Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action.
We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201-218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (K(D)) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity.
Allan
Gordon J
GJ
Hannah Research Institute, Ayr KA6 5HL, Scotland, United Kingdom. allang@hri.sari.ac.uk
Tonner
Elizabeth
E
Szymanowska
Malgorzata
M
Shand
John H
JH
Kelly
Sharon M
SM
Phillips
Kirsten
K
Clegg
Roger A
RA
Gow
Iain F
IF
Beattie
James
J
Flint
David J
DJ
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002942:Circular Dichroism; D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I