, Volume 3, Issue 3, pp 221–226 | Cite as

Lactogen enhances Nb2 cell GTPase activity after 4 hours incubation

  • Jennifer L Larsen
  • Tab W. Burkman


The lactogen receptor has been suggested to associate with one or more G proteins despite the absence of a 7-transmembrane spanning sequence. These studies were designed to determine whether lactogens acutely increase GTP binding to or GTPase activity in Nb2 cell membrane. Incubation of Nb2 cell membrane with either ovine PRL (10 ng/ml) or diluent for 0–1 h resulted in a decrease in total35S-GTP binding to both with no difference in GTP binding between PRL- and diluent-treated membranes. There was also no change in35S-GTP binding to Nb2 cell membrane incubated with increasing oPRL concentrations (0.001–100 ng/ml) for 60 min. α-32P-GTP photoaffinity labelling was used to evaluate changes in GTP binding to specific G proteins. Photoaffinity labelling of α-32P-GTP to no G protein was changed after preincubation with oPRL (10 ng/ml) for 0–60 min or with oPRL (0.01–10 ng/ml) for 60 min. Finally, it was determined whether oPRL had any acute effect on GTPase activity, as determined by release of32Pi from γ-32P-GTP. When Nb2 cell membrane was preincubated for 0–60 min with oPRL (10 ng/ml) or a range of oPRL concentrations (0–10 ng/ml), no change in GTPase activity was observed. However, when Nb2 cells were incubated with lactogen for 0–7 h, GTPase activity in equal quantities of Nb2 cell membrane prepared from those cells increased over time. Increased GTPase activity (64.9–74.4%;P<0.03 compared to 0 h) was observed after 4–7 h incubation with lactogen.

In summary, addition of lactogen to Nb2 cell membrane did not acutely increase either GTP binding or GTPase activity. Yet when Nb2 cells were incubated with lactogen for 4 h prior to preparation of membrane, GTPase activity was significantly increased. This evidence, in addition to our previous results showing that 4 h incubation with lactogen increased G protein β subunit concentration and pertussis toxin-stimulated ADP-ribosylation of Gi, support a role for delayed lactogen modulation of one or more G proteins in the Nb2 cell, requiring at least 4 h for maximal effect.


prolactin human growth hormone Nb2 cell, GTP binding protein beta subunit GTPase 


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  1. Baldini, G., Hohman, R., Charron, M.J. & Lodish, H.F. (1991).J. Biol. Chem.,266, 4037–4040.PubMedGoogle Scholar
  2. Barkey, R., Calvo, J. & Dufau, M. (1988).Biochem. Biophys. Res. Comm.,156, 776–782.PubMedCrossRefGoogle Scholar
  3. Birnbaumer, L., Abramowitz, J. & Brown, A. (1990).Biochim. Biophys. Acta.,1031, 163–224.PubMedGoogle Scholar
  4. Bokoch, G.M. (1987).J. Biol. Chem.,262, 589–594.PubMedGoogle Scholar
  5. Boutin, J.-M., Jolicoeur, C, Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J. & Kelly, PA. (1988).Cell,53, 69–77.PubMedCrossRefGoogle Scholar
  6. Burdett, E., Mills, G.B. & Klip, A. (1990).Am. J. Physiol.,258, (Cell physiol.,27): C99-C108.PubMedGoogle Scholar
  7. Cassel, D. & Selinger, Z. (1976).Biochim. Biophys. Acta.,452, 538–551.PubMedGoogle Scholar
  8. Church, J.G. & Buick, R.N. (1988).J. Biol. Chem.,263, 4242–4246.PubMedGoogle Scholar
  9. Edery, M., Jolicoeur, C, Levi-Meyrueis, C, Dusanter-Fourt, I., Petridou, B., Boutin, J.-M., Lesueur, L., Kelly, P.A. & Djiane, J. (1989).Proc. Natl. Acad. Sci. USA,86, 2112–2116.PubMedCrossRefGoogle Scholar
  10. Fleming, W., Murphy, P., Murphy, L., Hatton, T., Matusik, R. & Friesen, H. (1985).Endocrinology,117, 2547–2549.PubMedGoogle Scholar
  11. Freissmuth, M., Casey, P.J. & Gilman, A.G. (1989).FASEB J.,3, 2125–2131.6.PubMedGoogle Scholar
  12. Gilman, A., (1987).Ann. Rev. Biochem.,56, 615–649.PubMedCrossRefGoogle Scholar
  13. Goldsmith, P., Rossiter, K., Carter, A., Simonds, W., Unson, C, Vinitsky, R. & Spiegel, A. (1988).J. Biol. Chem.,263, 6476–6479.PubMedGoogle Scholar
  14. Im, M.-J. & Graham, R. (1990).J. Biol. Chem.,265, 18944–18951.PubMedGoogle Scholar
  15. Kim, D., Lewis, D.L., Graziadei, L., Neer, E.J., Bar-Sagi, D. & Clapham, D.E. (1989).Nature,337, 557–562.PubMedCrossRefGoogle Scholar
  16. Larsen, J. & Dufau, M. (1988).Endocrinology,123, 438–444.PubMedGoogle Scholar
  17. Larsen, J.L. (1992).J. Biol. Chem.,267, 10583–10587.PubMedGoogle Scholar
  18. Larsen, J. & Burkman, T. (1994).Endocrine,2, 1011–1016.Google Scholar
  19. Liang, M. & Garrison, J.C. (1991).J. Biol. Chem.,266, 13342–13349.PubMedGoogle Scholar
  20. Lotersztajn, S., Pavoine, C, Deterre, P.K.C., Mallat, A., LeNguyen, D., Dufour, M., Rouot, B., Bataille, D. & Pecker, F. (1992).J. Biol. Chem.,267, 2375–2379.PubMedGoogle Scholar
  21. Lowry, O., Rosebrough, N., Farr, A. & Randall, R. (1951).J. Biol. Chem.,193, 265–275.PubMedGoogle Scholar
  22. Kelly, P.A., Djiane, J. & Edery, M. (1992).Trends Endocrinol Metab.,3, 54–59.PubMedCrossRefGoogle Scholar
  23. Moises, R.S. & Heidenreich, K.A. (1990).J. Cell. Physiol.,144, 538–545.PubMedCrossRefGoogle Scholar
  24. Moriarty, T.M., Gillo, B., Carty, D.J., Premont, R.T., Landau, E.M. & Iyengar, R. (1988).Proc. Natl. Acad. Sci.,85, 8965–8869.CrossRefGoogle Scholar
  25. Neer, E.J., Lok, J.M. & Wolf, L.G. (1984).J. Biol. Chem.,259, 14222–14229.PubMedGoogle Scholar
  26. Nishimoto, I., Murayama, Y., Katada, T., Ui, M. & Ogata, E. (1989).J. Biol. Chem.,264, 14029–14038.PubMedGoogle Scholar
  27. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E. & Nishimoto, I. (1990).Cell,62, 709–717.PubMedCrossRefGoogle Scholar
  28. Srivastava, S.K. & Singh, U.S. (1990).Biochem. Biophys. Res. Comm.,173, 501–506.PubMedCrossRefGoogle Scholar
  29. Tang, W.-J. & Gilman, A.G. (1991).Science,254, 1500–1504.PubMedCrossRefGoogle Scholar
  30. Too, C, Murphy, P. & Friesen, H. (1989).Endocrinology,124, 2185–2192.PubMedCrossRefGoogle Scholar
  31. Too, C, Shiu, R. & Friesen, H. (1990).Biochem. Biophys. Res. Comm.,173, 48–52.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Jennifer L Larsen
    • 1
  • Tab W. Burkman
    • 1
  1. 1.Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaUSA

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