Biosensor Measurement of the Binding of Insulin-like Growth Factors I and II and Their Analogues to the Insulin-like Growth Factor-binding Protein-3

Abstract

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcore™ instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1–3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28–37 by Gly-Gly-Gly-Gly or deletion of residues 29–41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I. Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcore™ instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1–3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28–37 by Gly-Gly-Gly-Gly or deletion of residues 29–41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I. INTRODUCTIONInsulin-like growth factor-I and -II (IGF-I and IGF-II) 1The abbreviations used are: IGFinsulin-like growth factorIGFBPIGF-binding proteinNHSN-hydroxysuccinimideECDN-ethyl-N′-(3-diethylaminopropyl)carbodiimide. are small proteins that stimulate a variety of growth-promoting and metabolic effects via an interaction with the IGF-I receptor (1Lowe W.L. LeRoith D. Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press Inc., Boca Raton, FL1991: 49Google Scholar). The peptides consist of four regions: A- and B-regions, which are homologous to the A and B chains of insulin; a C-region, which is analogous to but unrelated to the C peptide of insulin that connects the A- and B-regions; and a short carboxyl-terminal D-region, with no counterpart in insulin (2LeRoith D. Clemmons D. Nissley P. Rechler M.M. Ann. Intern. Med. 1992; 116: 854-862Google Scholar, 3Sussenbach J.S. Steenbergh P.H. Holthuizen P. Growth Regul. 1992; 2: 1-9Google Scholar).IGF-I and -II form complexes with six different IGF-binding proteins (IGFBPs) in the circulation and in the extracellular environment (4Martin J.L. Baxter R.C. Curr. Opin. Endocrinol. Diabetes. 1994; 1: 16-21Google Scholar). In the circulation the majority of IGFs are bound in a 140-kDa complex consisting of IGFBP-3 of 40–50 kDa, an approximately 85-kDa so-called acid-labile subunit, and IGF-I or IGF-II (5Baxter R.C. Martin J.L. Be