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*

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly203 and Gln209, are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (1). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding.

[1]  R. Baxter,et al.  Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3. , 1999, The Journal of endocrinology.

[2]  D. Clemmons.  IGF binding proteins and their functions , 1993, Molecular reproduction and development.

[3]  Jen-Tsi Yang,et al.  β-II conformation of all-β proteins can be distinguished from unordered form by circular dichroism , 1992 .

[4]  J. Wallace,et al.  Alanine Screening Mutagenesis Establishes Tyrosine 60 of Bovine Insulin-like Growth Factor Binding Protein-2 as a Determinant of Insulin-like Growth Factor Binding* , 1998, The Journal of Biological Chemistry.

[5]  D. Clemmons,et al.  Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4 , 1995, The Journal of Biological Chemistry.

[6]  C A Ghiron,et al.  Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. , 1976, Biochemistry.

[7]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[8]  J. Shand,et al.  Growth hormone, acting in part through the insulin-like growth factor axis, rescues developmental, but not metabolic, activity in the mammary gland of mice expressing a single allele of the prolactin receptor. , 2002, Endocrinology.

[9]  J. Wallace,et al.  Localization of an Insulin-like Growth Factor (IGF) Binding Site of Bovine IGF Binding Protein-2 Using Disulfide Mapping and Deletion Mutation Analysis of the C-terminal Domain* , 1998, The Journal of Biological Chemistry.

[10]  W. C. Johnson,et al.  Sensitivity of circular dichroism to protein tertiary structure class , 1983, Nature.

[11]  M. Binoux,et al.  Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. , 1986, Analytical biochemistry.

[12]  C. Conover,et al.  Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. , 1989, The Journal of clinical investigation.

[13]  J. Wallace,et al.  The Insulin-like Growth Factor (IGF) Binding Site of Bovine Insulin-like Growth Factor Binding Protein-2 (bIGFBP-2) Probed by Iodination* , 1996, The Journal of Biological Chemistry.

[14]  D. Clemmons,et al.  Heparin, heparan sulfate, and dermatan sulfate regulate formation of the insulin-like growth factor-I and insulin-like growth factor-binding protein complexes. , 1994, The Journal of biological chemistry.

[15]  D. Clemmons,et al.  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* , 2000, The Journal of Biological Chemistry.

[16]  S. Provencher,et al.  Estimation of globular protein secondary structure from circular dichroism. , 1981, Biochemistry.

[17]  J. Wallace,et al.  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* , 2001, The Journal of Biological Chemistry.

[18]  M. Binoux,et al.  Molecular forms of human IGF binding proteins: physiological implications. , 1991, Acta endocrinologica.

[19]  D. Clemmons,et al.  Identification of the Extracellular Matrix Binding Sites for Insulin-like Growth Factor-binding Protein 5* , 1996, The Journal of Biological Chemistry.

[20]  J. Beattie,et al.  Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5. , 2000, Journal of molecular endocrinology.

[21]  D. Clemmons,et al.  Substitution of Specific Amino Acids in Insulin-like Growth Factor (IGF) Binding Protein 5 Alters Heparin Binding and Its Change in Affinity for IGF-I in Response to Heparin (*) , 1996, The Journal of Biological Chemistry.

[22]  C. Renner,et al.  Structure of the IGF‐binding domain of the insulin‐like growth factor‐binding protein‐5 (IGFBP‐5): implications for IGF and IGF‐I receptor interactions , 1998, The EMBO journal.

[23]  D. Clemmons,et al.  Binding of insulin-like growth factor (IGF)-binding protein-5 to smooth-muscle cell extracellular matrix is a major determinant of the cellular response to IGF-I. , 1998, Molecular biology of the cell.

[24]  D. Andress,et al.  Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells. , 1993, Biochemical and biophysical research communications.

[25]  A. Hoeflich,et al.  IGF-binding protein-5: flexible player in the IGF system and effector on its own. , 2002, The Journal of endocrinology.

[26]  R. Baxter,et al.  Ligand-binding characteristics of recombinant amino- and carboxyl-terminal fragments of human insulin-like growth factor-binding protein-3. , 2001, The Journal of endocrinology.

[27]  J. Shand,et al.  The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain. , 2001, Journal of molecular endocrinology.

[28]  J. Beattie,et al.  Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I. , 1999, Journal of molecular endocrinology.

[29]  R. Koistinen,et al.  Biologically active domain in somatomedin-binding protein. , 1986, Biochemical and biophysical research communications.

[30]  A. Sommer,et al.  Properties of glycosylated and non-glycosylated human recombinant IGF binding protein-3 (IGFBP-3). , 1993, Growth regulation.

[31]  M. Rechler,et al.  Isolation of a biologically active fragment from the carboxy terminus of the fetal rat binding protein for insulin-like growth factors. , 1988, Biochemical and biophysical research communications.