The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.

This article has been cited by other articles:

				X. Yan, R. C. Baxter, B. Perbal, and S. M. Firth The Aminoterminal Insulin-Like Growth Factor (IGF) Binding Domain of IGF Binding Protein-3 Cannot Be Functionally Substituted by the Structurally Homologous Domain of CCN3 Endocrinology, November 1, 2006; 147(11): 5268 - 5274. [Abstract] [Full Text] [PDF]

				A. M. Sorrell, J. H. Shand, E. Tonner, M. Gamberoni, P. A. Accorsi, J. Beattie, G. J. Allan, and D. J. Flint Insulin-like Growth Factor-binding Protein-5 Activates Plasminogen by Interaction with Tissue Plasminogen Activator, Independently of Its Ability to Bind to Plasminogen Activator Inhibitor-1, Insulin-like Growth Factor-I, or Heparin J. Biol. Chem., April 21, 2006; 281(16): 10883 - 10889. [Abstract] [Full Text] [PDF]

				B. Nicholas, R. Alberio, A.A. Fouladi-Nashta, and R. Webb Relationship Between Low-Molecular-Weight Insulin-Like Growth Factor-Binding Proteins, Caspase-3 Activity, and Oocyte Quality Biol Reprod, April 1, 2005; 72(4): 796 - 804. [Abstract] [Full Text] [PDF]

				X. Yan, B. E. Forbes, K. A. McNeil, R. C. Baxter, and S. M. Firth Role of N- and C-terminal Residues of Insulin-like Growth Factor (IGF)-binding Protein-3 in Regulating IGF Complex Formation and Receptor Activation J. Biol. Chem., December 17, 2004; 279(51): 53232 - 53240. [Abstract] [Full Text] [PDF]

				S. J. Headey, D. W. Keizer, S. Yao, G. Brasier, P. Kantharidis, L. A. Bach, and R. S. Norton C-Terminal Domain of Insulin-Like Growth Factor (IGF) Binding Protein-6: Structure and Interaction with IGF-II Mol. Endocrinol., November 1, 2004; 18(11): 2740 - 2750. [Abstract] [Full Text] [PDF]

				S. J Headey, K. S Leeding, R. S Norton, and L. A Bach Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding J. Mol. Endocrinol., October 1, 2004; 33(2): 377 - 386. [Abstract] [Full Text] [PDF]

				Q. Xu, B. Yan, S. Li, and C. Duan Fibronectin Binds Insulin-like Growth Factor-binding Protein 5 and Abolishes Its Ligand-dependent Action on Cell Migration J. Biol. Chem., February 6, 2004; 279(6): 4269 - 4277. [Abstract] [Full Text] [PDF]

				J. H. Shand, J. Beattie, H. Song, K. Phillips, S. M. Kelly, D. J. Flint, and G. J. Allan Specific Amino Acid Substitutions Determine the Differential Contribution of the N- and C-terminal Domains of Insulin-like Growth Factor (IGF)-binding Protein-5 in Binding IGF-I J. Biol. Chem., May 9, 2003; 278(20): 17859 - 17866. [Abstract] [Full Text] [PDF]

				S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [Abstract] [Full Text] [PDF]

				D. R. Clemmons Use of Mutagenesis to Probe IGF-Binding Protein Structure/Function Relationships Endocr. Rev., December 1, 2001; 22(6): 800 - 817. [Abstract] [Full Text] [PDF]

				G. R. Devi, D.-H. Yang, R. G. Rosenfeld, and Y. Oh Differential Effects of Insulin-Like Growth Factor (IGF)-Binding Protein-3 and Its Proteolytic Fragments on Ligand Binding, Cell Surface Association, and IGF-I Receptor Signaling Endocrinology, November 1, 2000; 141(11): 4171 - 4179. [Abstract] [Full Text] [PDF]

				R. C. Baxter Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E967 - E976. [Abstract] [Full Text] [PDF]

				V. Hwa, Y. Oh, and R. G. Rosenfeld The Insulin-Like Growth Factor-Binding Protein (IGFBP) Superfamily Endocr. Rev., December 1, 1999; 20(6): 761 - 787. [Abstract] [Full Text]

				Y. Imai, A. Moralez, U. Andag, J. B. Clarke, W. H. Busby Jr., and D. R. Clemmons Substitutions for Hydrophobic Amino Acids in the N-terminal Domains of IGFBP-3 and -5 Markedly Reduce IGF-I Binding and Alter Their Biologic Actions J. Biol. Chem., June 9, 2000; 275(24): 18188 - 18194. [Abstract] [Full Text] [PDF]

				F. E. Carrick, B. E. Forbes, and J. C. Wallace BIAcore Analysis of Bovine Insulin-like Growth Factor (IGF)-binding Protein-2 Identifies Major IGF Binding Site Determinants in Both the Amino- and Carboxyl-terminal Domains J. Biol. Chem., July 13, 2001; 276(29): 27120 - 27128. [Abstract] [Full Text] [PDF]