Characterization of insulin-like growth factor-free interaction between insulin-like growth factor binding protein 3 and acid labile subunit expressed from Xenopus oocytes

The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (Delta11-60) demonstrated a moderate reduction. D88 (Delta11-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

Regulation of the components of the 150 kDa IGF binding protein complex in cocultures of rat hepatocytes and Kupffer cells by 3',5'-cyclic adenosine monophosphate

In the circulation, most of IGFs are bound to a high molecular mass complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, biosynthesis of these components has been localized to different cell populations with hepatocytes as source of ALS and nonparenchymal cells (endothelial and Kupffer cells (KC)) as source of IGFBP-3. In the present study, the regulatory effects of the cAMP analogs dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP (8-br-cAMP) on IGF-I, ALS, and IGFBP expression were evaluated in primary cultures of rat hepatocytes, KC as well as in cocultures of hepatocytes and KC. In cocultures, biosynthesis of IGFBP-3 and ALS was inhibited dose-dependently by db-cAMP and 8-br-cAMP while that of IGF-I, IGFBP-1, and -4 was stimulated as demonstrated by ligand and Northern blotting. IGFBP-3 expression in primary cultures of pure KC did not respond to cAMP treatment indicating the importance of a cellular interaction between KC and hepatocytes for the decreased IGFBP-3 synthesis. The inhibition of IGFBP-3 in db-cAMP-treated cocultures was due to a decrease of IGFBP-3 mRNA level accompanied by a reduced cellular degradation of IGFBP-3. We conclude that cAMP stimulate the biosynthesis of IGF-I, IGFBP-1, and -4 in cocultures of hepatocytes and KC thereby enabling the formation of binary IGF/IGFBP complexes while the formation of the 150 kDa complex is impaired through downregulation of IGFBP-3 and ALS. This complex regulation may be a prerequisite for the effects of cAMP-dependent hormones on the transfer of IGFs from circulation to peripheral tissues.     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

Bovine mural granulosa cells, and not the oocyte, are the major source of proteases capable of IGFBP-2 degradation

Oocytes can regulate their own development, hence studies identifying and characterising oocyte-secreted factors are crucial to resolving the mechanisms by which oocytes can orchestrate follicular development. The insulin-like growth factor (IGF) system plays an important role during the development of a follicle; however, the regulation of IGF bioavailability is crucial throughout follicular growth. Proteolytic cleavage of the IGF/IGF binding protein (IGFBP) complex increases IGF bioavailability, hence, the production of IGFBP proteases by the oocyte and/or granulosa cells would provide a regulatory mechanism whereby they could regulate their own exposure to IGFs. The present study revealed mural granulosa cells (MGC), and not the oocyte, are the major source of proteases capable of cleaving IGFBP-2 in the developing bovine antral follicle. The addition of recombinant IGF-I or FSH had no effect in terms of modulating IGFBP-2 degradation. This work further supports the existence of a local regulatory mechanism modulating IGF bioavailability.     

The acid-labile subunit of the serum insulin-like growth factor-binding protein complexes. Structural determination by molecular modeling and electron microscopy.

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potential N-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.     

Role of N- and C-terminal residues of insulin-like growth factor (IGF)-binding protein-3 in regulating IGF complex formation and receptor activation

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu(77), Leu(80), and Leu(81) in the N terminus and Gly(217) and Gln(223) in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF.IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.     

Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.     

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.     

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.     

N-Linked glycosylation and sialylation of the acid-labile subunit. Role in complex formation with insulin-like growth factor (IGF)-binding protein-3 and the IGFs

Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

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.     

D440N mutation in the acid-labile subunit of insulin-like growth factor complexes inhibits secretion and complex formation

The acid-labile subunit (ALS) regulates IGF bioavailability by forming heterotrimeric complexes with IGFs and IGF-binding protein-3 (IGFBP-3). A homozygous missense mutation (D440N) resulting in undetectable circulating levels of ALS with a concomitant reduction in IGF-I and IGFBP-3 has been reported to cause mild growth retardation. To understand how this particular mutation affects ALS circulating levels and IGF-transport function, we expressed recombinant ALS and its variants, D440N-ALS, T442A-ALS, and D440N/T442A-ALS, using adenovirus vectors. Compared with wild-type ALS, the secretion of D440N-ALS was 80% lower. The D440N mutation was proposed to generate an N-glycosylation site additional to the seven existing motifs in ALS. D440N-ALS appeared larger than ALS, attributable to N-linked glycans because deglycosylation with N-glycosidase F reduced both proteins to the same molecular mass. When ALS was incubated with IGF-I and IGFBP-3, 70-80% of IGF-I was detected by gel-filtration chromatography in forms corresponding to the 150-kDa ternary complex. In contrast, when D440N-ALS was tested, less than 30% of IGF-I was found in high molecular mass complexes. Two other ALS variants mutated in the same putative glycosylation site, D440N/T442A-ALS and T442A-ALS, showed similar chromatographic profiles to wild-type ALS. The D440N mutation in ALS generates a hyperglycosylated form with impaired secretion and complex formation, potentially leading to dysregulation of endocrine IGF, thus contributing to the growth retardation observed in the affected patient. This is the first study to explain how a natural mutation, D440N, in ALS impairs its function.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Insulin-like growth factor binding proteins: new proteins, new functions.

The insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases regulate somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogens whose actions are determined by the availability of free IGFs to interact with IGF receptors. IGFBPs comprise a family of six proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs and thereby their actions. IGFBP-related proteins (IGFBP-rPs) bind IGFs with low affinity and also play important roles in cell growth and differentiation. The GH-IGF-IGFBP axis is complex and powerful. Future research on its physiology promises exciting insights into cell biology as well as therapies for diseases such as cancer and diabetes mellitus.     

Differential responses of IGF-I molecular complexes to military operational field training.

Insulin-like growth factor (IGF) I and IGF binding proteins (IGFBPs) modulate metabolic activity and tissue repair and are influenced by nutritional status. IGF-I circulates in free, ternary [IGF-I + IGFBP-3 + acid labile subunit (ALS)], and binary (IGF-I + IGFBP) molecular complexes, and the relative proportions regulate IGF-I extravascular shifting and bioavailability. This study examined the hypothesis that sustained physical activity and sleep deprivation superimposed on a short-term energy deficit would alter the IGFBP concentrations and alter the proportions of IGF-I circulating in ternary vs. binary molecular complexes. Components of the IGF-I system (total and free IGF-I; IGFBP-1, -3, and ALS; nonternary IGF-I and IGFBP-3), biomarkers of metabolic and nutritional status (transferrin, ferritin, prealbumin, glucose, free fatty acids, glycerol, beta-hydroxybutyrate), and body composition were measured in 12 men (22 +/- 3 yr, 87 +/- 8 kg, 183 +/- 7 cm, 20 +/- 5% body fat) on days 1, 3, and 4 during a control and experimental (Exp) period. During Exp, subjects performed prolonged work (energy expenditure of approximately 4500 kcal/day) with caloric (1600 kcal/day) and sleep (6.2 h total) restriction. IGF-I and IGFBP-3 were measured by immunoassay before and after immunoaffinity depletion of ALS-based complexes (i.e., ternary complex removal). Exp produced losses in body mass (-3.0%), lowered total IGF-I (-24%), free IGF-I (-42%), IGFBP-3 (-6%), nonternary IGF-I (-27%), and IGFBP-3 (-16%), and increased IGFBP-1 (256%). No Exp effects were observed for ALS. No changes were observed in the proportion of IGF-I circulating in free ( approximately 1.2%), ternary ( approximately 87.4%), or nonternary ( approximately 11.4%) molecular complexes. During Exp, glucose concentrations were lower on day 3, but days 1 and 4 were statistically similar. In conclusion, during a short-term energy deficit in young, healthy men, 1). IGF-I system components differentially respond (both in direction and magnitude) to a given metabolic perturbation and 2). the relative proportion of IGF-I sequestered in ternary vs. nonternary molecular complexes appears to be well maintained.