Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors

The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.     

High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.     

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa)

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.     

Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins

Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 X 10(10) l/mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs.     

A factor contained in plasma is required for IGF binding protein-1 to potentiate the effect of IGF-I on smooth muscle cell DNA synthesis.

One of the forms of the insulin-like growth factor (IGF) binding proteins present in human amniotic fluid has been shown to potentiate the growth-promoting effect of IGF-I markedly. This study was undertaken to determine the cellular and hormonal factors that modulate this potentiation and to determine whether this protein would potentiate the effects of other mitogens. Although the combination of the IGFBP-1 (20 ng/ml) and IGF-I (10 ng/ml) induced a five- to sixfold increase in DNA synthesis compared with IGF-I alone, this response required the simultaneous addition of IGF-I with 0.1% platelet-poor plasma (PPP). If PPP was omitted from the incubation medium, no increase above the effect that was obtained with IGF-I alone was noted. Substitution of cerebrospinal fluid (CSF) for PPP permitted a full mitogenic response, although substitution with amniotic fluid resulted in no enhancement. The factor contained in PPP was heat and acid stable. If the binding protein was co-incubated with fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), a slight inhibition of the cellular response to each of these factors was detected. Co-incubation of IGF-I with the IGF-binding protein plus these other peptide growth factors resulted in no further enhancement of DNA synthesis above the level observed with IGF-I and the binding protein alone. Likewise, addition of plasma proteins such as transferrin or albumin did not result in a further enhancement of the DNA synthesis response to IGF-I plus binding protein, and these proteins could not substitute for PPP or IGFBP-1. Transient exposure of the cultures (2 hr) to the binding protein plus IGF-I resulted in a submaximal DNA synthesis response, and the binding protein had to be present continuously to achieve a maximal effect. These studies indicate that a factor contained in plasma and CSF is required for a maximal cellular response to IGFBP-1 plus IGF-I, and this factor does not appear to be a well-defined mitogen.     

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a high molecular weight somatomedin binding protein from human plasma

The somatomedins insulin-like growth factor I and II (1,2) are in serum bound to high-molecular weight binding proteins (6,7,8). By use of a four step chromatographic procedure a somatomedin binding protein was isolated from outdated human plasma. Exclusion chromatography on Sephadex G-200 disclosed a molecular weight of 150 kDa. After lyophilization however, the binding activity was found in a lower molecular weight range of 35-45 kDa. A partial amino acid sequence analysis of the lyophilized material revealed a possible N-terminal sequence of Ala-Pro-Trp. This sequence is identical to the N-terminal sequence of the 35 kDa somatomedin binding protein previously isolated from human amniotic fluid (16).     

Insulin-like growth factor-binding protein from human plasma. Purification and characterization

Insulin-like growth factor (IGF)-binding protein (BP) has been purified from Cohn fraction IV of human plasma by acidification, ion exchange to remove endogenous ligands, and affinity chromatography on agarose-IGF-II. The pure protein appeared as a single peak by high performance reverse-phase and gel permeation chromatography (molecular mass, 45-50 kDa), but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band at 53 kDa and a minor band at 47 kDa, unreduced, or 43 and 40 kDa, respectively, reduced. The two bands stained for both protein and carbohydrate. After storage at 2 degrees C for 5 months at pH 3, two additional bands, at 26 and 22 kDa on unreduced gels, were also present. Autoradiography after affinity labeling with IGF-I or IGF-II tracer revealed a single labeled band of 61 kDa. BP, quantitated using a specific radioimmunoassay, was retained by agarose-immobilized IGF-I, IGF-II, concanavalin A, and wheat germ lectin, but not Helix pomatia lectin. Competitive binding curves using pure BP and human IGF-I and IGF-II as both labeled and unlabeled ligands indicated association constants of 2-3 X 10(10) liters/mol for both peptides, with a slightly higher affinity for IGF-II than IGF-I, and 0.9 binding sites for either peptide per 53-kDa protein. The exact relationship of this acid-stable IGF BP to the 150-kDa complex from which it is derived remains to be determined.     

Porcine insulin-like growth factor (IGF) binding protein blocks IGF-I action on porcine adipose tissue

A growth hormone-dependent insulin-like growth factor (IGF) binding protein (IGFBP) purified from porcine serum specifically blocked the acute insulin-like effects of IGF-I on lipogenesis and glucose oxidation in porcine adipose tissue. This inhibition was dose dependent with half-maximal effective concentrations of IGFBP of 530 ng/ml for lipogenesis and 590 ng/ml for glucose oxidation in the presence of 10(-8) M IGF-I. The IGFBP also caused decreased rates of lipogenesis following a 1-hr preincubation of tissue with IGF-I (10(-8) M). The IGFBP had no effect on insulin action on porcine adipose tissue. These findings demonstrate the inhibitory effects of a highly purified porcine serum IGFBP on the biologic effects of IGF-I in vitro, and provide evidence that the growth hormone-dependent IGFBP blocks the acute insulin-like actions of IGF-I in vivo.     

Purification of a 31,000-dalton insulin-like growth factor binding protein from human amniotic fluid. Isolation of two forms with different biologic actions

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.     

Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1).

IGF-I and IGF-II are growth-stimulating peptides with strong mitogenic properties. These polypeptide growth factors circulate in serum bound to specific binding proteins. We report the cloning and complete sequence of a cDNA encoding a low mol. wt IGF-binding protein from a human placenta cDNA library. We propose the designation IGF-binding protein 1 (IBP-1) for the gene and corresponding protein. Expression of the cDNA encoding IBP-1 in COS cells resulted in the synthesis of a 30-kd protein which binds IGF-I and is immunologically indistinguishable from the IGF-binding protein isolated from amniotic fluid or human serum. Northern blotting analysis demonstrated that expression of the IBP-1 gene is highly tissue specific and limited to placental membranes and fetal liver suggesting a rigid control. The IBP-1 gene is a single copy gene, located on chromosome 7. The results obtained suggest that most, if not all, lower mol. wt IGF-binding proteins originate from this gene.     

Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.     

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.     

Identification of a type 1 insulin-like growth factor binding protein (IGF BP) in serum from rats with diabetes mellitus

Circulating insulin-like growth factor binding protein (IGF BP) activity is increased in animals with streptozotocin-induced diabetes. Separation of BPs by SDS/PAGE for ligand and immunoblot analysis revealed that a 32,000 molecular weight BP is present and increased in diabetic serum. This BP is immunologically distinct from the low molecular weight fetal rat BP (rBP2) and is related to the human amniotic fluid BP (hBP1) that is increased in patients with insulin dependent diabetes mellitus.     

Monoclonal antibodies to the 27-34K insulin-like growth factor binding protein

Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12.     

Cloning, characterization, and expression of a human insulin-like growth factor binding protein

Insulin-like growth factors (IGFs) bind to specific proteins present in extracellular fluids. One of these binding proteins (IGF-BP) was purified from human amniotic fluid and was shown to potentiate the effects of IGF-I in vitro (10). In these studies, a polyclonal antibody to this protein was used to isolate a cDNA clone from a human decidua library. This clone encodes a polypeptide of 25,832 daltons that includes the sequences of 9 tryptic peptides that had been prepared from the purified IGF-BP. The protein has 15 cysteines that are clustered at the amino and carboxy ends of the molecule. The protein has an RGD sequence near its C-terminus, which may account for its ability to attach to cells and to potentiate the biological actions of IGF-I.     

Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid.

Using competitive ligand-binding studies, ligand blotting, and immunoprecipitation, we have characterized the insulin-like growth factor (IGF)-binding proteins (BPs) of porcine follicular fluid. Competitive ligand-binding studies revealed a preference of ovarian IGFBPs for IGF-II over IGF-I. Follicular fluid from small, 1-3-mm follicles had nearly twice the binding capacity for IGFs as that from large, 6-10-mm follicles. Ligand blots of porcine follicular fluid resolved 5 major bands of IGF-binding activity having apparent molecular sizes of 44, 40, 34, 29, and 22 kDa. The 40-44-kDa bands were immunoprecipitated by an antibody to porcine IGFBP-3, the acid-stable subunit of the 150-kDa growth hormone-dependent IGF-binding protein complex of porcine serum. The 34-kDa band was immunoprecipitated by an antibody to rat IGFBP-2, the major IGF-binding protein found in fetal rat serum. To date we have been unable to immunoprecipitate the 29- and 22-kDa bands with any of the antibodies tested, including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGF-binding protein. The 40-44-kDa species (IGFBP-3) was the predominant form and was equally abundant in fluid from large and small follicles. In contrast, the smaller forms, including IGFBP-2 and the 29- and 22-kDa forms were significantly more prominent in fluid from small follicles. In view of other studies indicating a significant effect of IGFBPs on ovarian cell function, follicular IGFBPs may play an important role in the IGF autocrine/paracrine regulatory system of the ovary.     

A novel human insulin-like growth factor binding protein secreted by osteoblast-like cells.

Insulin-like growth factor binding proteins (IGFBPs) modulate the cellular action of the insulin-like growth factors. Inhibition or enhancement of IGF effects by these cell-secreted binding proteins have been described. We have purified two IGFBPs (23 and 29 kDa) from media conditioned by U-2 human osteosarcoma cells using ligand-affinity chromatography and reversed phase HPLC. N-terminal amino acid analysis of the 23 kDa protein revealed a unique sequence with variable homology to IGFBPs 1-4. The 29 kDa IGFBP was found to be nearly identical to a recently reported IGFBP. Because the affinity purified U-2 IGFBPs enhanced IGF-I-stimulated osteoblast mitogenesis, we suggest that one or both of these binding proteins enhance IGF action in bone.     

Isolation and characterization of a folate receptor from human placenta

While folate binding proteins have been described in serum and a variety of tissues, the function of these proteins is unknown. A particulate folate binding protein from human placenta has been isolated and characterized following solubilization with Triton X-100. The protein was purified 61,000-fold using affinity chromatography on pteroylglutamic acid-Sepharose as the major purification step. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein gave a single band with a Mr = 38,500. Stoichiometry of binding indicated that 1 mol of folate was bound per mol of protein. The protein was a glycoprotein that contained 12% carbohydrate. Antiserum was raised in a rabbit, and on immunodiffusion, gave a single precipitin line with the purified placental folate binding protein. Immunoprecipitation studies using this antiserum indicated that the purified placental folate binding protein shared antigenic determinants with both the large Mr and small Mr folate binding proteins from human milk. Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.