Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10.

We have used the yeast two-hybrid system to study the interaction between the IGF-I receptor and two putative substrates, IRS-1 and Shc. In addition, we have identified Grb10 as a protein that binds to the insulin-like growth factor I (IGF-I) receptor. This two-hybrid system (the interaction trap) utilizes a hybrid protein containing the LexA DNA-binding domain fused to the intracellular portion of the IGF-I receptor (LexA-IGFIR beta) and hybrids containing an activation domain fused to either IRS-1 (Ad-IRS-1), Shc (Ad-Shc), or a cDNA library. A positive interaction of LexA-IGFIR beta with the activation domain hybrid results in activation of reporter genes, LacZ and LEU2, in the yeast. Western blotting of extracts from transformed yeast demonstrated that the LexA-IGFIR beta fusion protein was expressed and phosphorylated on tyrosine residues. Coexpression of LexA-IGFIR beta with Ad-IRS-1 resulted in strong activation of both reporter genes; activation did not occur with a kinase-negative receptor mutant. IRS-1 residues 160-516 were sufficient for this strong interaction. Coexpression of LexA-IGFIR beta with Ad-Shc also resulted in strong activation of both LacZ and LEU2 reporter genes. This interaction was also dependent upon a tyrosine kinase-active receptor and required tyrosine 950 in the juxtamembrane region of the receptor. An N-terminal fragment of Shc (amino acids 1-232) interacted almost as strongly as full-length Shc whereas the Shc SH2 domain only activated the more sensitive LEU2 reporter. Full-length Shc was phosphorylated on tyrosine when coexpressed with IGFIR beta but not when coexpressed with the kinase-negative receptor mutant. To identify additional proteins that interact with the IGFIRs, a human fetal brain cDNA library was screened using the interaction trap system. This analysis identified partial cDNAs for Grb10. Coexpression of LexA-IGFIR beta with Ad-Grb10 resulted in strong activation of both LacZ and LEU2 reporter genes; this interaction was dependent upon a tyrosine kinase-active receptor but did not require tyrosine 950.

Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15.

Most patients with deletion of the distal long arm of chromosome 15 have intrauterine growth retardation and postnatal growth deficiency in addition to developmental abnormalities. It has been proposed that the absence of one copy of the insulin-like growth factor I (IGF-I) receptor gene may play a role in the growth deficiency seen in this syndrome. To address this question we examined IGF-I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15 (15q26.1-->qter). Quantitative Southern blot analysis of the IGF-I receptor gene was performed on HindIII digests of fibroblast DNA. Radioactivity in the 1.7-kilobase receptor fragment in the two patients was 55% and 51% of the values in controls, consistent with the absence of one copy of the IGF-I receptor gene. IGF-I receptor messenger ribonucleic acid levels were quantitated by a solution hybridization/nuclease protection assay. Receptor messenger ribonucleic acid levels in the two patients were 45% and 52% of the values in controls. Northern blotting demonstrated normal size IGF-I receptor transcripts and affinity crosslinking of [125I]IGF-I to Triton X-100-solubilized fibroblasts demonstrated a normal size receptor in the patients. Analysis of placental membranes prepared from one patient revealed no difference in [125I]IGF-I binding. In the patients' fibroblasts, however, binding of [125I]long [R3]-IGF-I to the IGF-I receptor was significantly reduced, as assessed by the amount of radioactivity competed by the monoclonal antibody alpha IR-3 or insulin and Scatchard analysis of binding data. To assess IGF-I receptor function, stimulation of [alpha-1-14C]-methylaminoisobutyric acid transport and stimulation of [methyl-3H]thymidine incorporation into DNA by a full range of IGF-I concentrations was examined in patient and control fibroblasts. There was a significant decrease in the maximal response to IGF-I in both assays for one of the two patients when data were expressed as fold response over the basal value. However, there was no evidence for impairment of response to IGF-I in either patient's fibroblasts when data were expressed as net stimulation (maximal response minus basal). In conclusion, although IGF-I receptor expression was decreased in fibroblasts from two patients with deletion of the distal long arm of chromosome 15, we were unable to provide conclusive evidence for impairment of the biological response to IGF-I.

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.

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.

Insulin-like growth factor I activates insulin receptor substrate 1 and Ras in human osteosarcoma cells.

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63, by acting through IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras cycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 cells were (1) IGF-I stimulated and lysates were immunoprecipitated with polyclonal IRS-1 antibody or (2) metabolically labeled with [32P]orthophosphoric acid and then cells were treated with IGF-I. Cell lysates were immunoprecipitated with p21Ras antibody (Y13-259) and bound nucleotides were analysed by thin-layer chromatography. We demonstrated tyrosine phosphorylation of IRS-1/2 immunoprecipitated from MG-63 cells stimulated with IGF-I. We also showed an increased level of GTP in p21Ras immunoprecipitates from IGF-I treated cells. Nuclear extracts prepared from 32P-labeled cells before and after addition of IGF-I were immunoprecipitated with c-Jun antibody. After electrophoresis and autoradiography, phosphorylation of the c-Jun band was seen to be IGF-I independent. Phosphoamino acid analysis of the c-Jun band showed that phosphoserine was the major species.

Further studies on the quaternary structure of yeast casein kinase II.

Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.

[Serum apolipoprotein and lipoprotein levels in patients with diabetes mellitus type 2]. / Apolipoproteiny i lipoproteiny w surowicy krwi chorych na cukrzyce typu II.

Lipid disturbances were evaluated in type II diabetes comparing the results of determinations of total cholesterol and HDL-cholesterol with apolipoprotein A and B levels. The study was carried out in 32 diabetics with type II disease with mean duration 7 +/- 9 years, 15 males and 17 females, and in 30 controls. In all cases postprandial glycaemia, haemoglobin A1C, total cholesterol concentration (by the Liebermann-Burchardt method), HDL-cholesterol (by the Blaszczyszyn method), triglycerides (by the enzymatic method), an apolipoprotein A and B (by Mancini radial immunodiffusion method using a Bio-Merieux kit) were determined. A significant correlation was demonstrated between the concentrations of cholesterol and apolipoprotein A, on the one hand, and blood glucose level, on the other, and apolipoprotein A was found to be a better indicator of lipid disturbances in the aspect of diabetes control then apolipoprotein B. The latter was a better indicator of lipid disturbances in diabetes connected with obesity, than total cholesterol.

[Immunochemistry of eukaryotic ribosomes]. / Immunochemia rybosomów eukariotycznych.

Immunochemical investigations of ribosomes should correlate with basic knowledge of the function, structure and activity of organelles in the cell processes. Our paper presents data of immunochemical methods used to determine the structure, function and differences of ribosomes. We present the usefulness of immunochemical methods to test human ribosomes, diagnosis and therapy of many diseases.

Autophosphorylation of the insulin-like growth factor I receptor cytoplasmic domain.

The cytoplasmic domain of the beta subunit of the insulin-like growth factor I receptor (amino acids 936-1337) was overexpressed in Sf9 insect cells using a baculovirus expression system, and the 6-His tagged receptor was purified by metal-affinity chromatography. Autophosphorylation of the receptor was concentration dependent, consistent with a trans phosphorylation mechanism. Phosphoamino acid analysis of the autophosphorylated receptor showed predominantly phosphotyrosine, but phosphoserine and phosphothreonine were also present. However, when the receptor was further purified by gel filtration on Sephadex G-100 and then autophosphorylated, phosphoamino acid analysis showed only phosphotyrosine. We conclude that the IGF-I receptor tyrosine kinase is not a dual-specificity kinase and that autophosphorylation of the beta subunit is by a trans mechanism.

Effect of acute hyperthyroidism on insulin removal in the rats.

The aim of this work was to study secretion and removal of insulin in hyperthyroidism. The experiments were carried out on two groups of male Wistar rats: control and hyperthyroid. Acute hyperthyyreosis was induced by administration of L-thyroxine (Fluka A.G.) 0.8 mg/kg for 21 days. Each group was divided into three subgroups: fed, fasted 24 h, fasted 24 h-treated with glucose. The insulin concentration was determined in the portal and aortal blood and a difference between the two concentrations was considered as reflecting insulin removal. The plasma insulin concentrations in both vessels of fed as well fasted hyperthyroid group were higher from the respective values in the control one. The removal of the hormone in the fed rats was similar in the both groups. Fasting reduced insulin removal only in the control group. Treatment of the fasted rats with glucose abolishes this difference. The liver glycogen content was similar in the two fasted groups but the blood glucose level was higher in the hyperthyroid than in the control rats. The latter factor could account for the difference in insulin removal between the two groups. It is concluded that hyperthyroidism increases insulin secretion but does not affect its removal.

Effect of somatostatin and dopamine on the activity of cyclic AMP-independent protein kinase from human placenta.

Studies in the effect of somatostatin and dopamine on the incorporation of 32P from ATP to casein and ribosomes were carried out using purified protein kinase type II, isolated from the human placental cytosol. Low concentrations of somatostatin inhibited, while high ones of dopamine concentrations stimulated the activity of kinase.

Activation of extracellular signal-regulated kinases 1 and 2 by insulin-like growth factor I in human osteosarcoma MG-63 cells.

The activation of extracellular signal-regulated kinases 1 and 2 by insulin-like growth factor I in human osteosarcoma MG-63 cells was examined by Mono Q ion exchange chromatography of cell extracts and measurement of myelin basic protein kinase activity, and by immunoblotting of cell extracts with a phospho-specific extracellular signal-regulated kinase antibody. Extracellular signal-regulated kinase 1 appeared to be activated in resting cells and addition of insulin-like growth factor I resulted in the activation primarily of extracellular signal-regulated kinase 2. Extracellular signal-regulated kinase 2 was found in the nucleus after addition of insulin-like growth factor I.

Preprostatectomy: A clinical model to study stromal-epithelial interactions.

The preprostatectomy setting serves as a valuable clinical model for early developmental clinical trials for evaluating promising agents for chemoprevention. In the preprostatectomy model, study agents are administered between the diagnostic biopsy for prostate cancer and definitive therapy. The prostatic tissue that is available after prostatectomy allows for biomarker evaluation of all the components of the prostate, including the glandular epithelium, blood vessels, and the stroma. This provides an opportunity to study the reciprocal interactions between the stroma and the epithelium. Morphologic studies suggest that prostatic stromal cells play a critical role in affecting the growth and maturation of prostatic epithelium. Experimental studies in tissue culture show that carcinoma-associated stromal cells can promote prostatic carcinogenesis, and normal stromal cells may be able to inhibit prostatic carcinogenesis by inducing differentiation and decreasing the proliferation of the epithelium. Although the complex molecular mechanisms through which stroma modulates the epithelial cell phenotype remain to be elucidated, there are several well-characterized signaling pathways, such as for growth factors and steroid hormones, that are likely to contribute to the modulation of transformed epithelial cells. There is evidence of an association between increased serum levels of IGF-I and an increased risk of prostate cancer. The IGF system appears to play an important role in the development of prostate cancer by modulation of paracrine pathways, and also by modulation of the concentrations of different stromal and epithelial IGFBP, which are differentially expressed in the epithelium and stroma. Nerve growth factor is capable of stimulating a proliferative response via a high affinity Trk receptor present in normal and malignant prostate epithelia, and alternatively can mediate apoptosis via the low affinity p75NTR receptor that is progressively lost from the malignant prostate. As the role of each stromal element involved in carcinogenesis becomes further defined, these elements offer promising targets for new chemopreventive strategies.

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes.

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.

14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor.

We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR). This analysis identified 14-3-3beta and zeta proteins. 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain. 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro. The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions. Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue. 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation. Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities.

Insulin-like growth factor-II is a substrate for dipeptidylpeptidase I (cathepsin C). Biological properties of the product.

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP-I) caused the release of trichloroacetic-acid-soluble radioactivity from rat 125I-insulin-like growth factor-II (IGF-II). This activity could be blocked by dipeptide inhibitors of DAP-I, and was enhanced by chloride. Treatment of unlabeled rat IGF-II with DAP-I converted approximately 50% of the IGF-II to a species with a slightly shorter elution time on reverse-phase HPLC, whereas treatment of human IGF-II caused complete conversion to the species with the shorter elution time. Rat IGF-II purified from the rat BRL 3A cell line is a mixture of two molecules beginning with Ala-Tyr-Arg-Pro-Ser- and Tyr-Arg-Pro-Ser- [Marquardt, H., Todaro, G. J., Henderson, L. E. & Oroszlan, S. (1981) J. Biol. Chem. 256, 6859-6865] while human IGF-II begins with Ala-Tyr-Arg-Pro-Ser-. Determination of the N-terminal amino acid sequence of human IGF-II before and after digestion with DAP-I showed that DAP-I cleaved Ala-Tyr, terminating at Arg-Pro-; the rat IGF-II species beginning with Tyr-Arg-Pro-Ser- was resistant to digestion. In order to compare DAP-I-treated IGF-II with native IGF-II for binding to IGF receptors and IGF-binding proteins and in a bioassay, rat and human IGF-II were treated with DAP-I and the digested and undigested species were isolated by reverse-phase HPLC. The IGF-II/mannose 6-phosphate receptor was purified from rat placental membranes, the IGF-I receptor was solubilized from human placental membranes and IGF-binding proteins were partially purified from adult and three-day-old rat sera by sequential gel filtration on Sephadex G-200 (pH 8.0) and Sephadex G-50 (acid pH). The dose/response curves of the two IGF-II species were indistinguishable in radioreceptor assays utilizing the IGF-II/mannose 6-phosphate receptor and the IGF-I receptor and in IGF competitive binding assays utilizing partially purified IGF-binding proteins. The DAP-I-digested and native IGF-II species were also equipotent in stimulating [3H]thymidine incorporation into DNA in the human osteosarcoma cell line, MG-63. We conclude that DAP-I cleaves an N-terminal dipeptide from IGF-II and that this does not result in a change in the biological activity of the molecule.

Anchorage-independent growth of fibroblasts that express a truncated IGF-I receptor.

The purpose of this investigation was to study signaling by an insulin-like growth factor I receptor (IGF-I R) that lacks the extracellular portion of the receptor. We transfected IGF-I R-negative mouse embryo fibroblasts with a truncated IGF-I R consisting of only the transmembrane and cytoplasmic part of the beta subunit. Proliferation as assessed by counting cells was the same for vector only transfectants and the truncated receptor transfectants in defined medium containing EGF and PDGF. In contrast, anchorage-independent growth as measured by colony formation in soft agar was markedly increased for the truncated IGF-I R transfectants compared to the vector transfectants. MAP-kinase activity in the truncated IGF-I R transfectants was not higher than in the vector transfectants; however, PI 3-kinase activity was significantly higher in the IGF-I R transfectants. These results provide evidence that an IGF-I receptor consisting of only the transmembrane and cytoplasmic domain of the beta subunit can signal pathways leading to anchorage-independent growth.

Effects of the pineal gland on the growth processes of Guerin epithelioma in male Wistar rats.

The effects of pinealectomy (PX) and melatonin (Mel) administration on the growth processes of Guerin epithelioma, a malignant tumour derived from spontaneous cancer in Wistar rat uterus, was investigated in five groups of male rats. The mean life span of the rats bearing Guerin tumours (GT) and subjected to PX was shorter than in animals with an intact pineal gland. Mel did not affect the lifespan in rats with intact pineals or in those subjected to PX. Mel decreased the mitotic activity of GT cells in rats with and without the pineal gland. Pathomorphological examination revealed high malignancy of the primary tumour but no metastases. The results confirmed the important role of the pineal gland in 'oncostasis'.