The Insulin-like Growth Factor (IGF) Binding Site of Bovine Insulin-like Growth Factor Binding Protein-2 (bIGFBP-2) Probed by Iodination

Abstract

The insulin-like growth factors (IGF-I and IGF-II) 1 are polypeptide mitogens which play diverse roles in development and metabolism across a wide range of vertebrate species (recently reviewed in Ref.

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 either amino-or carboxylterminal residues from IGFBP-1 severely disrupted IGF binding (20,21), whereas deletion of amino acid residues 222-284 of bIGFBP-2 significantly altered the IGF binding capabilities of bIGFBP-2 (22). However, chemical iodination and sitedirected mutagenesis of bIGFBP-2 by Hobba et al (23,24) showed the amino-terminal residue Tyr-60 contributed to IGF binding. NMR analysis and site-directed mutagenesis confirmed the involvement of the equivalent residue of hIGFBP-5 (10,25), whereas recombinant amino-terminal fragments of rIGFBP-3 (26) and hIGFBP-4 (27) have also been shown to retain residual binding affinity.…”

“…Briefly, nonspecific interacting proteins were removed with 60 mM imidazole, and specifically bound protein was eluted with 1 M imidazole. Wild-type and truncated bIGFBP-2 preparations were further purified by rpHPLC as described by Hobba et al (23). The eluted protein was detected by absorbance at 215 nm.…”

“…The amino-terminal fusion protein was cleaved from the E. coli expressed proteins by enterokinase (0.5 units/mg of protein, Invitrogen EkMax), and the products were separated by rpHPLC. Purified protein samples were analyzed by separation on 12.5% polyacrylamide gels under nonreducing conditions and stained with Coomassie Blue R250 as previously described by Hobba et al (23). All protein samples were lyophilized for storage.…”

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

“…At the N-terminus of bovine IGFBP-2, it was shown that Tyr60 was protected against iodination upon binding of IGFBP-2 to IGF-II (Hobba et al 1996). Subsequently, the same group demonstrated that substitution of tyrosine-60 with alanine or phenyalanine leads to a 4.0-and 8.4-fold reduced affinity for IGF-I respectively, and a 3.5-and 4.0-fold reduced affinity for IGF-II respectively (Hobba et al1998).…”

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

“…Initially it was observed that Tyr 60 was protected against iodination upon binding of bovine IGFBP-2 to IGF-II (14), and subsequently, the same group demonstrated that substitution of this amino acid leads to reduced affinities for IGF-I and -II (15). In addition, NMR was carried out on a bacterially expressed N-terminal fragment of IGFBP-5 (residues 40 -92), and this clearly demonstrated that a major IGF-binding domain comprises Val 49 , Tyr 50 (equivalent to Tyr 60 in IGFBP-2), Pro 62 , and Lys 68 -Leu 75 , where the conserved Leu and Val residues localize in a hydrophobic patch on the surface of the IGFBP-5 protein (7).…”