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.
In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.
Carrick
F E
FE
Cooperative Research Centre for Tissue Growth and Repair, Department of Molecular Biosciences, Adelaide University, North Terrace, Adelaide, South Australia 5005, Australia. francine.carrick@adelaide.edu.au
Forbes
B E
BE
Wallace
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D019556:COS Cells; D002417:Cattle; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism*
D017931:DNA Primers; D018971:Insulin-Like Growth Factor Binding Protein 2; D011994:Recombinant Proteins; D013002:Somatomedins