Insulin-Like Growth Factor I Receptor Binding in Brains of Alzheimer''s and Alcoholic Patients

Patients with chronic alcoholism and/or Alzheimer's disease show degenerative changes in the cerebral cortex and hippocampus. To investigate possible changes in insulin-like growth factor I receptor binding sites in brain tissue of patients with these pathological conditions, the number of 125I-insulin-like growth factor I binding sites was determined in tissues obtained from control patients and those with Alzheimer's and/or with a history of alcoholism. The four experimental groups examined consisted of patients from similar age groups. Postmortem histology and a clinical history were used for the diagnosis of Alzheimer's disease and alcoholism, respectively. Careful clinical records were kept concerning other variables such as immediate cause of death and medications administered before death. Specific binding of 125I-insulin-like growth factor I to homogenates prepared from cerebral cortex of Alzheimer's, alcoholic, alcoholic Alzheimer's, and age-matched control patients was similar, although Alzheimer's patients tended to have slightly higher binding values. No significant differences in insulin-like growth factor I binding in cerebral cortex were found with regard to age of patients, the interval between death and autopsy, and CNS-active medications. No statistical differences in 125I-insulin-like growth factor I binding were noted in hippocampal tissue from the four patient groups. Thus, human insulin-like growth factor I binding sites in cerebral cortex and hippocampus appear unaffected by several variables.     

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Similarities in structure and function of bovine serum erythrotropin and human insulin-like growth factor II: two fetal erythroid cell stimulating factors

Serum erythrotropin (ET) was isolated from fetal bovine serum. Partial sequence analysis of the N-terminal portion of the peptide indicated that the first 20 amino acids were practically identical to those found in human insulin-like growth factor II (IGF II). The effect of IGF II on [3H] thymidine incorporation in cell cultures of fetal bovine liver was similar to the effect of ET. Both factors acted synergistically with erythropoietin but not with platelet derived growth factor. The stimulation of thymidine incorporation by ET and IGF II on cell cultures of fetal liver erythroid cells was at least 15 times higher than their effects on cultures of fetal calf intestine, lung and kidney cells.     

Identification of the types of insulin-like growth factor-binding proteins that are secreted by muscle cells in vitro

We have previously shown that muscle cells secrete insulin-like growth factor-binding proteins. In the present study, BC3H-1 cells were shown to secrete one binding protein of Mr 32,000, whereas L6 cells secreted two binding proteins of Mr 31,000 and 24,000, as determined by ligand blotting. Subconfluent proliferating L6 cells secrete more of the Mr 24,000 binding protein, relative to the Mr 31,000 form. In contrast, differentiated L6 myotubes secreted similar quantities of the two forms. Insulin-like growth factor I preferentially stimulated secretion of the Mr 31,000 versus the Mr 24,000 binding protein from L6 cells and caused an increase in the secretion of the Mr 32,000 binding protein from BC3H-1 cells. The Mr 31,000 binding protein from L6 cells had a greater affinity for insulin-like growth factor II compared with insulin-like growth factor I, as did the Mr 32,000 binding protein of BC3H-1 cells. In contrast, the Mr 24,000 binding protein of L6 cells preferred insulin-like growth factor I. Neither porcine insulin nor relaxin competed for 125I-IGF-I binding. In conclusion, these muscle cell lines secrete only one or two forms of insulin-like growth factor-binding proteins. L6 cell differentiation is associated with a relative increase in the secretion of the Mr 31,000 binding protein compared with the Mr 24,000 form. Insulin-like growth factor I stimulates the secretion of its own binding proteins from muscle cells, and this may be an important mechanism for modulating cellular responsiveness to this growth factor.     

Vitronectin—A major cell attachment-promoting protein in fetal bovine serum

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.     

A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II

An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor.     

Immobilized serotonin: a novel substrate for cell culture

Baby hamster kidney cells, bovine aortal endothelial cells, bovine smooth muscle cells, and chick embryo fibroblasts were all observed to attach and grow on serotonin which had been immobilized by covalent coupling to agarose beads. While growth and morphology of cells on immobilized serotonin appeared normal, a change in cell function may have occurred since the pattern of polypeptides expressed by these cells was different from that of cells grown on two other substrates: immobilized fibronectin and tissue culture plastic. By changing the composition of the fetal calf serum proteins in the growth medium it was shown that cells attach directly to immobilized fibronectin without mediation by medium components. In contrast, cells were found not to attach directly to immobilized serotonin but to attach indirectly via factors absorbed onto immobilized serotonin from fetal calf serum. The major component of this cell attachment activity was shown not to be fibronectin and was identified following separation by SDS-PAGE, electroblotting, and cell binding on nitrocellulose filters. The cell attachment activity compromises a major protein species of Mr 70,000 which is the molecular size of the recently identified serum spreading factor also called vitronectin.     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

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.     

Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking

The structure of receptors for insulin-like growth factors in rat liver plasma membranes and the BRL 3A2 rat liver cell line has been examined by chemical cross-linking with disuccinimidyl suberate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Two receptor subtypes have been identified: (i) 125I multiplication-stimulating activity cross-linked to liver membranes or intact cells appeared in a complex of Mr = 260,000 (reduced) and 220,000 (nonreduced) and (ii) 125I-insulin-like growth factor I cross-linked to BRL 3A2 cells appeared predominantly in two bands of Mr greater than 300,000 without disulfide reduction and in a Mr = 130,000 complex following reduction. The two subtypes of insulin-like growth factor receptors identified by structural analysis correspond to previously observed differences in their specificity for insulin and insulin-like growth factors.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

Characterization of the protein which binds insulin-like growth factor in human serum

The binding of the 125I-labelled insulin-like growth factors I and II (125I-IGF I and 125I-IGF II) to the high-molecular-mass binding protein of human serum was characterized. With diluted human serum both growth factors showed optimal specific binding at 4 degrees C and pH 5-6. When 0.1% Triton X-100 was present in the incubation buffer an increase in the affinity of the IGF-binding protein was induced, which produced an enhanced binding of IGF I and IGF II. Competition experiments with various peptide hormones revealed that the native IGF-binding protein complex binds both the IGF I and IGF II with high specificity. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF I and IGF II respectively, indicating that in human serum only a single class of non-interacting binding sites is present. At optimal binding conditions the dissociation constants were determined to be 0.28 x 10(-9) M for IGF I binding and 0.66 x 10(-9) M for IGF II. Human serum was gel-filtered on Sepharose CL-6B at neutral pH and the eluate was assayed for binding activity with both IGF I and IGF II. One peak with an apparent molecular mass of 175 kDa and a Stokes radius of 4.8 nm was determined for both growth factors. Thus, our data suggest that human serum contains one class of high-molecular-mass binding protein with comparable binding characteristics for IGF I and IGF II.     

The ligand binding subunit of the insulin-like growth factor 1 receptor has properties of a peripheral membrane protein

125I-insulin-like growth factor 1 was cross-linked to its receptor in human placenta microsomal membranes. The microsomes were treated with urea, with dithiothreitol or with both reagents prior to centrifugation at 100,000 X g. We found that greater than 80% of the label was membrane-associated following separate treatment with urea or dithiothreitol, but greater than 80% of the radioactivity remained in the supernatant after simultaneous exposure to both reagents. In identical experiments employing 125I-epidermal growth factor, no condition led to the release of greater than 10% of label from the membrane. We conclude that the ligand binding subunit of the insulin-like growth factor 1 receptor, like peripheral membrane proteins, lacks a membrane anchoring domain.     

Disruption of ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor by cancer-associated missense mutations.

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) carries out multiple regulatory and transport functions, and disruption of IGF2R function has been implicated as a mechanism to increase cell proliferation. Several missense IGF2R mutations have been identified in human cancers, including the following amino acid substitutions occurring in the extracytoplasmic domain of the receptor: Cys-1262 --> Ser, Gln-1445 --> His, Gly-1449 --> Val, Gly-1464 --> Glu, and Ile-1572 --> Thr. To determine what effects these mutations have on IGF2R function, mutant and wild-type FLAG epitope-tagged IGF2R constructs lacking the transmembrane and cytoplasmic domains were characterized for binding of insulin-like growth factor (IGF)-II and a mannose 6-phosphate-bearing pseudoglycoprotein termed PMP-BSA (where PMP is pentamannose phosphate and BSA is bovine serum albumin). The Ile-1572 --> Thr mutation eliminated IGF-II binding while not affecting PMP-BSA binding. Gly-1449 --> Val and Cys-1262 --> Ser each showed 30-60% decreases in the number of sites available to bind both (125)I-IGF-II and (125)I-PMP-BSA. In addition, the Gln-1445 --> His mutant underwent a time-dependent loss of IGF-II binding, but not PMP-BSA binding, that was not observed for wild type. In all, four of the five cancer-associated mutants analyzed demonstrated altered ligand binding, providing further evidence that loss of IGF2R function is characteristic of certain cancers.     

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.     

叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子系统表达变化

目的：叶酸是一种水溶性B族维生素,在体内氨基酸与核苷酸代谢中起重要作用,是胎儿生长发育所必须的营养素。本文通过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变化。方法：雌性C57BL／6J小鼠叶酸缺乏组6只、正常对照组6只,分别饲以不舍叶酸和含2mg叶酸／kg的纯合饲料。四周后与雄鼠交配,于怀孕第13．5天（13．5dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用Real-timePCR法检测胎鼠肝脏组织中胰岛素生长因子I（IGFI）、胰岛素生长因子I受体（IGFIR）、胰岛素生长因子II（IGFII）、胰岛素生长因子II受体（IGFIIR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13．5dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR比率显著升高,差异有统计学意义（P〈0．05）;叶酸缺乏组胎鼠肝脏组织中IGFII和IGFIIRmRNA的相对表达水平均低于正常对照组（P〈0．05）,IGFI、IGFIR、IGFBP-1和IGFBP-3mRNA的相对表达水平两组间没有差异（P〉0．05）。结论：叶酸缺乏会导致小鼠孕中期胎鼠IUGR比率升高及胎肝IGFII和IGFIIRmRNA的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠I-UGR发生机制有关。     

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.     

Mannose 6-phosphate/insulin like growth factor II receptor: the two types of ligands bind simultaneously to one receptor at different sites

Pentamannose 6-phosphate/trilysine substituted aprotinin (PMP-lys-aprotinin) and insulin like growth factor II (IGF II) were used as affinity ligands for the mannose 6-phosphate (M6P) and IGF II binding sites of the M6P/IGF II receptor. Both ligands were cross linked to intact receptor and tryptic fragments of the receptor. The pattern of receptor fragments with M6P and IGF II binding sites differed indicating that the two binding sites are located on different segments of the receptor. The receptor was incubated with [125I]IGF II and pentamannose 6-phosphate substituted bovine serum albumin (PMP-BSA). From these mixtures [125I]IGF II receptor complexes could be precipitated with antibodies against the PMP-BSA indicating that the M6P/IGF II receptor can bind simultaneously IGF II and M6P-containing ligands.