IGF binding proteins and their functions

The insulin-like growth factor (IGF) binding proteins (IGFBPs) have several functions, including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specific cell types, and modulating both IGF binding to receptors and growth-promoting actions. The functions of IGFBPs appear to be altered by posttranslational modifications. IGFBP-3, -4, -5, and -6 have been shown to be glycosylated. Likewise all the IGFBPs have a complex disulfide bond structure that is required for maintenance of normal IGF binding. IGFBP-2, -3, -4, and -5 are proteolytically cleaved, and specific proteases have been characterized for IGFBP-3, -4, and -5. Interestingly, attachment of IGF-I or II to IGFBP-4 results in enhancement of proteolysis, whereas attachment of either growth factor to IGFBP-5 results in inhibition of proteolytic cleavage. Cleavage of IGFBP-3 results in the appearance of a 31 kDa fragment that is 50-fold reduced in its affinity for the IGF-I or IGF-II. In spite of the reduction in its affinity, this fragment is capable of potentiating the effect of IGF-I on cell growth responses; therefore, proteolysis may be a specific mechanism that alters IGFBP modulation of IGF actions. Other processes that result in a reduction in IGF binding protein affinity are associated with potentiation of cellular responses to IGF-I and -II. Specifically, the binding of IGFBP-3 to cell surfaces is associated with its ability to enhance IGF action and with a ten- to 12-fold reduction in its affinity for IGF-I and IGF-II. Likewise, binding of IGFBP-5 to extracellular matrix (ECM) results in an eightfold reduction in its affinity and a 60% increase in cell growth in response to IGF-I. Another post-translational modification that modifies IGFBP activity is phosphorylation. IGFBP-1, -2, -3, and -5 have been shown to be phosphorylated. Phosphorylation of IGFBP-1 results in a sixfold enhancement in its affinity for IGF-I and -II. Following this enhancement of IGFBP-1 affinity, this binding protein loses its capacity to potentiate IGF-I growth-promoting activity. Future studies using site-directed mutagenesis to modify these proteins should enable us to determine the effect of these posttranslational modifications on the ability of IGFBPs to modulate IGF biologic activity. © 1993 Wiley-Liss, Inc.     

Cloning and sequence determination of bovine insulin-like growth factor binding protein-2 (IGFBP-2): comparison of its structural and functional properties with IGFBP-1.

Insulin-like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin-Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N-terminal sequence is similar to a form of IGFBP purified from rat BRL-3A cells that has recently been named IGFBP-2. These studies report the complete amino acid sequence of bovine IGFBP-2 and compare its functional properties with human IGFBP-1. The protein is 81% identical to rat IGFBP-2. When compared with both rat IGFBP-2 and human IGFBP-1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP-2 has a higher affinity for IGF-II than for IGF-I and its affinity for both forms of IGF is greater than for human IGFBP-1. Like IGFBP-1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF-I; however, IGFBP-2 was much less potent. The maximum potentiation of the IGF-mediated mitogenic response that could be achieved was approximately 42% that of IGFBP-1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP-2 inhibits DNA synthesis. The purification of IGFBP-2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.     

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.     

The role and regulation of IGFBP-1 phosphorylation in fetal growth restriction

Fetal growth restriction (FGR) increases the risk of perinatal complications and predisposes the infant to developing metabolic, cardiovascular, and neurological diseases in childhood and adulthood. The pathophysiology underlying FGR remains poorly understood and there is no specific treatment available. Biomarkers for early detection are also lacking. The insulin-like growth factor (IGF) system is an important regulator of fetal growth. IGF-I is the primary regulator of fetal growth, and fetal circulating levels of IGF-I are decreased in FGR. IGF-I activity is influenced by a family of IGF binding proteins (IGFBPs), which bind to IGF-I and decrease its bioavailability. During fetal development the predominant IGF-I binding protein in fetal circulation is IGFBP-1, which is primarily secreted by the fetal liver. IGFBP-1 binds IGF-I and thereby inhibits its bioactivity. Fetal circulating levels of IGF-I are decreased and concentrations of IGFBP-1 are increased in FGR. Phosphorylation of human IGFBP-1 at specific sites markedly increases its binding affinity for IGF-I, further limiting IGF-I bioactivity. Recent experimental evidence suggests that IGFBP-1 phosphorylation is markedly increased in the circulation of FGR fetuses suggesting an important role of IGFBP-1 phosphorylation in the regulation of fetal growth. Understanding of the significance of site-specific IGFBP-1 phosphorylation and how it is regulated to contribute to fetal growth will be an important step in designing strategies for preventing, managing, and/or treating FGR. Furthermore, IGFBP-1 hyperphosphorylation at unique sites may serve as a valuable biomarker for FGR.     

Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.     

Enhanced insulin-like growth factor-I (IGF-I) cell association at reduced pH is dependent on IGF binding protein-3 (IGFBP-3) interaction

The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Expression of IGF-binding protein-1 phosphoisoforms in fasted rat skin and its role in regulation of collagen biosynthesis

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen and glycosaminoglycan (GAG) biosynthesis in tissues. IGF-I activity is modulated by a family of IGF-binding proteins (IGFBPs) with different IGF-I binding affinities. At least IGFBP-1 and IGFBP-2 are known as inhibitors of IGF functions. Some IGFBPs (IGFBP-1, IGFBP-3 and IGFBP-5) may undergo phosphorylation that dramatically increase their affinity for IGF. During fasting of animals there is a significant decrease of the collagen and GAG content of the skin, accompanied by a reduction of plasma IGF-I levels. However, in previous studies we showed that in the skin of fasted rats IGF-I as well as IGFBP-1 and IGFBP-2 expressions were not different, compared to control rat skin, although collagen content was significantly decreased. In the present study we show that fasted rat skin contains similar amounts of IGF-I, IGFBP-3 and IGFBP-1, although extract from fasted rat skin induced inhibition of collagen biosynthesis in cultured fibroblasts, compared to control rat skin extract. Western immunoblot analysis of control and fasted rat skin extracts, using anti-phosphoserine antibodies for immunoprecipitated IGFBP-1 and IGFBP-3, revealed that both proteins are present in phosphorylated form. Although no differences were found in the expression of phosphorylated IGFBP-3 between control and fasted rat skins, that of phosphorylated IGFBP-1 in fasted rat skin extract was higher than in control one. We suggest that there is an increased level of IGFBP-1 phosphoisoform in fasted rat skin, associated with increased affinity for IGF-I. The increase of phosphorylated IGFBP-1 in fasted rat skin tissue may augment IGF-I binding affinity for IGF and decrease its bioavailability for receptor interaction. This mechanism may prevent IGF-I dependent stimulation of fibroblasts to produce extracellular matrix components. The specific expression of IGFBPs and their phosphoisoforms in tissues may play an important role in regulation of IGF-I action during physiologic and pathologic responses.     

Insulin-like growth factor-binding protein-3 binds fibrinogen and fibrin.

Following tissue injury, a fibrin network formed at the wound site serves as a scaffold supporting the early migration of stromal cells needed for wound healing. Growth factors such as insulin-like growth factor-I (IGF-I) concentrate in wounds to stimulate stromal cell function and proliferation. The ability of IGF-binding proteins (IGFBPs) such as IGFBP-3 to reduce the rate of IGF-I clearance from wounds suggests that IGFBP-3 might bind directly to fibrinogen/fibrin. Studies presented here show that IGFBP-3 does indeed bind to fibrinogen and fibrin immobilized on immunocapture plates, with K(d) values = 0.67 and 0.70 nM, respectively, and competitive binding studies suggest that the IGFBP-3 heparin binding domain may participate in this binding. IGF-I does not compete for IGFBP-3 binding; instead, IGF-I binds immobilized IGFBP-3.fibrinogen and IGFBP-3.fibrin complexes with affinity similar to that of IGF-I for the type I IGF receptor. In the presence of plasminogen, most IGFBP-3 binds directly to fibrinogen, although 35-40% of the IGFBP-3 binds to fibrinogen-bound plasminogen. IGFBP-3 also binds specifically to native fibrin clots, and addition of exogenous IGFBP-3 increases IGF-I binding. These studies suggest that IGF-I can concentrate at wound sites by binding to fibrin-immobilized IGFBP-3, and that the lower IGF affinity of fibrin-bound IGFBP-3 allows IGF-I release to type I IGF receptors of stromal cells migrating into the fibrin clot.     

Insulin-like growth factor-binding protein-3 activates a phosphotyrosine phosphatase. Effects on the insulin-like growth factor signaling pathway

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln(3),Ala(4),Tyr(15),Leu(16)]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42(MAPK) activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.     

Relevance of IGF-I, IGFBP-3, and IGFBP-2 measurements during GH treatment of GH-deficient and non-GH-deficient children and adolescents

BACKGROUND: Little information is available on the relevance of parameters representing the insulin-like growth factor (IGF) system with regard to growth hormone (GH) treatment during childhood. In adults, high IGF-I levels were found to be associated with side effects and long-term risks. AIM/METHOD: Our aim was to monitor the serum levels of IGF-I, IGF-binding protein (IGFBP) 3, and IGFBP-2 during long-term GH treatment of 156 patients with GH deficiency (GHD) and of 153 non-GHD patients. We determined the extent to which the IGF parameters exceed the normal ranges and identified those parameters which are predictive of 1st-year growth. RESULTS: In prepubertal GHD children, the levels of IGF-I, IGFBP-3, and IGF-I/IGFBP-3 exceeded the 95th centile of the reference values for this age group in 2.3, 0.3, and 7.9% of the cases, respectively, whereas in prepubertal non-GHD children, the same parameters exceeded the 95th reference centile in 20.1, 3.5, and 32.2%, respectively. In pubertal GHD children IGF-I, IGFBP-3, and IGF-I/IGFBP-3 levels exceeded the 95th reference centile in 11.1, 1.5, and 15.4%, respectively. In pubertal non-GHD children, these levels also exceeded the 95th centile in 26.7, 7.0, and 41.4%, respectively. In both GHD and non-GHD groups, however, some patients had IGF parameters which were below the reference values. Our analysis showed that, in both groups, in addition to maximum GH, all IGF parameters (IGF-I, IGFBP-3, IGF-I/IGFBP-3 ratio, IGFBP-2 or derivatives) significantly extend the scope of a calculated model for predicting 1st-year height velocity. CONCLUSION: For reasons of safety and optimization of GH therapy, it is essential to follow up IGF-I, IGFBP-3, and IGFBP-2 levels regularly during childhood.     

Discrete alterations of the insulin-like growth factor I molecule which alter its affinity for insulin-like growth factor-binding proteins result in changes in bioactivity.

Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity. They are present in extracellular fluids and modulate the interactions of their ligands with the type 1 IGF cell surface receptor. These studies utilized IGF-I analogs that have reduced binding affinity for either the type 1 IGF receptor or binding proteins to study the ligand specificity of IGF-BP-1 and the role of IGF-BP-1 in modulating the biological activity of IGF-I. The data indicate that the regions of IGF-I which are responsible for binding to IGF-BP-1 and to human serum-binding proteins are distinct but overlapping and are clearly distinct from the type I receptor binding sites. In the absence of exogenously added IGF-BP-1, the analogs with reduced affinity for IGF-BP-1 are more potent than IGF-I in stimulating DNA synthesis by porcine aortic smooth muscle cells. In contrast, when cells are concomitantly exposed to IGF-BP-1, two of the analogs with reduced affinity for binding protein give only 40-65% of the maximal IGF-I response. [Leu24, 1-62]IGF-I, which has a 100-fold reduced affinity for the type 1 IGF receptor, gave a value that was 62% of the maximal IGF-BP-1 potentiated response. A second biological response, that of stimulating binding protein secretion by IGF-I, was also examined. [Leu24, 1-62]IGF-I is more potent than IGF-I whereas the activity of the analogs with lower affinity for IGF-BP-1 is significantly reduced. Thus, the ability to activate DNA synthesis and binding protein secretion maximally in the presence of IGF-BP-1 is dependent on the affinity of IGFs for both type 1 receptors and binding proteins.     

Insulin-like growth factor (IGF) binding protein-5 blocks skeletal muscle differentiation by inhibiting IGF actions

Signaling through the IGF-I receptor by locally produced IGF-I or -II is critical for normal skeletal muscle development and repair after injury. In most tissues, IGF action is modulated by IGF binding proteins (IGFBPs). IGFBP-5 is produced by muscle cells, and previous studies have suggested that when overexpressed it may either facilitate or inhibit IGF actions, and thus potentially enhance or diminish IGF-mediated myoblast differentiation or survival. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5, we studied its effects in cultured muscle cells. Purified wild-type (WT) mouse IGFBP-5 or a variant with diminished extracellular matrix binding (C domain mutant) each prevented differentiation at final concentrations as low as 3.5 nm, whereas analogs with reduced IGF binding (N domain mutant) were ineffective even at 100 nm. None of the IGFBP-5 variants altered cell number. An IGF-I analog (R(3)IGF-I) with diminished affinity for IGFBPs promoted full muscle differentiation in the presence of IGFBP-5(WT), showing that IGFBP-5 interferes with IGF-dependent signaling pathways in myoblasts. When IGFBP-5(WT) or variants were overexpressed by adenovirus-mediated gene transfer, concentrations in muscle culture medium reached 500 nm, and differentiation was inhibited, even by IGFBP-5(N). As 200 nm of purified IGFBP-5(N) prevented activation of the IGF-I receptor by 10 nm IGF-II as effectively as 2 nm of IGFBP-5(WT), our results not only demonstrate that IGFBP-5 variants with reduced IGF binding affinity impair muscle differentiation by blocking IGF actions, but underscore the need for caution when labeling effects of IGFBPs as IGF independent because even low-affinity analogs may potently inhibit IGF-I or -II if present at high enough concentrations in biological fluids.     

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.     

Cooperativity of the N- and C-terminal domains of insulin-like growth factor (IGF) binding protein 2 in IGF binding

A family of six insulin-like growth factor (IGF) binding proteins (IGFBP-1-6) binds IGF-I and IGF-II with high affinity and thus regulates their bioavailability and biological functions. IGFBPs consist of N- and C-terminal domains, which are highly conserved and cysteine-rich, joined by a variable linker domain. The role of the C-domain in IGF binding is not completely understood in that C-domain fragments have very low or even undetectable IGF binding affinity, but loss of the C-domain dramatically disrupts IGF binding by IGFBPs. We recently reported the solution structure and backbone dynamics of the C-domain of IGFBP-2 (C-BP-2) and identified a pH-dependent heparin binding site [Kuang, Z., Yao, S., Keizer, D. W., Wang, C. C., Bach, L. A., Forbes, B. E., Wallace, J. C., and Norton, R. S. (2006) Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2), J. Mol. Biol. 364, 690-704]. Here, we have analyzed the molecular interactions among the N-domain of IGFBP-2 (N-BP-2), C-BP-2, and IGFs using cross-linking and nuclear magnetic resonance (NMR) spectroscopy. The binding of C-BP-2 to the IGF-I.N-BP-2 binary complex was significantly stronger than the binding of C-BP-2 to IGF-I alone, switching from intermediate exchange to slow exchange on the NMR time scale. A conformational change or stabilization of the IGF-I Phe49-Leu54 region and the Phe49 aromatic ring upon binding to the N-domains, as well as an interdomain interaction between N-BP-2 and C-BP-2 (which is also detectable in the absence of ligand), may contribute to this cooperativity in IGF binding. Glycosaminoglycan binding by IGFBPs can affect their IGF binding although the effects appear to differ among different IGFBPs; here, we found that heparin bound to the IGF-I.N-BP-2.C-BP-2 ternary complex, but did not cause it to dissociate.     

The heparin binding domain of insulin-like growth factor binding protein (IGFBP)-3 increases susceptibility of IGFBP-3 to proteolysis.

IGFBP-3 proteolysis clears IGFBP-3 from body fluids and increases IGF bioavailability. As shown here, native human IGFBP-3 was cleaved by proteases in media conditioned by hamster and insect cells. This proteolysis was less pronounced for IGFBP-3 containing a mutated heparin binding domain, and was prevented by purifying IGFBP-3 on an IGF-I affinity column in the presence of 2 M sodium chloride, suggesting that the responsible protease(s) binds the IGFBP-3 heparin binding domain. To determine if any human proteases act this way, we first studied plasma prekallikrein since it can copurify with IGFBP-3, and found: 1) [125]IGFBP-3 binds to prekallikrein immobilized either on nitrocellulose or on immunocapture plates; 2) the IGFBP-3 heparin binding domain participates in forming the IGFBP-3/prekallikrein complex; 3) the binary IGFBP-3/prekallikrein complex can bind IGF-I to form a ternary complex; and 4) activation of prekallikrein to alpha-kallikrein by Factor XIIa resulted in proteolysis of bound IGFBP-3. This work suggests: 1) cleavage of IGFBP-3 by a protease may be aided by the ability of the protease zymogen to directly bind the IGFBP-3 heparin binding domain; and 2) direct binding of protease zymogens to IGFBP-3 may explain some instances where IGFBP-3 is preferentially proteolyzed in the presence of other IGFBPs.     

IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3)

IGF-1 has been shown to promote proliferation of normal epithelial breast cells, and the IGF pathway has also been linked to mammary carcinogenesis in animal models. We comprehensively examined the association between common genetic variation in the IGF1, IGFBP1, and IGFBP3 genes in relation to circulating IGF-I and IGFBP-3 levels and breast cancer risk within the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). This analysis included 6,912 breast cancer cases and 8,891 matched controls (n = 6,410 for circulating IGF-I and 6,275 for circulating IGFBP-3 analyses) comprised primarily of Caucasian women drawn from six large cohorts. Linkage disequilibrium and haplotype patterns were characterized in the regions surrounding IGF1 and the genes coding for two of its binding proteins, IGFBP1 and IGFBP3. In total, thirty haplotype-tagging single nucleotide polymorphisms (htSNP) were selected to provide high coverage of common haplotypes; the haplotype structure was defined across four haplotype blocks for IGF1 and three for IGFBP1 and IGFBP3. Specific IGF1 SNPs individually accounted for up to 5% change in circulating IGF-I levels and individual IGFBP3 SNPs were associated up to 12% change in circulating IGFBP-3 levels, but no associations were observed between these polymorphisms and breast cancer risk. Logistic regression analyses found no associations between breast cancer and any htSNPs or haplotypes in IGF1, IGFBP1, or IGFBP3. No effect modification was observed in analyses stratified by menopausal status, family history of breast cancer, body mass index, or postmenopausal hormone therapy, or for analyses stratified by stage at diagnosis or hormone receptor status. In summary, the impact of genetic variation in IGF1 and IGFBP3 on circulating IGF levels does not appear to substantially influence breast cancer risk substantially among primarily Caucasian postmenopausal women.     

Synthesis and characterization of biotinylated forms of insulin-like growth factor-1: topographical evaluation of the IGF-1/IGFBP-2 AND IGFBP-3 interface

Activation of the insulin-like growth factor-1 (IGF)-1 receptor signaling pathways by IGF-1 and IGF-2 results in mitogenic and anabolic effects. The bioavailability of the IGFs is regulated by six soluble binding proteins, the insulin-like growth factor binding proteins (IGFBPs), which bind with approximately 0.1 nM affinity to the IGFs and often serve as endogenous antagonists of IGF action. To identify key domains of IGF-1 involved in the interaction with IGFBP-2 and IGFBP-3, we employed IGF-1 selectively biotinylated on residues Gly 1, Lys 27, Lys 65, and Lys 68. All monobiotinylated species of IGF-1 exhibited high affinity ( approximately 0.1-0.2 nM) for IGFBP-2 and IGFBP-3 in solid-phase-binding assays. However, different labeling intensities were observed in ligand blot analysis of IGFBP-2 and IGFBP-3. The N(epsilon)(Lys65/68)(biotin)-IGF-1 (N(epsilon)(Lys65/68b)-IGF-1) probe exhibited the highest signal intensity, while N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 demonstrated significantly lower signals. When taken together, these results suggest that, once bound to IGFBP-2 or IGFBP-3, the biotin moieties of N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 are inaccessible to NeutrAvidin-peroxidase, the secondary binding component. Ligand blots using IGF-1 derivatized with a long chain form of the N-hydroxysuccinimide biotin (NHS-biotin) to yield N(alpha)(Gly1)(LC-biotin)-IGF-1 and N(epsilon)(Lys27)(LC-biotin)-IGF-1 demonstrated increased signal intensity compared with their NHS-biotin counterparts. In BIAcore analysis, IGFBP-2 and IGFBP-3 bound only to the N(epsilon)(Lys65/68b)-IGF-1-coated flowcell of a biosensor chip, confirming the inaccessibility of Gly 1 and Lys 27 when IGF-1 is bound to IGFBP-2 and IGFBP-3. These data confirm the involvement of the IGFBP-binding domain on IGF-1 in binding to IGFBP-2 and IGFBP-3 and support involvement of the IGF-1R-binding domain in IGFBP binding.