Mutations in the C-terminal part of insulin-like growth factor (IGF)-binding protein-1 result in dimer formation and loss of IGF binding capacity.
In an attempt to define domains in insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) that are involved in IGF binding, we subjected the carboxyl end of the coding region of IGFBP-1 cDNA to mutagenesis. Mutant cDNAs were isolated, characterized by sequencing, and cloned in an expression vector under control of the simian virus-40 (SV40) early promoter. The constructs were transfected into COS-1 cells, and the mutant proteins, secreted into the culture medium, were analyzed for IGF binding by ligand blotting. The results obtained show that deletion of the C-terminal 20 amino acids or introduction of frame-shifts in this region resulted in loss of IGF binding and for some mutants in the formation of dimeric IGFBP-1 molecules. These dimers are probably formed when cysteine-226 (Cys-226) is missing, and its putative partner is able to form intermolecular disulfide bonds. Site-directed mutagenesis demonstrated that most of the introduced point mutations in the C-terminal region did not affect IGF binding. Only mutation of Cys-226 to tyrosine completely abolished IGF binding, as did the introduction of a negatively charged amino acid in the vicinity of this residue. Again, dimers were observed, supporting that Cys-226 is essential for the conformation of IGFBP-1. In addition, our data suggest that an IGF-binding domain may be located in the vicinity of the intramolecular disulfide bond formed by Cys-226 and its putative partner.
Brinkman
A
A
Department of Pediatrics, Erasmus University/Sophia Childrens Hospital, Rotterdam, The Netherlands.
Kortleve
D J
DJ
Zwarthoff
E C
EC
Drop
S L
SL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D046911:Macromolecular Substances; D007334:Insulin-Like Growth Factor I; D004247:DNA