Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers

In both NIH3T3 cells and HepG2 cells, insulin-like growth factor I (IGF-I) receptors possess two beta-subunits that display different electrophoretic mobilities. Increasing concentrations of IGF-I stimulated the phosphorylation of both beta-subunits to a similar extent, whereas insulin stimulated the phosphorylation of both subunits only at elevated concentrations. Both beta-subunits were immunoprecipitated with p5, an insulin receptor-specific anti-peptide antibody, or with A410, a polyclonal anti-insulin receptor antisera. However, if the tetrameric IGF-I receptor was first dissociated into alpha-beta heterodimers with 1 mM dithiothreitol, only the lower molecular weight beta-subunit was immunoprecipitated. These results suggested that p5 and A410 specifically recognized the lower molecular weight beta-subunit but immunoprecipitated the higher molecular weight beta-subunit because it was present in the same disulfide linked tetramer. Similarly, alpha-IR-3, an antibody specific for the alpha-subunit of the IGF-I receptor, immunoprecipitated both types of beta-subunit from the intact tetramer but only the higher molecular weight beta-subunit from the dissociated heterodimers, suggesting that there are two types of alpha-subunits in the same tetramer and that the alpha-subunit recognized by alpha-IR-3 is only associated with the higher molecular weight beta-subunit. Tryptic phosphopeptide maps of the lower molecular weight beta-subunit of IGF-I receptor were different from the higher molecular weight beta-subunit, but were similar to those of the insulin receptor beta-subunit. Thus, by immunochemical cross-reactivity and structural criteria, the lower molecular weight beta-subunit of the IGF-I receptor was similar to the beta-subunit of insulin receptor. These data suggest that there exists a species of IGF-I receptor that is a hybrid composed of an insulin receptor alpha-beta heterodimer and an IGF-I receptor alpha-beta heterodimer. The existence of such a hybrid receptor could have important functional consequences.     

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.     

Identification of determinants that confer ligand specificity on the insulin receptor.

We have previously shown, using truncated soluble recombinant receptors, that substituting the 62 N-terminal amino acids of the alpha subunit from the insulin-like growth factor I receptor (IGFIR) with the corresponding 68 amino acids from the insulin receptor (IR) results in a chimeric receptor with an approximately 200-fold increase in affinity for insulin and only a 5-fold decrease in insulin-like growth factor I (IGFI) affinity (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Sch?ffer, L., Balschmidt, P., M?ller, K. B., and M?ller, N. P. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408). We demonstrate that these 68 N-terminal amino acids of the IR also confer insulin affinity on the intact IGFI holoreceptor both in the membrane-bound state and when solubilized by Triton X-100. Furthermore, this domain can be subdivided into two regions (amino acids 1-27 and 28-68 of the IR alpha subunit) that, when replacing the corresponding IGFIR sequences, increases the insulin affinity of truncated soluble receptor chimeras 8- and 20-fold, respectively, with only minor effects on the IGFI affinity. Within the latter of these two regions, we found that amino acids 38-68 of the IR, representing 13 amino acid differences from IGFIR, confer the same 20-fold increase in insulin affinity on the IGFIR. Finally, the amino acids from position 42 to 50 are not responsible for this increase in insulin affinity. We thus propose that at least two determinants within the 68 N-terminal amino acids of the insulin receptor are involved in defining the ligand specificity of the insulin receptor, and that one or a combination of the remaining seven amino acid differences between position 38 and 68 are involved in conferring insulin affinity on the insulin receptor.     

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

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

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.     

Distinct structural elements of rab5 define its functional specificity.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.     

Insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the platelet-derived growth factor receptor

R^? cells are 3T3-like cells derived from mouse embryos in which the insulin-like growth factor I (IGF-I) receptor (IGF-IR) genes have been disrupted by targeted homologous recombination. These cells cannot grow in serum-free medium supplemented by the growth factors that sustain the growth of other 3T3 cell lines, and cannot be transformed by oncogenes that easily transform wild type mouse embryo cells. We have used these cells to study the role of the IGF-IR in the growth and transformation of cells overexpressing the platelet-derived growth factor (PDGF)-b?b? receptor. We report that an overexpressed PDGF-b?b? receptor fails to induce mitogenesis or transformation in cells lacking the IGF-IR, while capable of doing so in cells expressing the IGF-IR. We conclude that the ability of the activated PDGF-b?b? receptor to stimulate cell proliferation and transformation requires a funcitional IGF-IR. © 1995 Wiley-Liss, Inc.     

Insulin-like growth factor I receptor signaling is required for exercise-induced cardiac hypertrophy

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.     

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors.

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.     

Insulin-like growth factor I is the key growth factor in serum that protects neuroblastoma cells from hyperosmotic-induced apoptosis

Neuroblastoma is a childhood tumor of the peripheral nervous system that remains largely uncurable by conventional methods. Mannitol induces apoptosis in neuroblastoma cell types and insulin-like growth factor I (IGF-I) protects these cells from hyperosmotic-induced apoptosis by affecting apoptosis-regulatory proteins. In the current study, we investigate factors that enable SH-SY5Y neuroblastoma cells to survive in the presence of an apoptotic stimulus. When SH-SY5Y cells are exposed to high mannitol concentrations, more than 60% of the cells are apoptotic within 48 h. Normal CS prevents hyperosmotic-induced apoptosis in a dose-dependent manner, with 0.6% CS protecting 50% of the cells, and 3% CS rescuing more than 70% of the cells from apoptosis. Serum also delays the commitment point for SH-SY5Y cells from 9 h to 35 h. A survey of several growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), and IGF-I reveals that IGF-I is a component of serum necessary for protection of neuroblastoma cells from death. Mitochondrial membrane depolarization occurs in greater than 40% of the cells after mannitol exposure and caspase-3 activation is increased in high mannitol conditions after 9 h. IGF-I blocks both the mitochondrial membrane depolarization and caspase-3 activation normally induced by hyperosmotic treatment in neuroblastoma cells. Our results suggest that (1) IGF-I is a key factor in serum necessary for protection from death and (2) IGF-I acts upstream from the mitochondria and the caspases to prevent apoptosis in human neuroblastoma.     

Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.     

Insulin-like growth factor I binding and receptor kinase in red and white muscle

IGF-I receptors were partially purified from red and white skeletal muscle by lectin-affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I-IGF-I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF-I receptor preparation in the two fiber types was identical as determined by affinity cross-linking. The tyrosine kinase activity of the receptor from red muscle was 2-3-fold more active towards exogenous substrates in both the basal and ligand-activated states as compared to white muscle. These data show that there is IGF-I-dependent kinase activity intrinsic to IGF-I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF-I receptors in a parallel manner in vivo.