Janet L. Martin

Binding proteins for the insulin-like growth factors: Structure, regulation and function

Binding proteins for the insulin-like growth factors (IGF-I and IGF-II) are increasingly being recognized as modulators of IGF actions in both inhibitory and stimulatory ways. At least three distinct classes of binding protein are thought to exist, differing in their primary structures and binding characteristics, although all are able to bind both IGF-I and IGF-II. This review outlines the purification and characterization of the binding proteins that have been identified to date, and describes the regulation of their production and of their levels in the circulation. Current views on their potential biological roles are also discussed.

Growth hormone-dependent insulin-like growth factor (IGF) binding protein from human plasma differs from other human IGF binding proteins

A growth hormone-dependent binding protein for insulin-like growth factors (IGF-I and IGF-II) has been isolated from human plasma. Analyzed on SDS gels, the preparation contained a major protein band of 53 kDa, and a minor band of 47 kDa. After transfer to nitrocellulose, both species bound iodinated IGF-I, and could be detected using an antibody raised against the purified preparation. In contrast, an IGF binding protein purified from human amniotic fluid bound IGF-I but was not detectable immunologically. The amino acid comparison of the plasma binding protein preparation was different from that reported for amniotic fluid and HEP G2 hepatoma proteins, and the unique amino-terminal sequence, Gly-Ala-Ser-Ser-Ala-Gly-Leu-Gly-Pro-Val-, was different from that of the amniotic fluid and hepatoma proteins. This study indicates that the growth hormone-dependent IGF binding protein of human plasma is structurally and immunologically distinct from other IGF binding proteins.

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.

IGF Binding Proteins as Modulators of IGF Action

The insulin-like growth factors (IGFs) are able to modulate an extraordinarily wide variety of biological actions. First described as stimulators of proteoglycan biosynthesis in cartilage (1,2), IGF peptides (then known as somatomedins or nonsuppressible insulin-like activity) were soon recognized as having insulin-like stimulatory effects on the cellular uptake of glucose and other substrates (3),and were shown to increase protein, RNA, and DNA synthesis (1,4–6) in a variety of tissues. Subsequently IGFs have been found to act as positive regulators of an extensive range of differentiated functions in numerous tissues, often acting in synergy with other trophic or stimulatory factors, both hormones (e.g., adrenocorticotropic hormone [ACTH], follicle-stimulating hormone [FSH]) and growth factors (e.g.,platelet-derived growth factor, fibroblast growth factor) (7). Not all IGF actions, however, are stimulatory, and their ability to block lipolysis in adipocytes (8) and to inhibit adenylate cyclase activity (9,10) was recognized more than two decades ago. Among other important inhibitory effects are the attenuation of pituitary growth hormone (GH) secretion (11) and the prevention of apoptosis (12),a significant component of the “survival factor” role of the IGFs.

Regulation and Actions of Insulin-Like Growth Factor Binding Protein-3

Binding proteins for the insulin-like growth factors (IGFs), first described 15 years ago, are now recognized as playing important roles as carriers of IGFs in the circulation, and modulators of IGF actions on target cells 1. Three distinct structural classes of IGF binding protein (IGFBP) have been identified on the basis of cDNA cloning 2–6, and at least two other structurally distinct proteins appear to exist 7–9. The predominant circulating IGFBP is an acid-stable glycoprotein with the systematic name IGFBP-3 . This paper will discuss recent studies, mainly from the authors’ laboratory, which relate to the dual roles of IGFBP-3, as part of the growth hormone (GH) dependent, high molecular weight ternary IGF complex in serum, and as local cell regulators of mitogenic actions of the IGFs.

Abstract P5-07-05: Insulin-like growth factor binding protein-3 is a key component of the breast cancer cell response to DNA-damaging therapy

Introduction. Breast cancer cells frequently develop resistance to DNA-damaging therapies, such as radiation and the cytotoxic drugs doxorubicin and etoposide, and activation of the epidermal growth factor receptor (EGFR) may play an important role in this process. However many estrogen receptor (ER)-negative breast tumors respond poorly to DNA-damaging drugs even when combined with an EGFR inhibitor. EGFR can modulate the repair of DNA double strand breaks (DSB) by non-homologous end-joining (NHEJ), by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In previous studies we have shown that the growth-regulatory protein, insulin-like growth factor binding protein-3 (IGFBP-3), can translocate to the nucleus, is a substrate for DNA-PKcs, and can transactivate EGFR. The aim of this study was to delineate the role of IGFBP-3 in the response of breast cancer cells to DSB-inducing chemotherapeutic agents. Results. In the triple-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3, and of their complex as measured by coimmunoprecipitation (coIP), was enhanced by exposure to etoposide or doxorubicin, peaking 2 h after etoposide treatment. This effect was blocked by the EGFR kinase inhibitor gefitinib. Concomitantly, the co-location of EGFR-IGFBP-3 complexes with lipid rafts, visualized by proximity ligation assay (PLA) and confocal microscopy, decreased in response to drug treatment, consistent with the loss of this complex from the plasma membrane. Similar to the nuclear DNA-PKcs-EGFR complex, the nuclear DNA-PKcs-IGFBP-3 complex (seen by both coIP and PLA) was greatest 4 h after treatment. DNA-PKcs activation at serine-2056, required for the repair of DNA DSB and stimulated by etoposide treatment, was decreased by IGFBP-3 downregulation using two separate siRNAs, and this treatment also attenuated the enhanced DNA-PKcs-EGFR complexes seen in response to etoposide. Collectively these data suggest that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy through its interactions with EGFR and DNA-PKcs. Conclusion. IGFBP-3 co-translocation to the nucleus of breast cancer cells and its complex formation with DNA-PKcs and EGFR in response to DNA damage demonstrate its previously unrecognized involvement in the regulation of DNA DSB repair by NHEJ. These novel findings suggests the possibility of a therapeutic approach for sensitizing ER-negative breast cancers to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3. Supported by the Australian Research Council. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-05.