Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II.
Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.
Francis
G L
GL
Cooperative Research Centre for Tissue Growth and Repair, Adelaide, South Australia.
Aplin
S E
SE
Milner
S J
SJ
McNeil
K A
KA
Ballard
F J
FJ
Wallace
J C
JC
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005971:Glutamates; D018969:Insulin-Like Growth Factor Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D007335:Insulin-Like Growth Factor II; D001120:Arginine