Glucose starvation alters insulin but not IGF-I binding to Chinese hamster ovary (CHO) cells

The pattern of cellular protein glycosylation can be altered in CHO cells by glucose starvation. When wild type CHO cells are deprived of glucose, 125I-insulin binding increases from a B/F of 0.033 +/- 0.004 to 0.063 +/- 0.011, due to an increase in receptor affinity. The already elevated insulin binding to mutant B4-2-1 CHO cells, whose genetic defect causes abnormal glycosylation mimicking the pattern seen in the glucose starved normal cells, is not affected by glucose starvation. In neither cell line is 125I-IGF-I binding affected by glucose starvation. These data support the hypothesis that abnormal glycosylation can alter insulin binding to its receptor. Furthermore, there is a striking difference in the susceptibility of IGF-I and insulin receptors to alterations in glycosylation.     

Distinct receptors for IGF-I, IGF-II, and insulin are present on bovine capillary endothelial cells and large vessel endothelial cells

Endothelial cells were cultured from bovine fat capillaries, aortae and pulmonary arteries and their interactions with 125I-IGF-I, 125I-MSA (an IGF-II), 125I-insulin and the corresponding unlabeled hormones were evaluated. Each endothelial culture showed similar binding parameters. With 125I-insulin, unlabeled insulin competed with high affinity while IGF-I and MSA were approximately 1% as potent. With 125I-MSA, MSA was greater than or equal to IGF-I in potency and insulin did not compete for binding. Using 125I-IGF-I, IGF-I was greater than or equal to MSA whereas insulin decreased 125I-IGF-I binding by up to 72%. Exposing cells to anti-insulin receptor antibodies inhibited 125I-insulin binding by greater than 90%, did not change 125I-MSA binding, while 125I-IGF-I binding was decreased by 30-44%, suggesting overlapping antigenic determinants between IGF-I and insulin receptors that were not present on MSA receptors. We conclude that cultured capillary and large vessel endothelial cells have distinct receptors for insulin, IGF-I and MSA (IGF-II).     

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.     

Receptors for insulin-like growth factor-I and tumor necrosis factor-alpha are hormonally regulated in bovine granulosa and thecal cells.

Mastitis induces release of tumor necrosis factor-alpha (TNFalpha) and has been linked with reduced reproductive performance. To further elucidate the role and mechanism of action of TNFalpha on ovarian cells, the effect of TNFalpha on insulin-like growth factor-I (IGF-I)-induced steroidogenesis and IGF-I binding sites in granulosa and thecal cells as well as the hormonal regulation of TNFalpha receptors were evaluated. Granulosa and thecal cells were obtained from small (1-5mm) and large (> or =8mm) bovine ovarian follicles, respectively, and cultured for 3-4 days. During the last 2 days of culture, cells were treated with various hormones and steroid production and specific binding of 125I-IGF-I and 125I-TNFalpha was determined. Two-day treatment with 30 ng/ml of TNFalpha decreased (P<0.05) IGF-I-induced estradiol production by granulosa cells and IGF-I-induced androstenedione production by thecal cells. Two-day treatment with 10 and 30ng/ml of TNFalpha decreased (P<0.05) specific binding of 125I-IGF-I to thecal cells, but had no effect on specific binding of 125I-IGF-I to granulosa cells, or on specific binding of 125I-IGF-II to thecal cells. TNFalpha did not compete for 125I-IGF-I binding to granulosa or thecal cells whereas unlabeled IGF-I suppressed 125I-IGF-I binding. Insulin inhibited (P<0.10) whereas FSH had no effect on the number of specific 125I-TNFalpha binding sites in granulosa cells. In contrast, LH increased (P<0.10) whereas insulin had no effect on specific 125I-TNFalpha binding sites in thecal cells. These results suggest that IGF-I and TNFalpha receptors in granulosa and thecal cells are regulated by hormones differentially.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

Insulin receptors on Chinese hamster ovary (CHO) cells: altered insulin binding to glycosylation mutants

We have identified and characterized insulin receptors on Chinese hamster ovary (CHO) cells. Insulin binds in a time, temperature and pH dependent fashion and insulin analogues compete for 125I-insulin binding in order of their biological potencies. Furthermore, two CHO cell glycosylation mutants, B4-2-1, lacking high mannose containing glycoproteins, and Lec 1.3c, lacking complex carbohydrate containing glycoproteins, bind insulin with a much higher and lower affinity respectively than wild type cells. This is the first report of insulin receptors on CHO cells and the first to use glycosylation mutants to study the effects of abnormal carbohydrates on insulin binding.     

Insulin-like growth factor I receptors in retinal rod outer segments

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

Mitogen-potentiating action and binding characteristics of insulin and insulin-like growth factors in Chinese hamster fibroblasts

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung fibroblasts (CC139) as well as continued growth of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the growth-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around 15 h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like growth factors (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 fibroblasts by interacting with the IGF-I receptor.     

Characterization of Insulin-Like Growth Factor-I Receptors in PC 12 Pheochromocytoma Cells and Bovine Adrenal Medulla

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig.

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.     

Insulin-like growth factor I/somatomedin C action on 2-deoxyglucose and alpha-amino isobutyrate uptake in chick embryo fibroblasts

Maximum 125I-IGF-I/Sm-C total binding to chick embryo fibroblasts was 3% at +37 degrees C and decreased to less than 1% in presence of 2.8 X 10(-9) M unlabelled IGF-I/Sm-C. Insulin did not compete with IGF-I/Sm-C for the binding to cells. Biological action of IGF-I/Sm-C was evaluated on 2-deoxyglucose and alpha-aminoisobutyrate uptake. Results are compared with those obtained with insulin. Maximal peptide effects on the two transport processes were obtained at a 0.65 X 10(-7) M concentration and for a 120 minute association time, whereas cells were markedly less sensitive to insulin and time response curves were different. These results suggest that insulin action on nutrient uptake in chick embryo fibroblasts is not mediated by the binding of the hormone to IGF-I/Sm-C receptors.     

Type I insulin-like growth factor receptors in human ovarian stroma

Hyperandrogenism observed in a variety of hyperinsulinemic states is thought to be due to an effect of insulin mediated through the type I insulin-like growth factor (IGF) receptors. These receptors, however, have not yet been demonstrated in normal human ovarian cells capable of androgen production. We now report the presence of type I IGF receptors in membrane preparations of human ovarian stroma. The ovarian stromal tissue was obtained from women undergoing indicated oophorectomy. Stromal plasma membranes were prepared. Specific 125I-IGF-I binding was 6.6 +/- 0.2%/100 micrograms protein. The affinity constant estimated by Scatchard analysis was 4.6 X 10(-9) M. 50% inhibition of 125I-IGF-1 binding was observed at 5 ng/ml of IGF-1. Specificity of the 125I-IGF-I-binding sites was confirmed by analogue specificity studies and in experiments utilizing monoclonal antibody to the IGF-I receptor, alpha-IR-3. IGF-II and insulin competed with 125I-IGF-I for the binding sites, but with an affinity significantly lower than that of IGF-I: 50% inhibition was observed at approximately 60 ng/ml of IGF-II or insulin. alpha-IR-3, a monoclonal antibody with high specificity for the type I IGF receptor, effectively inhibited 125I-IGF-I binding in a dose-dependent manner, confirming that the 125I-IGF-I binding was indeed to the type I IGF receptor. We conclude that type I IGF receptors are present in human ovarian stroma. These receptors may mediate effects of insulin on the ovary in hyperinsulinemic insulin-resistant states.     

Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I.

Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 ± 0.30%/10⁵ cells (SEM, n=9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (αIR-3) inhibited ¹²⁵I-IGF-I but not ¹²⁵I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the αsubunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays.

IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.     

Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9).

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.     

Evidence for a subtype of insulin-like growth factor I receptor in brain

We examined the structure of receptors for insulin-like growth factor I (IGF-I), insulin, and epidermal growth factor (EGF) in human brain and human placenta using affinity cross-linking procedures and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In human brain, proteins specifically cross-linked to 125I-IGF-I, 125I-insulin, and 125I-EGF had apparent molecular weights of 120,000, 115,000 and 170,000, respectively. In human placenta, proteins cross-linked to 125I-IGF-I and 125I-insulin were 10 kDa larger than the corresponding subunits in brain. The receptor labeled by 125I-EGF in placenta was indistinguishable from the EGF receptor in brain. The size discrepancy of IGF-I receptors in brain and placenta was no longer apparent after removing the carbohydrate moieties of the proteins with endo-beta-N-acetylglucosaminidase F (EndoF). Furthermore, the brain IGF-I receptor was not cleaved by neuraminidase, whereas, the placental IGF-I receptor had increased mobility on SDS gels following neuraminidase treatment. The results indicate that receptors for IGF-I and insulin in human brain are structurally distinct from the corresponding receptors in human placenta, the structural heterogeneity of the receptors involves differences in N-linked glycosylation, particularly the terminal processing steps, and EGF receptors are present in human brain and human placenta but are structurally similar in these tissues. We conclude that there is a selective modification in the glycosylation of receptors for IGF-I and insulin in brain.     

Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells

Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.     

An altered IGF-I receptor is present in human leukemic cells.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.