Ligand-binding characteristics of recombinant amino- and carboxyl-terminalfragments of human insulin-like growth factor-binding protein-3

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a member of a fam ily of structurally conserved proteins (IGFBP-1 to -f,) which act as carrie rs and regulators of the mitogenic peptide hormones IGF-I and IGF-II. Membe rs of the IGFBP family share conserved cysteine-rich amino-and carboxyl-ter minal regions. The amino-terminal domain of these proteins is recognised to contain an IGF-binding. determinant, but evidence to support a binding sit e ill the carboxyl-terminal region of the protein is less rigorous. To furt her investigate this,we have synthesised both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of huma n IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG pep tide. Although only C-165 showed binding to IGF-I and -II by solution-bindi ng assays, both N-88 and C-165 demonstrated binding to ICF-I and -II by bio sensor analysis albeit with reduced affinities compared with full-length IC FBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero- trimeric complexes with IGF-I and the acid-labile subunit (ALS). We conclud e that the carbosyl-terminal domain of IGEBP-3 contains an IGF-binding dete rminant and can form ternary complexes with ALS.