Characterization and chromosomal localization of the human insulin-like growth factor-binding protein 6 gene

Insulin-like growth factor-binding protein 6 (IGFBP6), an extracellular protein with preferential affinity for insulin-like growth factor (IGF) II, belongs to a family of binding proteins with at least six members. We have characterized the genomic structure and the chromosomal location of the human IGFBP6, which is present in the human genome as a single-copy gene spanning 4.7 kb. It consists of four exons, encoding the translated regions, with sizes of 334, 146, 120, and 123 bp, while the intervening introns are 2661, 182, and 844 bp. Three mRNA cap sites were localized 101, 100, and 96 bp upstream of the ATG translation start codon as determined by S1 nuclease analysis. The proximal 5′-flanking region does not have any TATA or CAAT consensus sequences. The IGFBP6 was localized to Chr 12 by analysis of somatic cell hybrids and regionalized to 12q13 by fluorescence DNA in situ hybridization. Received: 14 October 1998 / Accepted: 1 December 1998     

Structure and functional expression of the acid-labile subunit of the insulin-like growth factor-binding protein complex.

Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). Full-length clones encoding ALS have been isolated from human liver cDNA libraries by using probes based on amino acid sequence data from the purified protein. These clones encode a mature protein of 578 amino acids preceded by a 27-amino acid hydrophobic sequence indicative of a secretion signal. Expression of the cDNA clones in mammalian tissue culture cells results in the secretion into the culture medium of ALS activity that can form the expected complex with IGF-I and IGF-binding protein-3. The amino acid sequence of ALS is largely composed of 18-20 leucine-rich repeats of 24 amino acids. These repeats are found in a number of diverse proteins that, like ALS, participate in protein-protein interactions.     

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.     

Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.

Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3(Ad)) was purified from the culture medium of virus-infected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3(Ad) was similar in size (43- to 45-kDa glycoform doublet) to IGFBP-3(Pl) derived from plasma. In addition, IGFBP-3(Ad) was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3(Ad) had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3(Pl). IGFBP-3(Ad) showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3(Pl), but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3(Ad) also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.     

Horse conceptuses secrete insulin-like growth factor-binding protein 3

Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radiolabeled proteins showed that cultured horse conceptuses secreted IGFBP3 into the culture medium. Endometrial samples from mares also contained IGFBP3 mRNA and protein; but there was no evidence of secretion of IGFBP3 into the uterine lumen by ligand blot analysis, and there was evidence of only very small amounts by Western blot analysis. These results indicate that the horse conceptus secretes significant quantities of IGFBP3 toward the conceptus capsule from as early as Day 10 after ovulation. Thus, most of the IGFBP3 contained within the capsule, which binds IGF-I to this special extracellular matrix of the preimplantation horse conceptus, is likely to be embryonic in origin. IGFBP3 in the horse conceptus capsule may enhance or modulate the action of IGFs on the developing conceptus.     

Glycosaminoglycans inhibit formation of the 140 kDa insulin-like growth factor-binding protein complex.

The 140 kDa insulin-like growth factor (IGF)-binding protein complex in human serum consists of three subunits: an acid-labile, non-IGF-binding glycoprotein (alpha-subunit), an IGF-binding glycoprotein known as BP-53 or IGFBP-3 (beta-subunit), and IGF-I or IGF-II (gamma-subunit). This study investigates the regulation, by salt and glycosaminoglycans, of ternary (alpha-beta-gamma) complex formation, measured by incubating radioiodinated alpha-subunit with a mixture of IGF-I and IGFBP-3 and precipitating bound radioactivity with an anti-IGFBP-3 antiserum. Increasing NaCl concentrations progressively decreased ternary complex formation without any effect on binary (beta-gamma) complex formation. In 0.15 M-NaCl, the association constant for the ternary complex was 0.318 +/- 0.092 nM-1, 100-fold lower than that for the binary complex. Glycosaminoglycans also inhibited ternary complex formation without affecting the binary complex. Heparin [50% inhibition at 0.27 +/- 0.08 units/ml (1.5 +/- 0.4 micrograms/ml)] was more potent than heparan sulphate (50% inhibition at 15 +/- 7 micrograms/ml), with chondroitin sulphate even less potent. The inhibition by heparin was due principally to a decrease in binding affinity, from 0.604 +/- 0.125 to 0.151 +/- 0.024 nM-1 in the presence of 0.25 units of heparin/ml, with a slight decrease in the number of apparent binding sites from 1.05 +/- 0.08 to 0.85 +/- 0.15 mol of alpha-subunit bound/mol of beta-subunit. Since the ternary IGF-binding protein complex cannot cross the capillary barrier, it is proposed that a decrease in the affinity of the complex, mediated by circulating or cell-associated glycosaminoglycans, may be important in the passage of IGFs and IGFBP-3 to the tissues.     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Binding between insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 is not influenced by glucose or 2-deoxy-D-glucose

A recent report (Zhong, D., Xiong, L., Liu, T., Liu, X., Liu, X., Chen, J., Sun, S. Y., Khuri, F. R., Zong, Y., Zhou, Q., and Zhou, W. (2009) J. Biol. Chem. 284, 23225-23233) details that 2-deoxy-D-glucose (2-DG), a well known inhibitor of glycolysis and a candidate antineoplastic agent, also induces insulin-like growth factor 1 receptor (IGF-1R) signaling through the inhibition of insulin-like growth factor 1-insulin-like growth factor-binding protein 3 (IGF-1-IGFBP-3) complex formation. Zhong et al. hypothesized that disrupted IGF-1/IGFBP-3 binding by 2-DG led to increased free IGF-1 concentrations and, consequently, activation of IGF-1R downstream pathways. Because their report suggests unprecedented off-target effects of 2-DG, this has profound implications for the fields of metabolism and oncology. Using ELISA, surface plasmon resonance, and novel "intensity-fading" mass spectrometry, we now provide a detailed characterization of complex formation between IGF-1 and IGFBP-3. All three of these independent methods demonstrated that there was no effect of glucose or 2-DG on the interaction between IGF-1 and IGFBP-3. Furthermore, we show examples of 2-DG exposure associated with reduced rather than increased IGF-1R and AKT activation, providing further evidence against a 2-DG increase in IGF-1R activation by IGF-1-IGFBP-3 complex disruption.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Structure and properties of the C-terminal domain of insulin-like growth factor-binding protein-1 isolated from human amniotic fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.