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Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM (125)I-labelled IGFBP-3 to occupy all binding sites present on 100 microg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a K(d) between 2.6x10(-10) and 8.0x10(-10) M and 1.51-4.89x10(11) binding sites/mg of platelet membrane. Kinetic analysis of (125)I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (k(on)) of 8.1x 10(8) per M per min. The reverse rate constant (k(off)) was calculated to be 1.6x10(-1) per min (t(1/2)=4.2 min) and confirmed experimentally to be 3.3x10(-1) per min (t(1/2)=2.1 min). Binding of (125)I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with (125)I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in (125)I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.
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Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.