Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

Insulin-like growth factor (IGF)-binding protein-5-(201—218) region regulates hydroxyapatite and IGF-I binding

1997 ◽  
Vol 273 (5) ◽  
pp. E1005-E1013 ◽  
Author(s):  
Phil G. Campbell ◽  
Dennis L. Andress
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Fluid Phase ◽  
Binding Domain ◽  
Free Fluid ◽  
Insulin Like Growth Factor ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Insulin-like growth factor-binding protein-5 (IGFBP-5), the major bone IGFBP, modifies the biological activity of IGFs within the osteoblastic pericellular environment. Because glycosaminoglycans modulate IGFBP-5 binding to osteoblast organic extracellular matrix (ECM), we assessed whether the heparin binding domain of IGFBP-5, IGFBP-5-(102—218), modifies the interaction of IGFBP-5 with the inorganic bone ECM hydroxyapatite (HA). Synthetic IGFBP-5-(201—218) peptide increased the binding of IGFBP-5 to HA as well as the binding of IGF-I to HA-bound IGFBP-5. This action was specific for the heparin-binding domain, because IGFBP-5-(130—138), IGFBP-5-(138—152), and IGFBP-5-(1—169) were without effect. IGFBP-5-(201—218) was found to bind directly to IGFBP-5 and cause a threefold enhancement of the IGF-I binding affinity for IGFBP-5, whether IGFBP-5 was bound to HA or was in a matrix-free fluid phase. Heparin inhibited the binding of IGFBP-5 to HA and blocked the interaction of IGFBP-5 with IGFBP-5-(201—218) in the fluid phase, suggesting that the primary heparin-binding domain of IGFBP-5 specifically enhances the binding of IGFBP-5 to HA and increases IGF-I binding to IGFBP-5.

Plasminogen binds the heparin-binding domain of insulin-like growth factor-binding protein-3

1998 ◽  
Vol 275 (2) ◽  
pp. E321-E331 ◽  
Author(s):  
Phil G. Campbell ◽  
Susan K. Durham ◽  
Adisak Suwanichkul ◽  
James D. Hayes ◽  
David R. Powell
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Binding Domain ◽  
Insulin Like Growth Factor ◽  
Heparin Binding ◽  
Binding Studies ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igfbp 3

Limited proteolysis lowers affinity of insulin-like growth factor (IGF)-binding protein (IGFBP)-3 for bound IGFs, resulting in greater IGF bioavailability. Plasmin is one of many proteases that cleave IGFBP-3, and the plasmin system may regulate IGFBP-3 proteolysis and IGF bioavailability in cultured cells in vitro. A role for the plasmin system in IGFBP-3 proteolysis in vivo is suggested by data presented here showing that IGFBP-3 binds plasminogen (Pg; Glu-Pg) with a dissociation constant ( Kd) ranging from 1.43 to 3.12 nM. IGF-I and Glu-Pg do not compete for IGFBP-3 binding; instead, the binary IGFBP-3/Glu-Pg complex binds IGF-I with high affinity ( Kd= 0.47 nM) to form a ternary complex. Competitive binding studies suggest that the kringle 1, 4, and 5 domains of Glu-Pg and the heparin-binding domain of IGFBP-3 participate in forming the IGFBP-3/Glu-Pg complex, and other studies show that Glu-Pg in this complex is activated at a normal rate by tissue Pg activator. Importantly, IGFBP-3/Glu-Pg complexes were detected in both human citrate plasma and serum, indicating that these complexes exist in vivo. Binding of IGFBP-3 to Glu-Pg in vivo suggests how Glu-Pg activation can specifically lead to IGFBP-3 proteolysis with subsequent release of IGFs to local target tissues.

scholarly journals Molecular Mapping and Functional Characterization of the VEGF164 Heparin-binding Domain

2007 ◽  
Vol 282 (38) ◽  
pp. 28045-28056 ◽  
Author(s):  
Dominik Krilleke ◽  
Andrea DeErkenez ◽  
William Schubert ◽  
Indrajit Giri ◽  
Gregory S. Robinson ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mapping ◽  
Functional Characterization ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Heparin Binding Domain

The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.

scholarly journals Localization of the IGF binding domain and evaluation of the role of cysteine residues in IGF binding in IGF binding protein-4

2001 ◽  
Vol 169 (1) ◽  
pp. 135-143 ◽  
Author(s):  
D Byun ◽  
S Mohan ◽  
DJ Baylink ◽  
X Qin
Keyword(s):  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Mutation ◽  
Structural Determinants ◽  
Fold Reduction ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.

Heparin binding domain of insulin-like growth factor binding protein-5 stimulates mesangial cell migration

1997 ◽  
Vol 273 (6) ◽  
pp. F899-F906 ◽  
Author(s):  
Christine K. Abrass ◽  
Anne K. Berfield ◽  
Dennis L. Andress
Keyword(s):  
Growth Factor ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Binding Protein ◽  
Interactive Effects ◽  
Insulin Like Growth Factor ◽  
Heparin Binding ◽  
Induction Mechanism ◽  
Igf I ◽  
Heparin Binding Domain

Insulin-like growth factor I (IGF-I) binding protein-5 (IGFBP-5) is produced by mesangial cells (MCs) and likely functions to modulate glomerular IGF-I activity. Although IGFBP-5 may be inhibitory for IGF-stimulated MC activity, preliminary studies suggested that IGFBP-5 acts directly on MCs. To investigate this further, we evaluated the effects of IGFBP-5 on rat MC migration. We found that the carboxy-truncated fragment, IGFBP-5-(1–169), inhibited IGF-I-stimulated migration, but intact IGFBP-5 simulated migration when IGF-I was not present. Demonstration that125I-labeled IGFBP-5 directly binds to MCs further supports an independent role for IGFBP-5. Because heparin inhibited MC binding of125I-IGFBP-5, we tested the heparin binding peptide, IGFBP-5-(201–218), for stimulatory activity. IGFBP-5-(201–218) stimulated MC migration, and this effect was inhibited by heparin. Because the disintegrin, kistrin, blocked IGF-I-induced migration but not migration induced by IGFBP-5-(201–218), the migratory induction mechanism for the two peptides is different. These data indicate that separate, specific regions of IGFBP-5 are responsible for interactive effects with IGF-I as well as direct effects on MC activity.

scholarly journals The domains of human fibronectin mediating the binding of α antigen, the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection

2000 ◽  
Vol 347 (3) ◽  
pp. 725-731 ◽  
Author(s):  
Mariko NAITO ◽  
Tomohiko FUKUDA ◽  
Kiyotoshi SEKIGUCHI ◽  
Takeshi YAMADA
Keyword(s):  
Recombinant Dna ◽  
Mycobacterial Infection ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Heparin Binding ◽  
Binding Domains ◽  
Immunodominant Antigen ◽  
Heparin Binding Domain ◽  
Recombinant Dna Techniques

We have recently shown that α antigen (α-Ag), the immunodominant antigen of mycobacteria, has a novel fibronectin (FN)-binding motif that is unique among mycobacteria [Naito, Ohara, Matsumoto and Yamada (1998) J. Biol. Chem. 273, 2905-2909]. In this study, we examined the domains of human FN that interacted with α-Ag. Fragments of FN generated by either proteolysis or recombinant DNA techniques were compared for their ability to bind to α-Ag. Fragments containing either the C-terminal heparin-binding domain or the central cell-binding domain consistently bound to α-Ag. The fragment of the C-terminal heparin-binding domain, upon mutation that resulted in the loss of its heparin-binding activity, could not bind with α-Ag. These findings suggested that the mutated site, i.e. the main heparin-binding site of FN, was also the principal site for binding to α-Ag. The α-Ag-binding domains of FN could bind whole mycobacterial bacilli, suggesting that these two domains are important contributors to mycobacterial infection.

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