helpful advice from M. Davies (Genetics Institute), EpoR and JAK2 clones from U. Klingmüller (Max Planck Institute for Immunobiology, Freiberg), and EMP1, Epo, and helpful discussion from D. Johnson and L. Jolliffe (R. W. Johnson Pharmaceutical Research Institute), and we thank M. Powell for timely advice. Supported by the Burroughs Wellcome Fund (S.W.M.) and NIH grant GM49497 (I.A.W.).