Biochemistry (Moscow)

, Volume 74, Issue 1, pp 29–35 | Cite as

Isolated compared to membrane-bound receptors exhibit altered insulin/IGF interaction

  • O. NedićEmail author
  • R. Masnikosa


Insulin and insulin-like growth factors (IGFs) bind to their cognate receptors with high affinities, but due to their homology they may cross-react with each other’s receptors. We performed a series of binding studies to reanalyze the cross-reactivity of insulin, IGF-I, and IGF-II to affinity-purified insulin (IR) and type 2 IGF receptors (IGF-2R) from human placental membranes. IR and IGF-2R were purified using insulin- and mannose-6-phosphate affinity chromatography (I-AC and M6P-AC). Binding studies were performed with 125I-labeled and unlabeled ligands. According to immunoblotting, the only receptor species isolated by I-AC was IR, whereas the only receptor isolated by M6P-AC was IGF-2R. Isolated IR reacted to similar extent with 125I-labeled insulin and 125I-labeled IGF-II and significantly less with 125I-labeled IGF-I, implicating predominance of IR-A. The affinity of IR towards heterologous ligands increased after its separation from other membrane proteins. Affinity-purified IGF-2R was almost unable to bind ligands under experimental conditions used in this work, but when incubated with 125I-labeled ligands prior to affinity chromatography, IGF-2R interacted not only with IGF-II, but to a certain extent with the other two ligands. In the competitive M6P-AC, the binding of labeled ligands was inhibited with either homologous or heterologous ligands, in a dose dependent manner. In competitive ligand-blotting, specific interactions between 125I-labeled insulin and IR, and 125I-labeled IGF-II and IGF-2R were also inhibited with all unlabeled ligands, although to a different extent. The results presented in this work imply that isolation of IR an IGF-2R from their membrane milieu increases their reactivity towards all members of the insulin/IGF ligand family.

Key words

insulin insulin-like growth factors receptors affinity chromatography ligand-binding studies 



cation-independent M6P receptor


dimethyl sulfoxide


disuccinimidyl suberate


enhanced chemiluminescence


horseradish peroxidase


hybrid receptor


insulin-affinity chromatography


insulin-like growth factor

IGF-1R and -2R

type 1 and 2 IGF receptors, respectively


IGF-binding protein


insulin receptor


ligand-binding assay




M6P-affinity chromatography


polyethylene glycol


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Juul, A. (2003) Growth Horm. IGF Res., 13, 113–170.PubMedCrossRefGoogle Scholar
  2. 2.
    Dominici, F. P., Argentino, D. P., Munoz, M. C., Miquet, J. G., Sotelo, A. I., and Turyn, D. (2005) Growth Horm. IGF Res., 15, 324–336.PubMedCrossRefGoogle Scholar
  3. 3.
    Werner, H. (1999) in The IGF System: Molecular Biology, Physiology and Clinical Applications (Rosenfeld, R. G., and Roberts, C. T., Jr., eds.) Humana Press, Totowa, pp. 63–88.Google Scholar
  4. 4.
    Harper, J., Burns, J. L., Foulstone, E. J., Pignatelli, M., Zaina, S., and Hassan, A. B. (2006) Cancer Res., 66, 1940–1948.PubMedCrossRefGoogle Scholar
  5. 5.
    Pandini, G., Frasca, F., Mineo, R., Sciacca, L., Vigneri, R., and Belfiore, A. (2002) J. Biol. Chem., 277, 39684–39695.PubMedCrossRefGoogle Scholar
  6. 6.
    Denley, A., Cosgrove, L. J., Booker, G. W., Wallace, J. C., and Forbes, B. E. (2005) Cytokine Growth Factor Rev., 16, 421–439.PubMedCrossRefGoogle Scholar
  7. 7.
    Firth, S. M., and Baxter, R. C. (2002) Endocr. Rev., 23, 824–854.PubMedCrossRefGoogle Scholar
  8. 8.
    Masnikosa, R., Baričević, I., Jones, D. R., and Nedić, O. (2006) Growth Horm. IGF Res., 16, 174–184.PubMedCrossRefGoogle Scholar
  9. 9.
    Bradford, M. M. (1976) Anal. Biochem., 72, 248–254.PubMedCrossRefGoogle Scholar
  10. 10.
    Hunter, W. M., and Greenwood, F. C. (1962) Nature, 194, 495–496.PubMedCrossRefGoogle Scholar
  11. 11.
    Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K. (1983) J. Biol. Chem., 258, 5045–5049.PubMedGoogle Scholar
  12. 12.
    Kiess, W., Blickenstaff, G. D., Sklar, M. M., Thomas, C. L., Nissley, S. P., and Sahagian, G. G. (1988) J. Biol. Chem., 263, 9339–9344.PubMedGoogle Scholar
  13. 13.
    Laemmli, U. K. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  14. 14.
    Baričević, I., Jones, D. R., Nikolić, J. A., and Nedić, O. (2006) Horm. Metab. Res., 38, 22–27.PubMedCrossRefGoogle Scholar
  15. 15.
    Kasuga, M., van Obberghen, E., Nissley, S. P., and Rechler, M. M. (1981) J. Biol. Chem., 256, 5305–5308.PubMedGoogle Scholar
  16. 16.
    Masnikosa, R., Nikolić, J. A., and Nedić, O. (2008) J. Biochem., 143, 813–820.PubMedCrossRefGoogle Scholar
  17. 17.
    Rebourcet, R., de Ceuninck, F., Deborde, S., Willeput, J., and Ferre, F. (1998) Biol. Reprod., 58, 37–44.PubMedCrossRefGoogle Scholar
  18. 18.
    Chisalita, S. I., Dekker Nitert, M., and Arnqvist, H. J. (2006) Growth Horm. IGF Res., 16, 258–266.PubMedCrossRefGoogle Scholar
  19. 19.
    Frasca, F., Pandini, G., Scalia, P., Sciacca, L., Mineo, R., Costantino, A., Goldfine, I. D., Belfiore, A., and Vigneri, R. (1999) Mol. Cell. Biol., 19, 3278–3288.PubMedGoogle Scholar
  20. 20.
    McKern, N. M., Lawrence, M. C., Streltsov, V. A., Lou, M. Z., Adams, T. E., Lovrecz, G. O., Elleman, T. C., Richards, K. M., Bentley, J. D., Pilling, P. A., Hoyne, P. A., Cartledge, K. A., Pham, T. M., Lewis, J. L., Sankovich, S. E., Stoichevska, V., da Silva, E., Robinson, C. P., Frenkel, M. J., Sparrow, L. G., Fernley, R. T., Epa, V. C., and Ward, C. V. (2006) Nature, 443, 218–221.PubMedCrossRefGoogle Scholar
  21. 21.
    Louvi, A., Accili, D., and Efstratiadis, A. (1997) Dev. Biol., 189, 33–48.PubMedCrossRefGoogle Scholar
  22. 22.
    Slaaby, R., Schaffer, L., Lautrup-Larsen, I., Andersen, A. S., Shaw, A. C., Stenfeldt Mathiasen, I., and Brandt, J. (2006) J. Biol. Chem., 281, 25869–25874.PubMedCrossRefGoogle Scholar
  23. 23.
    Hassan, A. B. (2003) Am. J. Pathol., 162, 3–6.PubMedGoogle Scholar
  24. 24.
    Braulke, T. (1999) Horm. Metab. Res., 31, 242–246.PubMedCrossRefGoogle Scholar
  25. 25.
    Linnell, J., Groeger, G., and Hassan, A. B. (2001) J. Biol. Chem., 276, 23986–23991.PubMedCrossRefGoogle Scholar
  26. 26.
    Byrd, J. C., Park, J. H. Y., Schaffer, B. S., Garmroudi, F., and MacDonald, R. G. (2000) J. Biol. Chem., 275, 18647–18656.PubMedCrossRefGoogle Scholar
  27. 27.
    Masnikosa, R., Nikolić, J. A., and Nedić, O. (2003) J. Serb. Chem. Soc., 68, 811–818.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  1. 1.Institute for the Application of Nuclear Energy (INEP)University of BelgradeBelgradeSerbia

Personalised recommendations