The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.     

Radioimmunoassay for insulin-like growth factor II (IGF-II)

Insulin-like growth factor II (IGF-II) levels in human plasma were measured in physiological and pathological conditions by radioimmunoassay (RIA) with biosynthetic IGF-II. This RIA was specific for IGF-II and cross-reactivity with IGF-I was 1%. The sensitivity was 15 pg/tube with 50% displacement at 50 pg/tube. The intra- and inter-assay coefficients of variation for IGF-II were 6.3 and 9.3%, respectively. The plasma IGF-II levels in normal adults, patients with hypopituitarism and patients with active acromegaly were 589.6 +/- 15.8, 800.9 +/- 45.6 and 330.3 +/- 24.3 ng/ml, respectively. After human growth hormone (hGH) treatment in hypopituitarism, IGF-II slightly increased, but not significantly. After adenomectomy in patients with acromegaly, IGF-II significantly decreased. These data indicate that IGF-II concentrations in plasma were partially GH dependent. This GH dependency was less than that of IGF-I. IGF-II was low in patients with anorexia nervosa and with liver cirrhosis and high in patients with renal failure. In two cases with extrapancreatic tumor-associated hypoglycemia, plasma IGF-II was increased to 1123.8 and 843.5 ng/ml, and returned to normal after tumor resection. These data showed that IGF-II was partly dependent on GH and nutritional conditions and that IGF-II was the most likely cause of some cases of hypoglycemia with extrapancreatic tumor. This specific and sensitive RIA of IGF-II would be useful in evaluating its physiological and pathological role in plasma and tissue.     

Structure and expression of the rat insulin-like growth factor II (rIGF-II) gene. rIGF-II RNAs are transcribed from two promoters

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5'-noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age.     

Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling

We have extended our previous yeast two-hybrid findings to show that 14-3-3beta also interacts with the insulin-like growth factor I receptor (IGFIR) in mammalian cells overexpressing both proteins and that the interaction involves serine 1283 and is dependent on receptor activation. Treatment of cells with the phorbol ester PMA stimulates the interaction of 14-3-3beta with the IGFIR in the absence of receptor tyrosine phosphorylation, suggesting that receptor activation leads to activation of an endogenous protein kinase that catalyzes the phosphorylation of serine 1283. To investigate the role of 14-3-3 proteins in IGF signal transduction, IGFIR structure-function studies were performed. Mutation of serine 1283 alone (S1283A) (a mutation that decreases but does not abolish the interaction of the IGFIR with 14-3-3) did not affect anchorage-independent growth of NIH 3T3 fibroblasts overexpressing the mutant receptor. However, the simultaneous mutation of this residue and the truncation of the C-terminal 27 residues of the receptor (Delta1310/S1283A) abolished the interaction of the receptor with 14-3-3 and reversed the enhanced colony formation observed with the IGFIR truncation mutation alone (Delta1310). The difference between the Delta1310 and Delta1310/S1283A transfectants in the soft agar assay was confirmed by tumorigenesis experiments. These findings suggest that 14-3-3 proteins interact with the IGFIR in vivo and that this interaction may play a role in a transformation pathway signaled by the IGFIR.     

Identification of domains of the insulin-like growth factor I receptor that are required for protection from apoptosis.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.     

Sulfated glycosaminoglycans are required for specific and sensitive fibroblast growth factor (FGF) 19 signaling via FGF receptor 4 and betaKlotho

Secreted from intestine, human fibroblast growth factor 19 (hFGF19) is an endocrine metabolic regulator that controls bile acid synthesis in the liver. Earlier studies have suggested that hFGF19 at 10-100 nM levels signals through FGF receptor 4 (FGFR4) in the presence of a co-receptor, betaKlotho, but its activity and receptor specificity at physiological concentrations (picomolar levels) remain unclear. Here we report that hFGF19 at picomolar levels require sulfated glycosaminoglycans (sGAGs), such as heparan sulfate, heparin, and chondroitin sulfates, for its signaling via human FGFR4 in the presence of human betaKlotho. Importantly, sGAGs isolated from liver are highly active in enhancing the picomolar hFGF19 signaling. At nanomolar levels, in contrast, hFGF19 activates all types of human FGFRs, i.e. FGFR1c, FGFR2c, FGFR3c, and FGFR4 in the co-presence of betaKlotho and heparin and activates FGFR4 even in the absence of betaKlotho. These results show that sGAGs play crucial roles in specific and sensitive hFGF19 signaling via FGF receptors and suggest that hepatic sGAGs are involved in the highly potent and specific signaling of picomolar hFGF19 through FGFR4 and betaKlotho. The results further suggest that hFGF19 at pathological concentrations may evoke aberrant signaling through various FGF receptors.     

Production of monoclonal antibodies to the rat insulin-like growth factor II (IGF-II) receptor

BALB/c mice were immunized with rIGF-II receptors purified from 18-54, SF cells by chromatography of solubilized receptors over agarose-immobilized rIGF-II. Two fusions of splenic lymphocytes with FO mouse myeloma cells yielded 27 stable hybrids which were positive by ELISA. Cloning of seven of these hybrids yielded 30 positive clones by ELISA. At least seven of these clones (minimum of one from each parent hybrid) were capable of specifically immunoprecipitating the rIGF-II receptor.     

The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.     

Association of the insulin-like growth factor II (IGF2) gene with human cognitive functions

Active search for candidate genes whose polymorphisms are associated with human cognitive functions has been in progress in the past years. The study focused on the role that the insulin-like growth factor II (IGF2) gene may play in the variation of cognitive processes related to executive functions. The ApaI polymorphism of the IGF2 gene was tested for association with selective attention during visual search, working memory/mental control, and semantic verbal fluency in a group of 182 healthy individuals. The ApaI polymorphism was associated with the general cognitive index and selective attention measure. Carriers of genotype AA displayed higher values of the two parameters than carriers of genotype GG. It was assumed that the ApaI polymorphism of the IGF2 gene influences the human cognitive functions, acting possibly via modulation of the IGF-II level in the central nervous system.     

Porcine insulin-like growth factor 1 (IGF1) gene polymorphisms are associated with body size variation

Previous studies have confirmed that insulin growth factor-1 (IGF1) plays important roles in growth and body size in humans and animals. However, whether single nucleotide polymorphisms (SNPs) within the IGF1 gene affects body size and growth in pigs has been unclear. We identified IGF1 SNPs among 5 pig breeds (Berkshire, Duroc, Landrace, Yorkshire and Korea Native Pig) and found that the G allele of SNP (c.G189A) was associated with higher body weight and was more predominant in western pig breeds, while the Korean Native Pig is the breed with the highest frequency of the A allele. Four haplotypes (–GA–, –GG–, –AG–, and –AA–) were constructed using the 2 identified SNPs. The GA haplotype was most frequently observed, except in the Berkshire breed. In addition, these SNPs and haplotypes were significantly associated with body size (final weight), average daily gain, and backfat thickness (P < 0.05) in 2 intercrossed F₂ pig populations (KNP × YS F₂ and KNP × LR F₂). Furthermore, the major GA haplotype had a significant additive effect on body size and average daily gain. In conclusion, specific SNPs within the porcine IGF1 gene may contribute to the smaller body size and lower growth rate of Korea Native Pigs.     

Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: Correlation between IGF-IIR mRNA and protein levels and heterochronic lung development

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2^b) and B10 (H-2^b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF IIR levels with progressive development in both strains. Further, slower developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns. © Wiley-Liss, Inc.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

Tumour Cannabinoid CB(1) receptor and phosphorylated epidermal growth factor receptor expression are additive prognostic markers for prostate cancer

Background

In cultured prostate cancer cells, down-regulation of epidermal growth factor receptor (EGFR) has been implicated in mediating the antiproliferative effect of the endogenous cannabinoid (CB) ligand anandamide. Using a well-characterised cohort of prostate cancer patients, we have previously reported that expression levels of phosphorylated EGFR (pEGFR-IR) and CB₁ receptor (CB₁IR) in tumour tissue at diagnosis are markers of disease-specific survival, but it is not known whether the two markers interact in terms of their influence on disease severity at diagnosis and disease outcome.

Methodology/Principal Findings

Data from a cohort of 419 patients who were diagnosed with prostate cancer at transurethral resection for voiding difficulties was used. Scores for both tumour CB₁IR and pEGFR-IR were available in the database. Of these, 235 had been followed by expectancy until the appearance of metastases. For patients scored for both parameters, Cox proportional-hazards regression analyses using optimal cut-off scores indicated that the two measures provided additional diagnostic information not only to each other, but to that provided by the tumour stage and the Gleason score. When the cases were divided into subgroups on the basis of these cut-off scores, the patients with both CB₁IR and pEGFR-IR scores above their cut-off had a poorer disease-specific survival and showed a more severe pathology at diagnosis than patients with high pEGFR-IR scores but with CB₁IR scores below the cut-off.

Conclusions/Significance

These data indicate that a high tumour CB₁ receptor expression at diagnosis augments the deleterious effects of a high pEGFR expression upon disease-specific survival.     

Analysis of insulin-like growth factor I gene expression in malignancy: evidence for a paracrine role in human breast cancer

Insulin-like growth factor I (IGF-I) activity has been reported to be produced by several human cancers. Identification of RNAs transcribed from the IGF-I gene has been complicated by the detection of multiple hybridizing bands on Northern analysis. To determine if any of these RNAs are transcribed from the IGF-I gene, we have used a sensitive and specific ribonuclease (RNAse) protection assay for IGF-I. We have also studied the breast cancer tissue expression of IGF-I using in situ hybridization histochemistry. We have found no IGF-I mRNA in breast (zero of 11) or colon cancer (zero of 9) cell lines; both of these tumors have been previously reported to express IGF-I mRNA. However, three of three neuroepithelioma and one of two Ewing's sarcoma cell lines express IGF-I mRNA; therefore, in these tumors IGF-I may be an autocrine growth factor. In contrast to breast cancer cell lines, RNA extracted from breast tissues has easily detectable IGF-I mRNA. In situ hybridizations show that IGF-I mRNA is expressed in the stromal cells, and not by normal or malignant epithelial cells. These findings suggest that although IGF-I is not produced by breast epithelial cells it may function as either a paracrine stimulator of epithelial cells or an autocrine stimulator of stromal cells.