Insulin-like Growth Factor Binding Proteins Superfamily Research Articles

Structural and biochemical characterization of native and recombinant single insulin‐like growth factor‐binding domain protein (SIBD‐1) from the Central American Hunting Spider Cupiennius salei (Ctenidae)

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.

IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells

Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells. DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1. Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection. Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.

Silencing of threeAmblyomma americanum(L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion

The insulin-like growth factor (IGF) binding proteins (IGFBP) family is the regulatory arm of the IGF signaling system that control mitogenic and anabolic actions of IGF peptide hormones. This study describes cloning and biological characterization of three Amblyomma americanum (L.) (Aam) proteins that show amino-terminal sequence and secondary structure similarity to the IGFBP superfamily. The three molecules here provisionally identified as AamIGFBP-rP1 and short (S) and long (L) AamIGFBP-rP6 are expressed in multiple tick organs and are responsive to tick feeding activity with the former being upregulated and the latter being downregulated. We show that they regulate tick physiological functions that may be related to A. americanum tick feeding success as revealed by RNAi-mediated dual silencing of AamIGFBP-rP6S and AamIGFBP-rP6L or AamIGFBP-rP1 alone, which caused a reduction in blood meal size compared to the controls. Additionally, in the case of AamIGFBP-rP1 silencing, 47% of ticks died while attempting to feed and those that did survive and spontaneously detached from the host failed to lay eggs. Although AamIGFBP-rP6S and AamIGFBP-rP6L show overall identities of 49% and 59%, respectively, to Rhipicephalus microplus C protein, the identity level jumps to ~84% when the comparison is restricted to first 70 amino acids of the mature protein. Similarly, the AamIGFBP-rP1 mature protein is ~72%, 87%, 88% and 92% identical to that of Ixodes scapularis S, R. microplus, R. appendiculatus N and A. variegatum F, respectively. The observed across-tick-species conservation suggests that the three molecules (AamIGFBP-rP1, AamIGFBP-rP6S and AamIGFBP-rP6L) represent target for development of vaccines to protect animals against multiple tick species. The data are discussed with reference to advances in tick molecular biology and the potential of the three proteins as targets for immunizing animals against tick feeding.

Insulin-like growth factor binding protein-2 is a novel mediator of p53 inhibition of insulin-like growth factor signaling

The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP’s, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.

Expression analysis of IGFBP-rP10, IGFBP-like and Mig30 in early Xenopus development

To date, five members of the insulin-like growth factor-binding protein (IGFBP) superfamily have been described in Xenopus laevis. Here, we report the isolation of two new IGFBPs: xIGFBP-rP10, and xIGFBP-like. The proteins share the same domain architecture, and together with Mig30, form a subgroup within the IGFBP superfamily. Temporal expression analysis shows that they are expressed differentially during early development. xIGFBP-rP10 is continuously expressed, whereas Mig30 expression peaks during gastrulation. IGFBP-like is expressed from neurulation onward. The three genes have characteristic spatial expression domains, which overlap in some regions. Both xIGFBP-rP10 and Mig30 are expressed on the dorsal side of the embryo during gastrulation. Later, xIGFBP-rP10 is expressed in the notochord, the floor plate, the somites, and the fin. xIGFBP-like expression is seen primarily in the developing central nervous system and overlaps with Mig30 expression at the end of neurulation in the developing somites and in tail bud stages in the eyes.

IGFBP-3 and IGFBP-10 (CYR61) up-regulation during the development of Barrett's oesophagus and associated oesophageal adenocarcinoma: potential biomarkers of disease risk

Dys-regulation of the insulin-like growth factor (IGF) system increases the risk of a number of malignancies. The aim of this study was to investigate the role of members of the IGF binding protein (IGFBP) superfamily in the development of oesophageal adenocarcinoma (EAC) and their possible use as markers of disease risk. Expression of IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 was assessed using Real-Time-polymerase chain reaction (PCR) and immunohistochemistry in oesophageal tissues from Barrett's oesophagus (BE) patients with and without associated EAC, and in control subjects. IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 mRNA levels were up-regulated in Barrett's (n=17) and tumour tissue of EAC patients (n=18) compared with normal tissue of control subjects without BE or EAC (n=18) (p<0.001). Over-expression of IGFBP-3 and IGFBP-10/CYR61 proteins was observed in Barrett's, dysplastic and tumour tissue of EAC cases (n=47 for IGFBP-10; n=39 for IGFBP-3) compared with adjacent normal epithelium (p<0.050). Notably, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 expression in Barrett's tissue of EAC cases (n=17) was significantly (p<0.001) higher than in Barrett's tissue of BE patients with no sign of progression to cancer (n=15). Overall, the results suggest that members of the IGFBP superfamily are up-regulated during oesophageal carcinogenesis and merit further investigation as markers of EAC risk.

CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily

Department of Cell Biology and Anatomy, University of Miami School of Medicine (G.R.G.), Miami, Florida 33136; Department of Molecular Genetics, University of Illinois College of Medicine (L.F.L.), Chicago, Illinois 60607; Laboratoire d’Oncologie Virale et Moleculaire, UFR de Biochimie, Universite Paris 7 (B.P.), 75005 Paris, France; and INSERM U-515, Hopital Saint Antoine (B.P.), Paris 75012, France

CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily

CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily

CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily

CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily

CCN proteins are distinct from and should not be considered members of the insulin-like growth factor-binding protein superfamily.

Department of Cell Biology and Anatomy, University of Miami School of Medicine (G.R.G.), Miami, Florida 33136; Department of Molecular Genetics, University of Illinois College of Medicine (L.F.L.), Chicago, Illinois 60607; Laboratoire d’Oncologie Virale et Moleculaire, UFR de Biochimie, Universite Paris 7 (B.P.), 75005 Paris, France; and INSERM U-515, Hopital Saint Antoine (B.P.), Paris 75012, France

CCN Proteins Are Distinct from and Should Not Be Considered Members of the Insulin-Like Growth Factor-Binding Protein Superfamily

CCN Proteins Are Distinct from and Should Not Be Considered Members of the Insulin-Like Growth Factor-Binding Protein Superfamily

The insulin-like growth factor-binding protein superfamily

The insulin-like growth factor-binding protein superfamily

The insulin-like growth factor-binding protein (IGFBP) superfamily.

Over the last decade, the concept of an IGFBP family has been well accepted, based on structural similarities and on functional abilities to bind IGFs with high affinities. The existence of other potential IGFBPs was left open. The discovery of proteins with N-terminal domains bearing striking structural similarities to the N terminus of the IGFBPs, and with reduced, but demonstrable, affinity for IGFs, raised the question of whether these proteins were "new" IGFBPs (22, 23, 217). The N-terminal domain had been uniquely associated with the IGFBPs and has long been considered to be critical for IGF binding. No other function has been confirmed for this domain to date. Thus, the presence of this important IGFBP domain in the N terminus of other proteins must be considered significant. Although these other proteins appear capable of binding IGF, their relatively low affinity and the fact that their major biological actions are likely to not directly involve the IGF peptides suggest that they probably should not be classified within the IGFBP family as provisionally proposed (22, 23). The conservation of this single domain, so critical to high-affinity binding of IGF by the six IGFBPs, in all of the IGFBP-rPs, as well, speaks to its biological importance. Historically, and perhaps, functionally, this has led to the designation of an "IGFBP superfamily". The classification and nomenclature for the IGFBP superfamily, are, of course, arbitrary; what is ultimately relevant is the underlying biology, much of which still remains to be deciphered. The nomenclature for the IGFBP related proteins was derived from a consensus of researchers working in the IGFBP field (52). Obviously, a more general consensus on nomenclature, involving all groups working on each IGFBP-rP, has yet to be reached. Further understanding of the biological functions of each protein should help resolve the nomenclature dilemma. For the present, redesignating these proteins IGFBP-rPs simplifies the multiple names already associated with each IGFBP related protein, and reinforces the concept of a relationship with the IGFBPs. Beyond the N-terminal domain, there is a lack of structural similarity between the IGFBP-rPs and IGFBPs. The C-terminal domains do share similarities to other internal domains found in numerous other proteins. For example, the similarity of the IGFBP C terminus to the thyroglobulin type-I domain shows that the IGFBPs are also structurally related to numerous other proteins carrying the same domain (87). Interestingly, the functions of the different C-terminal domains in members of the IGFBP superfamily include interactions with the cell surface or ECM, suggesting that, even if they share little sequence similarities, the C-terminal domains may be functionally related. The evolutionary conservation of the N-terminal domain and functional studies support the notion that IGFBPs and IGFBP-rPs together form an IGFBP superfamily. A superfamily delineates between closely related (classified as a family) and distantly related proteins. The IGFBP superfamily is therefore composed of distantly related families. The modular nature of the constituents of the IGFBP superfamily, particularly their preservation of an highly conserved N-terminal domain, seems best explained by the process of exon shuffling of an ancestral gene encoding this domain. Over the course of evolution, some members evolved into high-affinity IGF binders and others into low-affinity IGF binders, thereby conferring on the IGFBP superfamily the ability to influence cell growth by both IGF-dependent and IGF-independent means (Fig. 10). A final word, from Stephen Jay Gould (218): "But classifications are not passive ordering devices in a world objectively divided into obvious categories. Taxonomies are human decisions imposed upon nature--theories about the causes of nature's order. The chronicle of historical changes in classification provides our finest insight into conceptual revolutions

The Insulin-like Growth Factor Binding Protein Superfamily: New Perspectives

The insulin-like growth factor (IGF) binding proteins (IGFBPs) were initially identified as carrier proteins for IGF-I and IGF-II in a variety of biologic fluids. Their presumed function was to protect IGF peptides from degradation and clearance, increase the half-life of the IGFs, and deliver them to appropriate tissue receptors. The concept of IGFBPs as simple carrier proteins has been complicated, however, by a number of observations: 1) the six IGFBPs vary in their tissue expression and their regulation by other hormones and growth factors; 2) the IGFBPs are subjected to proteolytic degradation, thereby altering their affinities for the IGFs; 3) IGFBP-3 and IGFBP-5, in addition to binding IGFs, also can associate with an acid-labile subunit, thereby increasing further the half-life of the IGFs; 4) in addition to modifying the access of IGF peptides to IGF and insulin receptors, several of the IGFBPs may be capable of increasing IGF action; 5) some of the IGFBPs may be capable of IGF-independent regulation of cell growth; 6) some of the IGFBPs are associated with cell membranes or possibly with membrane receptors; and 7) some of the IGFBPs have nuclear recognition sites and may be found within the nucleus. Additionally, a number of cDNAs identified recently have been found to encode proteins that bind IGFs, but with substantially lower affinities than is the case with IGFBPs. The N-terminal regions of the predicted proteins are structurally homologous to the classic IGFBPs, with conservation of the cysteine-rich region. These observations suggest that these low-affinity binders are members of an IGFBP superfamily, capable of regulating cell growth by both IGF-dependent and IGF-independent mechanisms.insulin-like growth factor, insulin-like growth factor binding proteins.

Binding properties and distribution of insulin-like growth factor binding protein-related protein 3 (IGFBP-rP3/NovH), an additional member of the IGFBP Superfamily.

The protein product of the novH oncogene, a member of the CCN family, is structurally related to the insulin-like growth factor (IGF) binding proteins (IGFBPs). We have characterized aspects of structure, function, and distribution of this protein, which, as IGFBP-related protein 3 (IGFBP-rP3), is a proposed member of the IGFBP Superfamily. Affinity cross-linking experiments performed with baculovirus synthesized recombinant human IGFBP-rP3 established that rhIGFBP-rP3 binds IGF-I, IGF-II, and insulin with low affinity. Specificity of binding was shown by competitive cross-linking experiments; binding to IGF-I and -II was also demonstrated by nondenaturing Western ligand blots. Northern blot analysis indicated the presence of IGFBP-rP3 messenger RNA (mRNA) in a broad range of human tissues. Western immunoblotting studies, using a polyclonal rabbit anti-rhIGFBP-rP3 antibody, demonstrated that IGFBP-rP3 protein is synthesized in vitro by several breast and prostate cancer cell lines: Hs578T, PC3, P69, and LNCaP cells. Western immunoblotting studies of human biological fluids identified that IGFBP-rP3 was present in normal serum, pregnancy serum, serum from patients with growth hormone receptor deficiency, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and follicular fluid, while IGFBP-rP3 fragments were identified in cerebrospinal fluid, amniotic fluid, and prepubertal and pubertal urine samples. Our studies demonstrate that IGFBP-rP3 exhibits IGF binding, albeit at low affinity, and IGFBP-rP3 thus merits inclusion in the IGFBP Superfamily. The low affinity IGF binding suggests that IGFBP-rP3 may act primarily independently of the IGFs. The synthesis of IGFBP-rP3 by several malignant cell lines and its presence in human biological fluids suggest that this protein possesses other interesting roles, potentially in cell growth regulation.

Insulin-like growth factor binding proteins: a proposed superfamily.

The conventional concept is that the insulin-like growth factor binding proteins (IGFBPs) are cysteine-rich proteins, with conserved N- and C-domains, that are capable of binding insulin-like growth factors (IGFs) with high affinity. This dogma was recently challenged by the discovery of a group of cysteine-rich proteins that share important structural similarities with the IGFBPs, but have demonstrably lower affinity for IGFs. It is therefore proposed that these IGFBP-related proteins (IGFBP-rPs) and the IGFBPs constitute an IGFBP superfamily. We speculate that the IGFBP superfamily is derived from an ancestral gene/protein that was critically involved in the regulation of cell growth and was capable of binding IGF peptides. Over the course of evolution, some members (IGFBPs) evolved into high-affinity IGF binders and others (IGFBP-rPs) into low-affinity IGF binders, thereby conferring on the IGFBP superfamily the ability to influence cell growth by both IGF-dependent and IGF-independent means.

Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-beta in Hs578T human breast cancer cells.

Connective Tissue Growth Factor (CTGF) is a cysteine-rich peptide involved in human atherosclerosis and fibrotic disorders such as scleroderma. CTGF has considerable N-terminal sequence similarity with the insulin-like growth factor binding proteins (IGFBPs), including preservation of cysteines, and has been postulated to be a member of the IGFBP superfamily. Indeed, recent studies have shown that baculovirus generated CTGF, a secreted 38-kDa protein, binds IGFs in a specific manner, leading to the provisional renaming of CTGF as IGFBP-8 (or IGFBP-rP2). With immunoprecipitation and immunoblotting, using polyclonal anti-IGFBP-rP2 antibody generated against recombinant human IGFBP-rP2bac, IGFBP-rP2 can be identified in the serum-free conditioned media of Hs578T human breast cancer cells, as well as in various human biological fluids, such as normal sera, pregnancy sera, and cerebrospinal, amniotic, follicular and peritoneal fluids. Glycosylation studies with endoglycosidase F reveal that endogenous human IGFBP-rP2 is a secreted, glycosylated, approximately 32-38-kDa protein with 2-8-kDa of N-linked sugars and a 30-kDa core. There are 18- and 24-kDa proteins that appear to be IGFBP-rP2 degradation products. In Hs578T human breast cancer cells, transforming growth factor (TGF)-beta 2, a potent growth inhibitor for these cells, upregulates IGFBP-rP2 mRNA and protein levels. Expression of Hs578T IGFBP-rP2 is significantly increased by TGF-beta 2 treatment in a dose-dependent manner, with 2.5- and 6-fold increases in mRNA and protein levels, respectively, at a TGF-beta 2 concentration of 10 ng/ml. Our studies indicate that IGFBP-rP2 appears to be an important endocrine factor, and one of the critical downstream effectors of the critical downstream effectors of TGF-beta, similar to the role of IGFBP-3 in TGF-beta-induced growth inhibition in human breast cancer cells.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40-60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an "IGFBP motif' (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1-6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

Biological roles of insulin-like growth factor binding proteins (IGFBPs)

The insulin-like growth factor binding protein (IGFBP) family is a critical component of the insulin-like growth factor (IGF) system which regulate the biological actions of the IGFs and may also be capable of IGF-independent actions. To date, seven distinct IGFBPs have been described. Among these IGFBPs, IGFBPs-1-6 bind IGFs with high affinity, while only IGFBP-7 binds with low affinity. Recently, we have demonstrated that connective tissue growth factor (CTGF) also binds IGFs with low affinity, suggesting that a family of low-affinity IGFBPs, distinct from the high-affinity members, may exist, and together these constitute an IGFBP superfamily. IGFBPs have various biological roles. IGFBPs act not only as a carrier proteins, but also as a modulators of IGF actions by involving in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, some IGFBPs (IGFBPs-1, -3, and -5) appears to have intrinsic activity independent of IGFs. This review will focus on recent studies on the biological roles of IGFBPs in IGF-dependent and IGF-independent modes.