A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.
A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011384:Proinsulin / Q000235:genetics*; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D002352:Carrier Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D009838:Oligodeoxyribonucleotides; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D011384:Proinsulin; D017526:Receptor, IGF Type 1