Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.     

Hybrid receptors formed by insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) have low insulin and high IGF-1 affinity irrespective of the IR splice variant

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.     

Nuclear translocation of insulin receptor substrate-1 by the insulin receptor in mouse embryo fibroblasts

Translocation of the insulin receptor substrate-1 (IRS-1) to the nuclei has been reported to occur in cells stimulated by insulin-like growth factor-1 (IGF-I) or expressing certain viral and cellular oncogenes. We show here that insulin can also induce nuclear translocation of IRS-1 in mouse embryo fibroblasts (MEF), that do not express the type 1 insulin-like growth factor receptor (IGF-IR). Only the A isoform of the insulin receptor (IR) can induce IRS-1 nuclear translocation, which is significant when the receptor is over-expressed. At physiological receptor levels, translocation occurs only in a fraction of cells, and only at high concentrations of ligand.     

Insulin and insulin-like growth factor II differentially regulate endocytic sorting and stability of insulin receptor isoform A

The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3-10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R-/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R-/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R-/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyr(B26)]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli.     

Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.     

Insulin-like growth factor (IGF)/somatomedin receptor subtypes: structure, function, and relationships to insulin receptors and IGF carrier proteins

The insulin-like growth factors IGF-I and IGF-II are mitogenic polypeptides with a high degree of chemical homology. Two distinct subtypes of receptors for the IGFs have been identified on the basis of structure and binding specificity. Type I IGF receptors bind IGF-I with equal or greater affinity than IGF-II, and also bind insulin with a low but definite affinity. They are structurally homologous to insulin receptors, containing disulfide-linked a-subunits that bind the peptides and beta-subunits that have intrinsic tyrosine-specific kinase activity. Type II IGF receptors typically bind IGF-II with greater affinity than IGF-I, and do not interact with insulin. They consist of a single polypeptide and lack tyrosine kinase activity. Because of the extensive cross-reactivity of IGF-I and IGF-II with both type I and type II receptors, we believe that potentially either receptor may mediate the biological responses of either peptide. Type I IGF receptors have been shown to mediate the mitogenic effects of the IGFs in some cell types. Whether type II IGF receptors mediate the same or different functions remains to be elucidated.     

Expression and function of insulin-like growth factor receptors on anti-CD3-activated human T lymphocytes.

The pattern of expression of receptors for insulin-like growth factors (IGF-I and IGF-II) and insulin was studied on monocyte-depleted human peripheral blood T cells activated via anti-CD3. Binding assays demonstrated the sequential appearance of receptors for IGF-I, IGF-II, and insulin on activated T cells. IGF-IR appeared early, their expression reaching maximum levels at or before the peak of cellular proliferation. IGF-IIR expression generally followed that of the IGF-IR and was more transient, with increases and decreases in expression paralleling the rise and decline of cellular proliferation. Insulin receptor expression remained low throughout the activation time course. The identity of the IGFR on anti-CD3-activated T cells was confirmed in affinity cross-linking experiments. These data demonstrated a 135,000 Mr peptide that specifically binds radiolabeled IGF-I and corresponds to the alpha subunit of the type I IGF-IR, and a 260,000 Mr peptide that specifically binds radiolabeled IGF-II and corresponds to the type II IGFR. We have additionally found that IGF-I and IGF-II (in nanomolar concentrations) produce as much as a threefold enhancement of T cell proliferation early in the activation process, correlating with the early appearance of IGF-IR. The effect of both IGF appeared to be mediated through the type I receptor, since an antibody (alpha IR3), which blocks binding to the alpha subunit of this receptor, inhibited enhancement by up to 83%. Furthermore, we have found expression of IGF-IR on T cells after activation to be associated with both CD4+ and CD8+ T cell subpopulations. These observations provide a foundation for investigating the contribution of IGF in regulating T cell proliferation, differentiation, and effector function.     

LONGTMR3IGF-I as a more potent alternative to insulin in serum-free culture of HEK293 cells

LONG^TMR³IGF-I, an analogue of insulin-like growth factor (IGF)-I, was specifically engineered for use in biopharmaceutical protein production in mammalian cells. LONG^TMR³IGF-I is capable of supporting the growth and survival of Chinese hamster ovary cells in serum-free media at concentrations at least 200-fold lower than required for insulin. LONG^TMR³IGF-I also acts as a more potent growth and survival factor than either insulin or native IGF-I in SF culture of human embryonic kidney (HEK293) cells. To investigate the basis of the enhanced potency of LONG^TMR³IGF-I we have examined the mechanism of action of these mitogens in HEK293 cells. All mitogens tested were found to activate the TypeI IGF receptor (IGF-IR) and insulin receptor (IR) in a dose-responsive manner. However, the level of activation of both receptors after stimulation with LONG^TMR³IGF-I, at lower concentrations, was greater than with either insulin or IGF-I. The greater potency of LONG^TMR³IGF-I in activating the IR, despite having a low affinity for IRs, suggests the presence of heterotetrameric IGF-IR/IR dimers. Interestingly, the decrease in IGF-IR activation at higher concentrations of LONG^TMR³IGF-I suggests that the dose-response curve may be bell-shaped.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.     

Primary structure of a putative receptor for a ligand of the insulin family

Nucleotide sequence analysis of human and guinea pig genomic DNA encoding a new member of the insulin receptor (IR) family revealed that the predicted primary structure of this IR-related protein is as similar to the IR and insulin-like growth factor (IGF) I receptor as the IR and IGF-IR are to each other. The conservation of this IR-related sequence among mammals and with the IR and IGF-IR suggests that this IR-related protein is a novel receptor for insulin, IGF-I, IGF-II, or an as yet unidentified peptide hormone or growth factor belonging to the insulin family.     

Insulin-like growth factor-binding protein-3 activates a phosphotyrosine phosphatase. Effects on the insulin-like growth factor signaling pathway

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln(3),Ala(4),Tyr(15),Leu(16)]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42(MAPK) activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

Differential regulation of signaling pathways for insulin and insulin-like growth factor I.

The insulin receptor (IR) and the insulin-like growth factor receptor I (IGF-IR) have different functions in cell growth, apoptosis, differentation, and transformation. Although some of these differences may be explained by the relative level of receptor expression and receptor structure (alpha and beta subunits), they may also be attributed to differences in intracellular signals generated by insulin and IGF-I. The presence of hybrid receptors (IR alphabeta subunits and IGF-IR alphabeta subunits) making up the heterotetramers has added a new dimension to our understanding of the functional roles of these receptors. However, to date the results of efforts to understand the differences between these two closely related receptors have indicated mostly similarities. For example, both receptors utilize IRS-1/IRS-2 and Shc as immediate downstream adaptors, leading to activation of the Ras, Raf, ERK kinases and PI-3 kinase pathways. We have used the yeast two hybrid system to identify proteins which bind to the activated IGF-IR but not to the IR. The cytoplasmic domain of the IGF-IR was used to screen a human fetal brain library and two isoforms of the 14-3-3 family were identified. 14-3-3 proteins are a highly conserved family of proteins which have recently been shown to interact with other components of the mitogenic and apoptotic signaling pathways, including Raf, BAD, Bcr/Bcr-Abl, middle-T antigen, Ksr, PKC, PI-3 kinase, ASK1 kinase, and cdc25C phosphatase. We also identified human Grb10, an adaptor protein with SH2 domain associated with the IGF-IR beta subunit. Smith's laboratory showed that Grb10 preferentially binds to the IR in intact cells. Using the interaction trap screen (active cytoplasmic domain of the IGF-IR) 55PIK and SOCS-2 proteins were also identified. However, 55PIK and SOCS-2 also interact with the IR in the yeast two hybrid system. These studies raise the possibility that 14-3-3 and Grb10 may play a role in insulin and IGF-I signal transduction and may underlie the observed differences.     

The role of insulin receptors and IGF-I receptors in cancer and other diseases

There is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression of human cancer. In particular, the insulin-like growth factor receptor (IGF-IR), a tyrosine kinase receptor for IGF-I and IGF-II, has been well documented in cell culture, animal studies, and humans to play a role in malignant transformation, progression, protection from apoptosis, and metastasis. In addition, the hormone insulin (which is very closely related to the IGFs) and its tyrosine kinase receptor (the IR, which is very closely related to the IGR-IR) have been documented both in vitro and in vivo to play a key role in cancer biology. Indeed, several epidemiological studies have shown that insulin resistance status, characterized by hyperinsulinaemia, is associated with an increased risk for a number of malignancies, including carcinomas of the breast, prostate, colon and kidney. Recent data have elucidated some molecular mechanisms by which IR is involved in cancer. IR is over-expressed in several human malignancies. Interestingly, one of the two IR isoform (IR-A) is especially over-expressed in cancer. IR-A is the IR foetal isoform and has the peculiar characteristic to bind not only insulin but also IGF-II. In addition, the IR contributes to formation of hybrid receptors with the IGF-IR (HR). By binding to hybrid receptors, insulin may stimulate specific IGF-IR signalling pathways. Over-expression of IR-A is, therefore, a major mechanism of IGF system over-activation in cancer. In this respect, IR-A isoform and hybrid receptors should be regarded as potential molecular targets, in addition to IGF-IR, for novel anti-cancer therapy. These findings may have important implications for both the prevention and treatment of common human malignancies. They underline the concept that hyperinsulinaemia, associated with insulin resistance and obesity, should be treated by changes in life style and/or pharmacological approaches to avoid an increased risk for cancer. Moreover, native insulin and insulin analogue administration should be carefully evaluated in terms of the possible increase in cancer risk.     

Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells

Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.     

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.     

Role of the insulin-like growth factor system on an estrogen-dependent cancer phenotype in the MCF-7 human breast cancer cell line

We previously established that exposure of the estrogen receptor (ER) positive MCF-7 human breast cancer cell line to 17-β-estradiol (E₂) results in the post-confluent development of multilayered cellular aggregates (foci) which is consistent with the in vivo cancer phenotype of uncontrolled cellular proliferation. In this investigation, the interaction between the insulin-like growth factor receptor (IGF-IR) and ER-signaling systems in regard to post-confluent focus development was studied. We demonstrated that focus development requires the presence of E₂ and insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II), as well as intact ER and IGF-IR.

Focus development in MCF-7 cultures, which occurs only after formation of a confluent monolayer, coincides with E₂ regulation of key members of the IGF-signaling system such as IGF-IR, IGF-II, insulin receptor substrate 1 (IRS-1), and insulin-like growth factor binding protein 3 (IGFBP-3), as demonstrated by real-time polymerase chain reaction (PCR). To establish the relevancy of an intact IGF-signaling system for foci formation, we generated stable clones from MCF-7 with IGF-IR suppressed by siRNA. Results from these studies implicate signaling through the IGF-IR to be an integral requirement for E₂-dependent post-confluent proliferation and focus formation. In summary, these studies establish the interactive roles of IGFs and E₂ in the post-confluent development of foci, and will allow subsequent identification of targets for therapeutic intervention in the control and treatment of estrogen-dependent breast cancer.     

Affinity modulation of human placental insulin and insulin-like growth factor receptors by lectins

The ability of plant lectins to modify the interactions of the insulin receptor (IR) and insulin-like growth factor (IGF) receptors (IGFRs) with their ligands was investigated. The lectins profoundly affected the competition-binding curves for (125)I-labelled IGF-I and insulin, causing an increase in the affinity of placental IGF1R and IR towards their ligands. This increment was of such a magnitude that it could affect the receptors' specificity towards these ligands. The lower the ligand concentration, the greater was the lectin-induced affinity shift, which suggests potential physiological significance of the effect. The affinity modulation occurred in a lectin-specific and dose-dependent manner. In contrast to IGF1R and IR, the binding of (125)I-labelled IGF-II to its receptors resisted lectin modulation. Here we provide evidence of the possibility of external modulation of the affinity of placental IGF1R and IR via interactions of the receptors' carbohydrate moieties with lectins. The existence of modulators that would selectively inhibit or enhance the binding of IGFs or insulin to their corresponding receptors may have important implications for placental cell responses to these molecules.     

Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination

The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.     

Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor

The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.