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Endocrine

, Volume 23, Issue 2–3, pp 199–213 | Cite as

Stepwise posttranslational processing of progrowth hormone-releasing hormone (proGHRH) polypeptide by furin and PC1

  • Samuel F. Posner
  • Charles A. Vaslet
  • Michelle Jurofcik
  • Alisson Lee
  • Nabil G. Seidah
  • Eduardo A. NillniEmail author
Original Articles

Abstract

Through a posttranslational processing mechanism, progrowth hormone releasing hormone (proGHRH) gives rise to an amidated GHRH molecule, which in turn stimulates the synthesis and release of growth hormone. We have previously proposed a model for the biochemical processing of proGHRH [Nillni et al. (1999), Endocrinology 140, 5817–5827]. We demonstrated that the proGHRH peptide (10.5 kDa, 104 aa) is first processed to an 8.8 kDa intermediate form that is later cleaved to yield two products: the 5.2 kDa GHRH and the 3.6 kDa GHRH-RP. However, the proteolytic enzymes involved in this process are unknown. Therefore, in this study we determined which proconverting enzymes are involved in this process. We transfected different constructs in cell lines carrying different PC enzymes followed by analysis of the peptide products after metabolic labeling or Western blots. We found that in the absence of furin (LoVo cells) or CHO cells treated with BFA, only one moiety was observed, and that corresponds to the same electrophorectic mobility to the GHRH precursor. This finding strongly supports an initial role for furin in the processing of proGHRH. The results from transfections with preproGHRH alone or double or triple transfections with PC1 and PC2 in AtT-20, GH3, and GH4C1 cells indicated that PC1 is the primary enzyme involved in the generation of GHRH peptide from the 8.8 kDa intermediate form. We found that AtT-20 cells (high PC1, very low PC2) were able to generate GHRH. However, GH3 cells (high PC2, but not PC1) were able to process the 8.8 kDa peptide to GHRH only after the cotransfection with the PC1 enzyme. Transfections with preproGHRH-GFP and preproGHRH-V5 provided similar results in all the cell lines analyzed. These data support the hypothesis that proGHRH is initially cleave by furin at preproGHRH29–30, followed by a second cleavage at preproGHRH74 primarily by PC1 to generate GHRH and GHRH-RP peptides, respectively.

Key Words

proGHRH GHRH prohormone processing convertases PC1 PC2 furin peptide sorting 

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References

  1. 1.
    Steiner, D. F. (1998). Curr. Opinion Chem. Biol. 2, 31–39.CrossRefGoogle Scholar
  2. 2.
    Seidah, N. G. and Chretien, M. (1999). Brain Res. 848, 45–62.PubMedCrossRefGoogle Scholar
  3. 3.
    Arvan, P. and Castle, D. (1998). Biochem. J. 332, 593–610.PubMedGoogle Scholar
  4. 4.
    Nillni, E. A., Steinmetz, R., and Pescovitz, O. H. (1999). Endocrinol. 140, 5817–5827.CrossRefGoogle Scholar
  5. 5.
    Seidah, N. G., Chretien, M., and Day, R. (1994). Biochimie 76, 197–209.PubMedCrossRefGoogle Scholar
  6. 6.
    Guillemin, R., Brazeau, P., Bohlen, P., Esch, F., Ling, N., and Wehrenberg, W. B. (1982). Science 218, 585–587.PubMedCrossRefGoogle Scholar
  7. 7.
    Rivier, J., Spiess, J., Thorner, M., and Vale, W. (1982). Nature 300, 276–278.PubMedCrossRefGoogle Scholar
  8. 8.
    Hofman, P. L. and Pescovitz, O. H. (1998). In: The cellular basis of pediatric endocrinology. Handwerger, S. (ed.), Humana Press: Totowa, NJ.Google Scholar
  9. 9.
    Loh, Y. P., Beinfeld, M. C., and Birch, N. P. (1993). In: Mechanisms of intracellular trafficking and processing proproteins. Loh, Y. P. (ed.), CRC Press: Boca Raton, FL, pp. 179–224.Google Scholar
  10. 10.
    Mains, R. E., Dickerson, I. M., May, V., et al. (1990). Front. Neuroendocrinol. 11, 52–89.Google Scholar
  11. 11.
    Seidah, N., Marcinkiewicz, M., Benjannet, S., et al. (1991). Mol. Endocrinol. 5, 111–122.PubMedGoogle Scholar
  12. 12.
    Seidah, N. G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M., and Chretien, M. (1990). DNA 9, 415–424.CrossRefGoogle Scholar
  13. 13.
    Lusson, J., Vieau, D., Hamelin, J., Day, R., Chretien, M., and Seidah, N. G. (1993). Proc. Natl. Acad. Sci. USA 90, 6691–6695.PubMedCrossRefGoogle Scholar
  14. 14.
    Schafer, M.-H., Day, R., Cullinan, W. E., Chretien, M., Seidah, N., and Watson, S. (1993). J. Neurosci. 13, 1258–1279.PubMedGoogle Scholar
  15. 15.
    Schaner, P., Todd, R. B., Seidah, N. G., and Nillni, E. A. (1997). J. Biol. Chem. 272, 19958–19968.PubMedCrossRefGoogle Scholar
  16. 16.
    Pu, L. P., Ma, W., Barker, J., and Loh, Y. P. (1996). Endocrinol. 137, 1233–1241.CrossRefGoogle Scholar
  17. 17.
    Nillni, E. A., Friedman, T. C., Todd, R. B., Birch, N. P., Loh, Y. P., and Jackson, I. M. D. (1995). J. Neurochem. 65, 2462–2472.PubMedCrossRefGoogle Scholar
  18. 18.
    Friedman, T. C., Loh, Y. P., Huang, S. S., Jackson, I. M. D., and Nillni, E. A. (1995). Endocrinol. 136, 4462–4472.CrossRefGoogle Scholar
  19. 19.
    Steiner, D. F., Smeekens, S. P., Ohag, S., and Chan, S. J. (1992). J. Biol. Chem. 267, 23435–23438.PubMedGoogle Scholar
  20. 20.
    Smeekens, S. P., Montag, A. G., Thomas, G., et al. (1992). Proc. Natl. Acad. Sci. USA 89, 8822–8826.PubMedCrossRefGoogle Scholar
  21. 21.
    Rouille, Y., Duguay, S. J., Lund, K., et al. (1995). Front. Neuroendocrinol. 16, 332–361.CrossRefGoogle Scholar
  22. 22.
    Brakch, N., Galanopoulou, A. S., Patel, Y. C., Boileau, G., and Seidah, N. G. (1995). FEBS Lett. 362, 143–146.PubMedCrossRefGoogle Scholar
  23. 23.
    Benjannet, S., Rondeau, N., Day, R., Chretien, M., and Seidah, N. G. (1991). Proc. Natl. Acad. Sci. USA 88, 3564–3568.PubMedCrossRefGoogle Scholar
  24. 24.
    Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. P., Steiner, D. F., and Thomas, G. (1991). Proc. Natl. Acad. Sci. USA 88, 5297–5301.PubMedCrossRefGoogle Scholar
  25. 25.
    Benjannet, S., Reudelhuber, T., Rondeau, N., Chretien, M., and Seidah, N. G. (1992). J. Biol. Chem. 267, 11417–11423.PubMedGoogle Scholar
  26. 26.
    Breslin, M. B., Lindberg, I., Benjannet, S., Mathis, J. P., Lazure, C., and Seidah, N. G. (1993). J. Biol. Chem. 268, 27084–27093.PubMedGoogle Scholar
  27. 27.
    Hoflehner, J., Eder, U., Laslop, A., Seidah, N. G., Fischer, R., and Winkler, H. (1995). FEBS Lett. 360, 294–298.PubMedCrossRefGoogle Scholar
  28. 28.
    Perez de la Cruz, I. and Nillni, E. A. (1996). J. Biol. Chem. 271, 22736–22745.CrossRefGoogle Scholar
  29. 29.
    Nillni, E. A., Luo, L. G., Jackson, I. M. D., and McMillan, P. (1996). Endocrinol. 137, 5651–5661.CrossRefGoogle Scholar
  30. 30.
    Mayo, K. E., Vale, W., Rivier, J., Rosenfeld, M. G., and Evans, R. M. (1983). Nature 306, 86–88.PubMedCrossRefGoogle Scholar
  31. 31.
    Mayo, K. E., Cerelli, G. M., Rosenfeld, M. G., and Evans, R. M. (1985). Nature 314, 464–467.PubMedCrossRefGoogle Scholar
  32. 32.
    Benjannet, S., Rondeau, N., Paquet, L., et al. (1993). Biochem. J. 294, 735–743.PubMedGoogle Scholar
  33. 33.
    Anderson, E. D., Molloy, S. S., Jean, F., Fei, H., Shimamura, S., and Thomas, G. (2002). J. Biol. Chem. 277, 12879–12890.PubMedCrossRefGoogle Scholar
  34. 34.
    Muller, L., Zhu, X., and Lindberg, I. (1997). J. Cell Biol. 139, 625–638.PubMedCrossRefGoogle Scholar
  35. 35.
    Muller, L. and Lindberg, I. (1999). Prog. Nucleic Acid Res. Mol. Biol. 63, 69–108.PubMedCrossRefGoogle Scholar
  36. 36.
    Zhu, X., Zhou, A., Dey, A., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 10293–10298.PubMedCrossRefGoogle Scholar
  37. 37.
    Elagoz, A., Benjannet, S., Mammarbassi, A., Wickham, L., and Seidah, N. G. (2002). J. Biol. Chem. 277, 11265–11275.PubMedCrossRefGoogle Scholar
  38. 38.
    Benjannet, S., Elagoz, A., Wickham, L., et al. (2001). J. Biol. Chem. 276, 10879–10887.PubMedCrossRefGoogle Scholar
  39. 39.
    Nillni, E. L., Xie, W., Mulcahy, L., Sanchez, V. C., and Wetsel, W. C. (2002). J. Biol. Chem. 277, 48587–48595.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Samuel F. Posner
    • 1
  • Charles A. Vaslet
    • 1
  • Michelle Jurofcik
    • 1
  • Alisson Lee
    • 1
  • Nabil G. Seidah
    • 2
  • Eduardo A. Nillni
    • 1
    Email author
  1. 1.Division of Endocrinology, Department of MedicineBrown Medical School, Rhode Island HospitalProvidence
  2. 2.Laboratory of Biochemical NeuroendocrinologyClinical Research Institute of MontrealMontrealCanada

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