Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease.

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.

Structural basis of the activation of type 1 insulin-like growth factor receptor.

Type 1 insulin-like growth factor receptor (IGF1R) is a receptor tyrosine kinase that regulates cell growth and proliferation, and can be activated by IGF1, IGF2, and insulin. Here, we report the cryo-EM structure of full-length IGF1R-IGF1 complex in the active state. This structure reveals that only one IGF1 molecule binds the Γ-shaped asymmetric IGF1R dimer. The IGF1-binding site is formed by the L1 and CR domains of one IGF1R protomer and the α-CT and FnIII-1 domains of the other. The liganded α-CT forms a rigid beam-like structure with the unliganded α-CT, which hinders the conformational change of the unliganded α-CT required for binding of a second IGF1 molecule. We further identify an L1-FnIII-2 interaction that mediates the dimerization of membrane-proximal domains of IGF1R. This interaction is required for optimal receptor activation. Our study identifies a source of the negative cooperativity in IGF1 binding to IGF1R and reveals the structural basis of IGF1R activation.

Physiological concentrations of insulin induce cellular desensitization to the mitogenic effects of insulin-like growth factor I.

Insulin and insulin-like growth factor I (IGF-I) are structurally related peptides capable of stimulating a variety of metabolic and mitogenic processes. In this study, we investigated the interaction between these peptides and their receptor-mediated pathways in an untransformed cell line. Cultured bovine fibroblasts specifically bound IGF-I and insulin, and each peptide could stimulate DNA synthesis and cell replication through its own receptor. Preincubation of bovine fibroblasts with concentrations of insulin that did not bind to the IGF-I receptor resulted in complete but reversible cellular desensitization to IGF-I-stimulated mitogenesis. Preincubation with as little as 0.1 nM insulin was sufficient to inhibit subsequent IGF-I action. Various insulin analogs produced desensitization in direct relation to the affinity of the insulin for the insulin receptor. Desensitization required > 4 h of cell exposure to insulin and was blocked in the presence of cycloheximide. Neither serum-stimulated mitogenesis nor IGF-I-stimulated glucose uptake were affected by insulin pretreatment. 125I-labeled IGF-I affinity cross-linking experiments indicated that preincubation with insulin did not affect labeling of the 130,000-M(r) alpha-subunit of the IGF-I receptor, but was associated with the loss of IGF-I- and insulin-inhibitive bands at M(r) = 100,000, 85,000, 58,000, and 34,000. These studies suggest that insulin, via interaction with insulin receptors on bovine fibroblasts, regulates IGF-I action at a step distal to IGF-I receptor binding and are consistent with desensitization occurring at an intracellular step in the mitogenic pathway shared by insulin and IGF-I.

[Prokaryotic expression, purification and activity detection of the extracellular and intracellular domains of human IGF1R beta subunit].

OBJECTIVE: To construct the prokaryotic expression vectors of extracellular domain (ß-ED) and intracellular protein kinase domain (ß-PKD) of insulin-like growth factor 1 receptor beta (IGF1R-ß) subunit, purify the fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD, and detect their activities. METHODS: Human GST-IGF1R ß-ED and GST-IGF1R ß-PKD coding regions were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD were expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2 (ERBB2)-driven cell motility (MEMO) protein were identified by GST pull-down assay. RESULTS: GST-IGF1R ß-ED and GST-IGF1R ß-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R ß-PKD could interact with MEMO in vitro. CONCLUSION: GST-IGF1R ß-ED and GST-IGF1R ß-PKD were successfully cloned and purified. In addition, GST-IGF1R ß-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.

Functional characterization of hybrid receptors composed of a truncated insulin receptor and wild type insulin-like growth factor 1 or insulin receptors.

To assess the characteristics of hybrid receptors composed of one kinase-inactive alpha beta-insulin half-receptor and one endogenous alpha beta-insulin-like growth factor 1 (IGF-1) or insulin half-receptor, a cell line expressing an insulin receptor truncated by 365 amino acids (HIR delta 978) was studied, which lacks most of the cytoplasmic beta-subunit. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions revealed four distinct receptor species: endogenous receptors, the more rapidly migrating HIR delta 978 homodimer, and two intermediate species representing HIR delta 978/IGF-1 hybrid receptors and HIR delta 978/IR hybrid receptors. In vivo ligand-binding affinity of the hybrid receptors was studied by receptor-ligand cross-linking, and the delta 978/IGF-1R hybrid receptor was found to have a high affinity for IGF-1, whereas its affinity for insulin was low. Autophosphorylation studies of lectin-purified receptors revealed that neither the HIR delta 978 holoreceptor nor the hybrid receptors underwent autophosphorylation in response to either ligand, despite the presence of intact IGF-1 or insulin half-receptors in the hybrids. Neither hybrid receptor underwent ligand-induced endocytosis, as assessed with the bioactive photoaffinity probes B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin and N-epsilon B28-monoazidobenzoyl-IGF-1. In conclusion, the HIR delta 978/IGF-1R hybrid receptor has a high in vivo affinity for IGF-1 but not for insulin. Neither the delta 978/IGF-1R nor the delta 978/IR hybrids undergo autophosphorylation or ligand-induced endocytosis in response to either ligand, indicating that intramolecular trans-, rather than cis-, signal transduction is important in mediating autophosphorylation and endocytosis.

Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system.

Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic beta domain (wild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C-SH2, N-SH2, C-SH2, SH3 + N-SH2). The results of his3 activation indicated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta. Interaction of p85 and N+C-SH2 with IR beta (delta C-43) or IGF-1R beta(delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF-1R. Activation of lacZ upon interaction of p85 with IR beta(delta C-43) was 4-fold less as compared to IR beta. This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased (2-fold) for IGF-1R beta (delta C-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway.

Localization of [125I]IGF-I binding on the ovine pars tuberalis.

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin-stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF-I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF-I may influence the function of the ovine pars tuberalis the presence of receptors for IGF-I has been investigated. Using in vitro autoradiography specific [125I]IGF-I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [125I]IGF-I with a mean dissociation constant (Kd) of 0.5 +/- 0.1 nM (n = 4). Competition studies revealed a rank order of potency of IGF-I > IGF-II > > > insulin, in displacing [125I]IGF-I binding, indicative of a mixed population of IGF-I and IGF-II/mannose-6-phosphate receptors and insulin-like growth factor binding proteins (IGFBPs). Cross-linking of [125I]IGF-I to pars tuberalis membrane homogenates and analysis by SDS-PAGE under reducing conditions confirmed the presence of both IGF-I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the beta-sub-unit of the IGF-I receptor, was increased in the presence of IGF-I, indicating the existence of functional IGF-I receptors on the ovine pars tuberalis. In contrast in the rat [125I]IGF-I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.

Insulin-like growth factor-I (IGF-I) dependent phosphorylation of the IGF-I receptor in MG-63 cells.

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63. by acting through the IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of the IGF-I receptor in this cell line. Serum starved MG-63 cells were metabolically labeled with [32P]orthophosphoric acid and the cells were treated with IGF-I. Phosphotyrosine containing proteins were immunoprecipitated from the cell lysates with antiphosphotyrosine-Agarose and eluted with phenyl phosphate. Further immunoprecipitation with IGF-I receptor monoclonal antibodies (alpha IR-3, 18E9) and analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography demonstrated IGF-I dependent autophosphorylation of the IGF-I receptor. Phosphoamino acid analysis of the IGF-I receptor beta subunit and the observation that antiphosphotyrosine-Agarose did not immunoprecipitate [35S]methionine-labeled receptor from unstimulated cells, demonstrated that in the absence of IGF-I, the receptor was not phosphorylated on tyrosine residues. Western blotting of cell lysates with a monoclonal phosphotyrosine antibody did not identify the IGF-I receptor or pp185 but demonstrated IGF-I dependent phosphorylation on tyrosine residues in three other proteins, p110, p70 and p40.

Characterization of type I IGF receptor and IGF-I mRNA expression in cultured human and bovine glomerular cells.

Glomerular hypertrophy is reported in several endocrine disorders such as acromegaly and diabetes mellitus, where abnormalities of growth hormone and insulin-like growth factor (IGF-I) have been reported. In the present report, we have cultured bovine and human glomerular endothelial cells, and bovine glomerular epithelial and mesangial cells, and characterized the expression of IGF-I mRNA and its receptor in these cells. High affinity, specific receptors for IGF-I were identified in all three types of cells by radioreceptor assays. Receptor number (Ro) derived by Scatchard analysis revealed an unusually high number of Type I IGF receptors, approx. 1.2 x 10(5) receptors/cell in glomerular endothelial cells. Affinity crosslinking studies and immunoprecipitation with antibodies against the Type I IGF receptor identified the alpha-subunit of the IGF-I receptor as having a molecular mass of 140 kDa. Biologically, IGF-I was more potent than insulin or IGF-II in stimulating DNA synthesis in glomerular endothelial cells. Northern blot analysis showed that glomerular and aortic endothelial cells expressed IGF-1 mRNA of 1.7 kb. In contrast, renal glomeruli showed several IGF-1 mRNAs of 7.5, 1.7 and 1.2 kb. Thus, the demonstration of both a prepondence of Type I IGF receptors coupled with the growth promoting effects of IGF-I in glomerular endothelial and epithelial cells, as well as the local production of IGF-I mRNA suggests that IGF-I serves an important role as an autocrine or paracrine regulator of the growth of renal glomeruli.

Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins.

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-receptor kinase is enhanced in placentas from non-insulin-dependent diabetic women with large-for-gestational-age babies.

The function of insulin receptor and IGF-1 receptor was investigated in placentas from 10 healthy control mothers, 8 diabetic mothers with appropriate-for-gestational-age babies (AGA group) and 9 diabetic mothers with large-for-gestational-age babies (LGA group). None of the diabetic mothers were obese before pregnancy; their blood glucose was well controlled during pregnancy and glycosylated HbA1c was 6.52 +/- 0.71% (M +/- S.E.). Insulin and IGF-1 receptors were partially purified from placentas using wheat germ agglutinin chromatography. The insulin-binding capacity was significantly increased in both the AGA and the LGA groups compared to the control, whereas the IGF-1 binding capacity was similar in the three groups. Autophosphorylation studies were performed with partially purified receptors equalized for similar binding capacity, then immunoprecipitated with anti-insulin receptor antibody or anti-IGF-1 receptor antibody. Insulin-stimulated 32P-incorporation into the insulin receptor beta-subunit was increased by 133% in the LGA group versus the control, whereas incorporation in the AGA group was equivalent to the control. Insulin-stimulated tyrosine kinase activity of the receptor preparation for histone H2B phosphorylation was also significantly increased in the LGA group compared to the control. 32P-incorporation into beta-subunit IGF-1 receptor and IGF-1-stimulated tyrosine kinase activity did not show any significant differences among the three groups. The data in the present study suggest that elevated insulin receptor kinase might be involved in fetal overgrowth in diabetic mothers.

Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue.

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Fine details of IGF-1R activation, inhibition, and asymmetry determined by associated hydrogen /deuterium-exchange and peptide mass mapping.

The structural features of the asymmetric activated states of the insulin receptor family are still poorly understood. We investigated hydrogen/deuterium (H/D)-exchange within the extracellular domain of the type-I insulin-like growth factor receptor (IGF-1R) in the absence and presence of IGF-1 (active state) and in the presence of antibody inhibitors (inactive state). Near complete coverage of the 210 kDa receptor sequence was obtained by mass mapping of proteolytically derived peptides at all H/D-exchange time points. The data provide details regarding solvent exposure and dynamics across the extracellular region as well as conformational changes induced by activation or inactivation. Multiple peptides, distant in structure, individually demonstrated two distinct H/D-exchange rates, suggesting that each of these peptides exists in two separate environments in IGF-1R. The dual-exchange behavior of these peptides was enhanced on ligand binding and eliminated on inhibitor binding, clearly associating these regions with active state asymmetry and enabling them to serve as reporters of receptor activity.

Simultaneous protein expression and modification: an efficient approach for production of unphosphorylated and biotinylated receptor tyrosine kinases by triple infection in the baculovirus expression system.

Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin-biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies.

Insulin is a potent myeloma cell growth factor through insulin/IGF-1 hybrid receptor activation.

Insulin and insulin growth factor type 1 (IGF-1) and their receptors are closely related molecules, but both factors bind to the receptor of the other one with a weak affinity. No study has presently documented a role of insulin as a myeloma growth factor (MGF) for human multiple myeloma cells (MMCs), whereas many studies have concluded that IGF-1 is a major MGF. IGF-1 receptor (IGF-1R) is aberrantly expressed by MMCs in association with a poor prognosis. In this study we show that insulin receptor (INSR) is increased throughout normal plasma cell differentiation. INSR gene is also expressed by MMCs of 203/206 newly diagnosed patients. Insulin is an MGF as potent as IGF-1 at physiological concentrations and requires the presence of insulin/IGF-1 hybrid receptors, stimulating INSR(+)IGF-1R(+) MMCs, unlike INSR(+)IGF-1R(-) or INSR(-)IGF-1R(-) MMCs. Immunoprecipitation experiments indicate that INSR is linked with IGF-1R in MMCs and that insulin induces both IGF-1R and INSR phosphorylations and vice versa. In conclusion, we demonstrate for the first time that insulin is an MGF as potent as IGF-1 at physiological concentrations and its activity necessitates insulin/IGF-1 hybrid receptor activation. Further therapeutic strategies targeting the IGF/IGF-1R pathway have to take into account neutralizing the IGF-1R-mediated insulin MGF activity.

Immunohistochemical localization of insulin-like growth factor-I receptor (IGF-IR) in the developing and mature rat testes.

It has been suggested that insulin-like growth factor-I (IGF-I) plays an important role in the regulation of spermatogenesis in the testes. Its signal is mediated predominantly by the IGF-I receptor (IGF-IR). Signalling through IGF-IR has been shown to have a potent survival function. IGF-IR, a transmembrane tyrosine kinase, is widely expressed across many cell types. In this study, we demonstrated the distribution of IGF-IR in testes of differently aged rats. Anti-IGF-IR is a rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus of the IGF-IR of human origin. Testicular specimens were fixed in Bouin's solution and embedded in paraffin. The paraffin-embedded sections were processed for standard immunohistochemistry by the labelled streptavidin-biotin technique. At postnatal day 19, IGF-IR immunoreactivity was seen moderately in spermatogonia, and slightly both in leptotene and zygotene primary spermatocytes. At postnatal day 35, immunoreactivity was seen slightly both in the pachytene primary spermatocytes and Leydig cells. Although there was intense immunoreactivity in the Leydig cells and in the elongated spermatids on days 50 and 70, the intensity of reaction was decreased in the elongated spermatids in the 10th month. Our results suggest that IGF-IR may play significant roles in testicular function and germ cell development.

Monitoring the activation state of insulin/insulin-like growth factor-1 hybrid receptors using bioluminescence resonance energy transfer.

In cells expressing both the insulin receptor isoform A (IRA) and the insulin-like growth factor-1 receptor (IGF1R), the presence of hybrid receptors, made up of an alphabeta-IRA chain associated with an alphabeta-IGF1R chain, has been demonstrated. These heterodimers are found in normal cells, and they also seem to play crucial roles in a number of cancers. However, they remain difficult to study, due to the concomitant presence of IRA and IGF1R homodimers. Using bioluminescence resonance energy transfer (BRET), we have developed assays to specifically monitor the activation state of IRA/IGF1R hybrids, both in vitro and in living cells. The first assay allowed the study of ligand-induced conformational changes within hybrid receptors purified from cells cotransfected with one type of receptor fused to Renilla reniformis luciferase (Rluc), and the other type of receptor fused to yellow fluorescent protein (YFP). In these conditions, only hybrid receptors were BRET-competent. In the second assay, the activation state of IRA/IGF1R hybrids was monitored in real time, in living cells, by cotransfection of kinase-dead versions of IRA-Rluc or IGF1R-Rluc, wild-type untagged IRA or IGF1R, and a YFP-tagged soluble version of the substrate-trapping mutant of protein tyrosine phosphatase 1B (YFP-PTP1B-D181A-Cter). In hybrid receptors, trans-phosphorylation of the kinase-dead alphabeta-Rluc moiety by the wild-type alphabeta moiety induced the recruitment of YFP-PTP1B-D181A-Cter, resulting in a hybrid-specific ligand-induced BRET signal. Therefore, both methods allow monitoring of the activity of IRA/IGF1R hybrid receptor and could be used to detect molecules of therapeutic interest for the treatment of cancer.

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.