Structural Determinants of Ligand and Cell Surface Binding of Insulin-like Growth Factor-binding Protein-3

Abstract

The three deletion variants were incapable of binding ALS; but of the site-specific variants, the MDGEA mutant bound ALS with 90% lower affinity (K a ‫؍‬ 2.5 ؎ 0.9 liters/nmol) than seen for rhIGF-BP-3 (K a ‫؍‬ 24.3 ؎ 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity (K a ‫؍‬ 21.7 ؎ 4.5 liters/nmol). The ability of IGFBP-3 to associate with the cell surface was lost in variants lacking residues 185-264 and in the 228 KGRKR 3 MDGEA mutant. We conclude that residues 228 -232 of IGFBP-3 are essential for cell association and are required for normal ALS binding affinity.

Genomewide meta‐analysis identifies loci associated with IGF ‐I and IGFBP ‐3 levels with impact on age‐related traits

“…The IGFALS encodes the insulin‐like growth factor‐binding protein complex, acid labile subunit protein (ALS) which forms a ternary complex with IGF‐I and IGFBP‐3 (Firth et al ., 1998; Twigg & Baxter, 1998). Like in most GWAS, our analyses cannot establish which is the causative SNP or gene of a locus.…”

Nuclear Import of Insulin-like Growth Factor-binding Protein-3 and -5 Is Mediated by the Importin β Subunit

“…(35). However, we have also shown that this IGFBP-3 mutant is unable to bind at the cell surface (38), leading to the speculation that transport to the nucleus may be blocked at the level of the plasma membrane rather than at the level of entry into the nucleus. To address this issue, we compared nuclear uptake of wild-type and mutant IGFBP-3 (in cells were the plasma membrane had been permeabilized) using the fully reconstituted in vitro nuclear transport assay.…”

“…human IGFBP-3 and IGFBP-3[ 228 KGRKR 3 MDGEA] were produced by a replication-deficient adenovirus-mediated expression system, as described previously (37). IGFBP-3 was purified from conditioned media by IGF-I affinity chromatography and reverse-phase high pressure liquid chromatography (38). In studies requiring fluorescently labeled IGFBP-3, the protein was conjugated to dichlorotriazinylaminofluorescein I HCl as described previously for Cy3 (35).…”

“…Construction of EGFP Fusion Proteins-A 1080-base pair EcoRIPvuII fragment containing the full coding sequence of human IGFBP-3 was inserted into pSELECT (Promega) to generate pSF106 (38). Sitedirected mutagenesis of pSF106 was carried out with the following oligonucleotides to introduce specific mutations: 5Ј-CCCAACTGTGAC-AAGAACGGATTTTATAAGAAAAAGC was used to generate pSF184 ( 216 K 3 N); 5Ј-GTGACAAGAAGGGATTTTATCACTCCCGCCAGTGT-CGCCCTTCCAAAGG was used to generate pSF170 ( 220 KKK 3 HSR) and 5Ј-TTTTATAAGAAAAAGCAGTGTCGCCCTTCCATGGACGGGG-AGGCGGGCTTCTGCTGGTGTGTGGATAAGTATGGG to generate pSF110 ( 228 KGRKR 3 MDGEA).…”

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

“…Removal of 14, 36, and 48 amino acid residues from the carboxyl terminus appeared to have no effect on IGF binding, whereas removal of a further 14 amino acid residues dramatically reduced IGF binding. Other studies have shown that removal of most or all of the carboxyl-terminal domains of IGFBP-3 (16,41), , and IGFBP-5 (8) reduces IGF binding significantly.…”