Variable accumulation of insulin-like growth factor II in mouse tissues deficient in insulin-like growth factor II receptor

The insulin-like growth factor II receptor mediates endocytosis of insulin-like growth factor II, resulting in growth factor degradation in lysosomes. This degradation is an important regulator of growth factor activity in vivo, as shown by the phenotype of receptor deficient mice. Recent evidence suggests that the insulin-like growth factor II receptor functions as a tumour supressor in humans, and that loss of receptor function leads to increased levels of the growth factor in tumours. It is difficult to establish such a causal relationship in human tumours however, since most tumours have undergone several genetic changes by the time they are examined. Using mouse embryos deficient in receptor expression, and an insulin-like growth factor II-specific radioimmunoassay, we tested the hypothesis that lack of receptor function leads to local accumulation of insulin-like growth factor II. We found that mutant blood and skeletal muscle had excess insulin-like growth factor II, but that mutant lungs and liver had no accumulation. Mutant hearts had less growth factor than wild-type hearts, an unexpected observation, since the normal embryonic heart expresses very high levels of insulin-like growth factor II receptor, and mutant mice apparently die of congestive heart failure. The placentas of mutant mice were larger than those of wild-type, but this did not correlate with an excess of placental insulin-like growth factor II. These results indicate that lack of insulin-like growth factor II receptor can lead to local excess of the growth factor but that such excess is not a necessary consequence of receptor-deficiency.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

The binding of insulin-like growth factor II to the erythroleukemia cell line K562

The erythroleukemia cell line K562 was previously shown to have specific binding sites for insulin but not for insulin-like growth factor I (IGF-I). In this study the presence of specific receptors for insulin-like growth factor II (IGFqI) is established. Scatchard analysis of the competition curve for IGF-II disclosed a non-cooperative binding kinetic with a calculated affinity constant of 2.4×108 M^–1 and a receptor number of 4.8×l0⁴ sites/cell. IGF-I displayed 10% crossreactivity over the IGF-II receptor but insulin did not crossreact at all. Instead insulin, present in high concentrations, enhanced the binding of IGF-II. The presence of IGF II but not IGF-I receptors makes t h e K562 cell line suitable for studying properties of the type-2 receptor.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.     

Synthetic insulin-like growth factor II

Human insulin-like growth factor II with 67 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. Homogeneity of the synthetic product is ascertained by chromatofocusing, high performance liquid chromatography and amino acid analysis. In both radioimmunoassay and radioreceptor assay, the synthetic product is indistinguishable from the natural hormone.     

Solution structure of human insulin-like growth factor II; recognition sites for receptors and binding proteins.

The three-dimensional structure of human insulin-like growth factor II was determined at high resolution in aqueous solution by NMR and simulated annealing based calculations. The structure is quite similar to those of insulin and insulin-like growth factor I, which consists of an alpha-helix followed by a turn and a strand in the B-region and two antiparallel alpha-helices in the A-region. However, the regions of Ala1-Glu6, Pro31-Arg40 and Thr62-Glu67 are not well-defined for lack of distance constraints, possibly due to motional flexibility. Based on the resultant structure and the results of structure-activity relationships, we propose the interaction sites of insulin-like growth factor II with the type 2 insulin-like growth factor receptor and the insulin-like growth factor binding proteins. These sites partially overlap with each other at the opposite side of the putative binding surface to the insulin receptor and the type 1 insulin-like growth factor receptor. We also discuss the interaction modes of insulin-like growth factor II with the insulin receptor and the type 1 insulin-like growth factor receptor.     

Chemical synthesis of insulin-like growth factor II

Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.     

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.     

Purification of the type II insulin-like growth factor receptor from rat placenta

The membrane receptor for insulin-like growth factor II (IGF II) has been purified to near homogeneity from rat placenta by chromatography of crude plasma membranes solubilized in Triton X-100 on agarose-immobilized IGF II. Elution of the IGF II receptor from the matrix at pH 5.0 in the presence of 1.5 M NaCl resulted in a receptor purification of 1100-fold from isolated plasma membranes, or 340-fold from the Triton extract with an average yield of about 50% in five separate purifications. Analysis of 125I-IGF II binding to the solubilized receptor in the Triton extract and in purified form by the method of Scatchard demonstrated no change in receptor affinity (Kd = 0.72 nM). Sodium dodecyl sulfate electrophoresis of the purified receptor showed one major band at Mr = 250,000 with only minor contamination. Affinity labeling of the receptor in isolated placenta membranes and in purified form using 125I-IGF II and the cross-linking agent disuccinimidyl suberate resulted in covalent labeling of only the Mr = 250,000 band. Such labeling was abolished by unlabeled IGF II but was unaffected by insulin, consistent with the previously reported specificity of IGF II receptor (Massague, J., and Czech, M.P. (1982) J. Biol. Chem. 257, 5038-5045). These results establish a one step affinity method for the purification of the type II IGF receptor that is rapid and highly efficient.     

Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor II stimulation of DNA synthesis in human fibroblasts

To investigate the role of insulin-like growth factor II in the control of DNA synthesis in human fibroblasts, dose-response curves for insulin-like growth factor I and II stimulation of [3H]thymidine incorporation were compared in the absence and presence of alpha IR-3, a highly specific monoclonal antibody directed against the type I insulin-like growth factor receptor. Specific binding of [125I]insulin-like growth factor I to human fibroblast monolayer cultures was inhibited 60-70% in the presence of alpha IR-3. alpha IR-3 had no effect on [125I]insulin-like growth factor II binding to human fibroblasts. However, alpha IR-3 inhibited both insulin-like growth factor I and II stimulated [3H]thymidine incorporation. These data indicate that the type II insulin-like growth factor receptor does not function as a transducer of insulin-like growth factor II's mitogenic effect in human fibroblasts.     

Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor.

With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.     

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.     

Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and appears to arise from developing striated muscle-forming cells. Since insulin-like growth factor II (IGF-II) is involved in normal muscle growth and maturation and elevated IGF-II mRNA levels have previously been reported in rhabdomyosarcomas, we have been studying the possible role of IGF-II in the unregulated growth and invasive potential of these embryonal tumors. In this study, we demonstrate that 13 of 14 rhabdomyosarcoma tumors express high levels of IGF-II mRNA relative to normal adult muscle and also express mRNA for the type I IGF receptors on their cell surface, the receptor thought to mediate the effects of IGF-II on muscle cells. We have established several rhabdomyosarcoma cell lines in mitogen-free media and demonstrate that these cells express type I IGF receptors on their cell surface and secrete IGF-II into the media. Exogenous IGF-II is able to stimulate cellular motility in these cell lines as assayed in a modified Boyden chamber. Finally, alpha IR-3, a type I receptor antagonist, inhibits the growth of these cell lines in serum-free media but does not inhibit IGF-II-induced motility of these cells. These data suggest that endogenously produced IGF-II functions as an autocrine growth and motility factor in many rhabdomyosarcoma tumors. The mitogenic actions of IGF-II are mediated through a domain of the type I IGF receptor that is blocked by alpha IR-3. IGF-II-induced motility may be mediated through an alternative signaling pathway.     

Spatiotemporal distribution of insulin-like growth factor receptors during nephrogenesis in fetuses from normal and diabetic rats

Exposure to hyperglycemia in utero impairs rat nephrogenesis. The effect of maternal diabetes on insulin-like growth factors and their receptors in the fetal kidney is associated with an increase in both mRNA and protein of the insulin-like growth factor II/mannose 6-phosphate receptor. However, this receptor has never been localized in the fetal kidney. The spatial and temporal distribution of the three insulin-like growth factor receptors (insulin-like growth factor I receptor, insulin-like growth factor II/mannose 6-phosphate receptor and insulin receptor) in rat metanephros during both normal and streptozotocin-induced diabetic renal development was investigated using in situ hybridization and immunohistochemistry. All receptors were found in the fetal kidney from the start of nephrogenesis. Insulin-like growth factor I receptor expression was ubiquitous and continuously present during metanephric development. Insulin receptor expression was developmentally regulated during kidney maturation with an enhanced expression in proximal tubules at the late stages of development. Insulin-like growth factor II/mannose 6-phosphate receptor expression was ubiquitous in the early stages of development and was dramatically decreased at the late stages of normal kidney development. Insulin receptor and insulin-like growth factor I receptor expressions were unchanged in diabetic metanephroi. Although the spatial expression of insulin-like growth factor II/mannose 6-phosphate receptor was unaffected by hyperglycemia, its expression was not downregulated in the mesenchyme of the nephrogenic zone of diabetic fetuses on gestational day 20. This study suggests a crucial role of insulin-like growth factor II/mannose 6-phosphate receptor in the pathogenesis of the impaired nephrogenesis in fetuses of diabetic mothers.     

Evidence for the phosphorylation of the type II insulin-like growth factor receptor in cultured cells

The ATP pools of monolayer cultures of rat embryo fibroblasts and rat liver cells (BRL-3A2) were labeled with [32P]H3PO4. The type II insulin-like growth factor (IGF) receptor was purified by affinity chromatography on wheat germ lectin-Sepharose and IGF-II-Sepharose columns. A phosphorylated species having the expected size of the type II receptor (Mr = 220,000 without reduction, Mr = 260,000 with reduction) was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. IGF-II stimulated phosphorylation of the type II receptor in BRL-3A2 rat liver cells. Lability of the receptor phosphate bonds to alkaline pH suggests that the bulk of phosphorylation was occurring on serine residues.     

The cysteine-rich domains of the insulin and insulin-like growth factor I receptors are primary determinants of hormone binding specificity. Evidence from receptor chimeras

To delineate the structural determinants of the insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) which affect hormone binding specificity we have constructed seven chimeric receptor cDNAs and stably expressed them in Chinese hamster ovary cells. Clonal cell lines expressing high levels of each receptor chimera were analyzed for insulin and insulin-like growth factor I (IGFI) binding activity. Measurements of hormone binding and immunoprecipitation of metabolically labeled receptors showed that all chimeras were properly processed and expressed at the cell surface. The binding data indicate that 56 amino acids of the IR and 52 amino acids of the IGFIR located in corresponding regions of the cysteine-rich domains are the primary determinants of hormone binding specificity. These regions are located between amino acids Asn-230 and Ile-285 on the IR and between His-223 and Met-274 on the IGFIR. In addition, the alpha IR-3 antibody, which competes for IGFI binding, was found to interact with the same 52 amino acids of the IGFIR which determines hormone specificity. Other antibodies which interfere with insulin binding (5D9, MC51, and MA20) interact with epitopes in the COOH-terminal 288 amino acids of the alpha-subunit. We conclude that 56 and 52 amino acids of the cysteine-rich domains of the IR and IGFIR contain the major determinants of hormone binding specificity although other more COOH-terminal regions of both receptors contribute to hormone binding.     

Isolation and characterization of mannose 6-phosphate/insulin-like growth factor II receptor from bovine serum.

A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor.     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.