Inhibition of the biologic response to insulin-like growth factor I in MCF-7 breast cancer cells by a new monoclonal antibody to the insulin-like growth factor-I receptor. The importance of receptor down-regulation.

We developed a mouse monoclonal antibody (4G11) against insulin-like growth factor I receptor by immunizing mice with mouse embryo fibroblasts overexpressing the human insulin-like growth factor-I receptor. Not only did the 4G11 antibody inhibit the binding of [ (125)I]insulin-like growth factor-I to the fibroblast receptor, but 4G11 antibody also potently down-regulated the insulin-like growth factor-I receptor. 4G11 Fab fragment inhibited ligand binding, but did not down-regulate the receptor, suggesting that receptor aggregation is required for down-regulation. 4G11 antibody also down-regulated the receptor in MCF-7 breast cancer cells, a panel of colon cancer cells and MG-63 osteosarcoma cells. Receptor recovery in MCF-7 cells after down-regulation by 4G11 antibody was slow, requiring 32 - 48 h for full recovery. Receptor down-regulation in MCF-7 cells by 4G11 antibody was confirmed by FACS analysis of intact and permeabilized cells. In contrast to 4G11 antibody, insulin-like growth factor-I did not down-regulate the receptor in MCF-7 cells. Down-regulation of the receptor by 4G11 antibody in MCF-7 cells resulted in inhibition of Akt and MAPK activation by insulin-like growth factor-I. We conclude that the ability of a monoclonal antibody to down-regulate the receptor may be an important antibody property in targeting the insulin-like growth factor-I receptor for the treatment of certain cancers.

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.

Suppressor of cytokine signaling (SOCS)-3 protein interacts with the insulin-like growth factor-I receptor.

SOCS proteins are a class of proteins that are negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In a yeast two-hybrid screen of a human fetal brain library, we have previously identified SOCS-2 as a binding partner of the activated IGF-I receptor (IGFIR). To test whether or not SOCS-3 also binds to the IGFIR, we cloned human SOCS-3 by reverse transcription-polymerase chain reaction from human skeletal muscle mRNA. SOCS-3 mRNA was expressed in many human fetal and adult tissues and in some human cancer cell lines (Hela, A549 pulmonary adenocarcinoma and G361 human melanoma). We found that human SOCS-3 protein interacts directly with the cytoplasmic domains of the activated IGFIR and the insulin receptor (IR) in the yeast two-hybrid assay. In GST-SOCS-3 pull-down experiments using IGFIR from mammalian cells and in immunoprecipitation experiments in which IGFIR and FLAG-SOCS-3 were transiently expressed in human embryonic kidney 293 cells, we found that SOCS-3 interacts constitutively with IGFIR in vitro and in intact cells. Unlike SOCS-2, hSOCS-3 was phosphorylated on tyrosines in response to IGF-I addition to 293 cells. We conclude that SOCS-3 binds to the IGFIR and may be a direct substrate for the receptor tyrosine kinase.

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.

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.

Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor.

SOCS (suppressor of cytokine signaling) proteins have been shown to be negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We have cloned a member of this family (hSOCS-2) by utilizing the insulin-like growth factor I receptor (IGF-IR) cytoplasmic domain as bait in a yeast two-hybrid screen of a human fetal brain library. The hSOCS-2 protein interacted strongly with the activated IGF-IR and not with a kinase negative mutant receptor in the two-hybrid assay. Mutation of receptor tyrosines 950, 1250, 1251, and 1316 to phenylalanine or deletion of the COOH-terminal 93 amino acids did not result in decreased interaction of the receptor with hSOCS-2 protein. hSOCS-1 protein also interacted strongly with IGF-IR in the two-hybrid assay. Glutathione S-transferase-hSOCS-2 associated with activated IGF-IR in lysates of mouse fibroblasts overexpressing IGF-IR. Human embryonic kidney cells (293) were transiently transfected with vectors containing IGF-IR and FLAG epitope-tagged hSOCS-2. After IGF-I stimulation, activated IGF-IR was found in anti-FLAG immunoprecipitates and, conversely, FLAG-hSOCS-2 was found in anti IGF-IR immunoprecipitates. Thus, hSOCS-2 interacted with IGF-IR both in vitro and in vivo. HSOCS-2 mRNA was expressed in many human fetal and adult tissues with particularly high abundance in fetal kidney and adult heart, skeletal muscle, pancreas, and liver. These results raise the possibility that SOCS proteins may also play a regulatory role in IGF-I receptor signaling.

Insulin-like growth factor II modulates the routing of cathepsin D in MCF-7 breast cancer cells.

A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P-II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells.

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.

Evidence against roles for pertussis toxin sensitive G proteins or diacylglycerol generation in insulin-like growth factor-1 stimulated DNA synthesis in MG-63 osteosarcoma cells.

A pertussis toxin-sensitive G protein has been reported to play a role in the mitogenic response to insulin-like growth factor-I (IGF-I) in mouse fibroblasts, and diacylglycerol generation has been shown to accompany growth stimulation by IGF-I of several cell lines. We have examined the roles of pertussis toxin sensitive G proteins and diacylglycerol generation in signaling by the insulin-like growth factor-I receptor in a cell line that is very responsive to IGF-I, the human osteosarcoma cell line, MG-63. Pertussis toxin failed to inhibit IGF-I induced [3H]-thymidine incorporation into DNA. Furthermore, the stable analog GTP gamma S had no effect on the binding of 125I-labelled IGF-I to MG-63 membrane preparations. Following addition of IGF-I to growth-arrested MG-63 cells there was no increase in diacylglycerol levels over 30 min. We conclude that the activated IGF-I receptor does not use pertussis toxin sensitive G proteins or diacylglycerol generation in a pathway leading to DNA synthesis in MG-63 cells.

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.

Developmental expression of the IGF-II/mannose 6-phosphate receptor.

The first indication that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) is developmentally regulated came from studies of the serum form of the receptor in the rat. By immunoblotting, the circulating form of the receptor, which was 10 kDa smaller than the tissue receptor, was high in 19 day fetal and 3, 10, and 20 day postnatal sera and then declined sharply. We next used quantitative immunoblotting to measure the total tissue IGF-II/M6PR in the rat. The receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. The rank order of receptor expression was heart > placenta > lung = intestine > muscle = kidney > liver > brain. In heart, the receptor was 1.7% of total protein in the extract. More recently, we have examined the expression of IGF-II/M6PR mRNA using Northern blotting and a solution hybridization/RNase protection assay. The rank order of receptor mRNA concentration among fetal tissues agreed with the rank order of receptor protein. The concentration of receptor mRNA was significantly lower in postnatal tissue than in fetal tissue. Thus IGF-II/M6PR mRNA concentration is an important determinant of receptor protein in most tissues. What is the function of the IGF-II/M6PR in embryonic and fetal tissues? The M6PR in birds and frogs does not bind IGF-II. It is intriguing that the rat IGF-II/M6PR is prominent during the embryonic and fetal periods, times at which the differences between mammals, on the one hand, and frogs and birds, on the other, are most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental expression of rat insulin-like growth factor-II/mannose 6-phosphate receptor messenger ribonucleic acid.

We have examined the developmental pattern of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor mRNA in various rat tissues from 20-day gestation fetuses and 20-day postnatal animals by Northern blotting and solution hybridization/RNase protection assays. The major mRNA species in all fetal and postnatal tissues was 9.0 kilobases. The rank order of receptor mRNA concentrations among the fetal tissues was heart greater than limb/muscle, lung, intestine, kidney, liver greater than brain, which agrees with the previously reported rank order of the tissue concentrations of receptor protein. The concentration of IGF-II/M6P receptor mRNA was significantly lower in postnatal tissues, again reflecting the relative levels of receptor protein in fetal and postnatal tissues. We measured IGF-II/M6P receptor mRNA copy number in fetal heart, the tissue with the highest concentration of receptor protein and mRNA, by including in the solution hybridization/RNase protection assay known amounts of a sense strand transcript of the receptor cDNA. This sense strand standard was quantitated by incorporating a tracer amount of [32P]UTP into the transcript and measuring the radioactivity in the product purified by gel electrophoresis. The receptor mRNA copy number in fetal heart was 74 molecules/cell. We conclude that the IGF-II/M6P receptor mRNA concentration is an important determinant of the level of receptor protein in most tissues.

NIH conference. Insulin-like growth factors in health and disease.

The insulin-like growth factor (IGF) family of peptides, binding proteins, and receptors are ubiquitous and important for normal human growth and development. Modern techniques including specific radioimmunoassays, radioreceptor assays and recombinant DNA technology have improved our understanding of the role of IGFs in growth and development. In addition to enhancing our understanding of normal physiology, these techniques assess changes in these hormones, binding proteins, and receptors in pathologic conditions including growth retardation, acromegaly, malnutrition, diabetes, and malignancy. Further, these studies have led to improvement in the assessment of responses to certain therapies used in the treatment of these diseases and may lead to improvements in these therapies.

Evidence against a role for insulin-like growth factor II in the autonomous growth of rat 18,54-SF cells.

The 18,54-SF rat cell line multiplies in serum-free medium and has been reported to produce insulin-like growth factor II (IGF-II) and to possess IGF-II/mannose 6-phosphate (Man-6-P) receptors, raising the possibility of autocrine growth stimulation by IGF-II acting through this receptor. When serum-free medium was changed every 24 h the 18,54-SF cells multiplied at the same rate as when the medium was not changed. An antibody (No. 3637) which blocks the binding of IGF-II to the IGF-II/Man-6-P receptor did not decrease or increase the growth rate of the 18,54-SF cells when medium was changed every 24 h. When the medium was changed every 12 h the rate of accumulation of cells in the culture was decreased. Addition of IGF-I or IGF-II at 10, 50, and 100 ng/ml every 12 h did not correct this decrease in cell number. When the medium was replaced every 12 h, cells in the periphery of the cultures gradually became nonviable as assessed by trypan blue uptake. However, the percentage of cells synthesizing DNA in the center of the cultures (approximately 50% during a 3 h pulse with tritiated thymidine) was the same whether the medium was changed every 12 h or left unchanged. Addition of IGF-I or IGF-II to the fresh medium change every 12 h did not increase the percentage of cells synthesizing DNA.

The insulin-like growth factor II/mannose-6-phosphate receptor is present in monkey serum.

We recently reported that the insulin-like growth factor II (IGF-II)/mannose-6-phosphate (Man-6-P) receptor is present in fetal and postnatal rat serum and that its serum content declined dramatically postnatally between days 20 and 40 . We now provide evidence that the IGF-II/Man-6-P receptor is also present in monkey serum. Serum was gel filtered on Sephadex G-200, and the column fractions were assayed for binding of radiolabeled IGF-II. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 150K and 40K carrier proteins. Binding to the void volume fractions was greatest in cord serum and decreased with age. Competitive binding studies with [125I]IGF-II and the void volume pools from monkey serum demonstrated that IGF-I competed less potently than IGF-II, and insulin did not compete. Radiolabeled IGF-I did not bind specifically to the void volume pools. Chemical cross-linking of [125I]IGF-II to aliquots of the void volume pools from monkey cord serum samples and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol demonstrated a specific band at about 240K. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of the Sephadex G-200 void volume pools of monkey serum. A band of approximately the same size as that found with human fibroblast members (approximately 215 K without dithiothreitol) was detected. The IGF-II/Man-6-P receptor band was more intense in cord serum than in the postnatal samples. When cord serum Sephadex G-200 pools were gel filtered on Sephadex G-50 in 1 mol/L acetic acid to separate binding components from free IGF, and IGF-II was measured by RRA, approximately 20% of the circulating IGF-II was found to be associated with this IGF-II/Man-6-P receptor in monkey serum. We conclude that the IGF-II/Man-6-P receptor present in serum may be a significant carrier for IGF-II in the monkey.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.