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

Abstract

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.

Insulin-like Growth Factor-binding Protein-5 Activates Plasminogen by Interaction with Tissue Plasminogen Activator, Independently of Its Ability to Bind to Plasminogen Activator Inhibitor-1, Insulin-like Growth Factor-I, or Heparin

“…5f). Details of the properties of these mutants have been reported previously (22,23). Similar observations were made for the C-term E and C-term F, two additional non-heparin-binding mutants (results not shown).…”

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

“…There has been a considerable body of work to delineate the determinants of IGFBPs binding to IGFs and vice versa (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(21)(22)(23)(24)(25)(26)(27)(28)(29). The structural information presented in this work is broadly in agreement with these data, but disagrees with reports of a critical role of the completely conserved Gly-187(C) and Gln-193(C) (in the IGFBP4 sequence) for binding of C domains to IGFs (2, 6, 29, and references cited therein): These residues are not in contact with IGF1, although they are close to the IGF1͞CBP4 interface surface.…”

“…Each IGFBP can be divided into three distinct domains of approximately equal lengths: highly conserved cysteinerich N and C domains and a central linker domain unique to each IGFBP species. Both the N and C domains participate in the binding to IGFs, although the specific roles of each of these domains in IGF binding have not been decisively determined (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The C-terminal domain may be responsible for preferences of IGFBPs for one species of IGF over the other (2,(3)(4)(5)(6)(7)(9)(10)(11)(12)(13); the C-terminal domain is also involved in regulation of the IGF-binding affinity through interaction with extracellular matrix components (1,2,14) and is most probably engaged in mediating IGF1-independent actions (1,4,14).…”

IGFBP-5 enhances epithelial cell adhesion and protects epithelial cells from TGFβ1-induced mesenchymal invasion

“…Our studies were almost exclusively done in the absence of exogenous IGF-I, although we cannot rule out endogenous production of IGFs by the cells. However, our studies in MCF-7 cells included the use of a mutant form of IGFBP-5 which could not bind to IGFs (Allan et al , 2006) and this was fully active in inducing adhesion and inhibiting migration (Sureshbabu, Okajima, 2012) suggesting that the actions of IGFBP-5 are indeed IGF-independent.…”