IGF-I Receptor Beta-subunit Research Articles

Expression and function of receptors for insulin-like growth factor-I and insulin in human coronary artery smooth muscle cells

Hyperinsulinaemia and insulin resistance, as well as low IGF-I, have been implicated in the pathogenesis of cardiovascular disease. Little is known about direct effects of IGF-I and insulin on human coronary artery smooth muscle cells (HCASMCs). Our aim was to characterise the expression and function of IGF-I receptor (IGF-IR) and insulin receptor (IR) in HCASMCs. Cultured HCASMCs were used. mRNA expression was measured by quantitative real-time RT-PCR analysis. Receptor proteins, phosphorylation of beta-subunits and the presence of hybrid IR/IGF-IR were analysed by immunoprecipitation and western blotting. DNA synthesis and glucose metabolism were assessed using [3H]thymidine incorporation and D-[U-14C]glucose accumulation respectively. The mRNA expression of IGF-IR was approximately eight-fold higher than that of IR in HCASMCs. The presence of IGF-IR and IR could be demonstrated by immunoprecipitation and western blot analysis. Phosphorylation of the IGF-IR beta-subunit was obtained by IGF-I at 10(-10)-10(-8) mol/l and insulin at 10(-8) mol/l. Insulin and IGF-I at 10(-10)-10(-9) mol/l phosphorylated the IR beta-subunit. When immunoprecipitated with monoclonal anti-IR alpha-subunit or IGF-IR alpha-subunit antibodies, we found bands in slightly different positions, suggesting the presence of hybrid IR/IGF-IR. IGF-I at 10(-9)-10(-8) mol/l significantly stimulated [3H]thymidine incorporation and at a concentration of 10(-9)-10(-7) mol/l also D-[U-14C]glucose accumulation in HCASMCs. Insulin at 10(-9)-10(-7) mol/l had no effect on DNA synthesis, but increased glucose accumulation at 10(-7) mol/l. Our study provides experimental evidence that IGF-IR and possibly hybrid IR/IGF-IR play a role in HCASMCs.

Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.

Micro- and macroangiopathy are major causes of morbidity and mortality in patients with diabetes. Our aim was to characterize IGF-I receptor (IGF-IR) and insulin receptor (IR) in human micro- and macrovascular endothelial cells. Cultured human dermal microvascular endothelial cells (HMVEC) and human aortic endothelial cells (HAEC) were used. Gene expression was measured by quantitative real-time RT-PCR and receptor protein by ligand-binding assay. Phosphorylation of IGF-IR beta-subunit was analyzed by immunoprecipitation and Western blot. Glucose metabolism and DNA synthesis was assessed using [(3)H]glucose and [(3)H]thymidine incorporation, respectively. We detected gene expression of IGF-IR and IR in HAEC and HMVEC. IGF-IR gene expression was severalfold higher than that of IR. The specific binding of (125)I-IGF-I was higher than that of (125)I-insulin in HAEC and HMVEC. Insulin and the new, long-acting insulin analog glargine interacted with the IGF-IR with thousand- and hundred-fold less potency than IGF-I itself. Phosphorylation of the IGF-IR beta-subunit was shown in HAEC for IGF-I (10(-8) M) and insulin (10(-6) M) and in HMVEC for IGF-I and glargine (10(-8) M, 10(-6) M). IGF-I 10(-7) M stimulated incorporation of [(3)H]thymidine into DNA, and 10(-9)-10(-7) M also the incorporation of [(3)H]glucose in HMVEC, whereas glargine and insulin had no significant effects at 10(-9)-10(-7) M. Human micro- and macrovascular endothelial cells express more IGF-IR than IR. IGF-I and high concentrations of glargine and insulin activates the IGF-IR. Glargine has a higher affinity than insulin for the IGF-IR but probably has no effect on DNA synthesis at concentrations reached in vivo.

Suppression of insulin-like growth factor signalling pathway and collagen expression in keloid-derived fibroblasts by quercetin: its therapeutic potential use in the treatment and/or prevention of keloids.

Keloids are characterized by abnormal proliferation of fibroblasts and overproduction of collagen. Insulin-like growth factor (IGF)-I is mitogenic for fibroblasts and a stimulatory factor for collagen synthesis. We have assessed the in vitro effects of quercetin on proliferation, collagen synthesis and the expression of the IGF system in keloid-derived fibroblasts. Fibroblasts were isolated from earlobe keloids and exposed to quercetin at different concentrations. The inhibitory effects of quercetin on fibroblast proliferation were assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western and Northern blot analyses. Quercetin inhibited keloid fibroblast (KF) proliferation in a dose-dependent manner. Significant growth inhibition was observed on day 2 of culture. The dose required for 50% growth inhibition was approximately 25 microg mL-1. Collagen 1 expression was significantly decreased while collagen 3 was almost undetectable following quercetin treatment. Basal levels of IGF-I receptor (IGF-IR) beta subunits, p85 subunit of phosphatidylinositol 3-kinase, c-Raf, phospho-Raf-1, phospho-MEK 1/2, phospho-mitogen-activated protein kinase, phospho-Elk-1 and phospho-Akt-1 were significantly reduced when KF cells were exposed to quercetin for 24 h. Blocking IGF-IR activity with IGF-IR antibody or neutralizing endogenous IGF-I activity with IGF-I antibody led to significant growth inhibition suggesting the role of IGF-I in regulation of KF proliferation. Because the IGF system plays an important part in fibroblast cell proliferation and collagen production, the described activities of quercetin on the IGF system and collagen expression may provide a novel approach for the use of quercetin in treatment and/or prevention of hypertrophic scar and keloid.

The Effects of Insulin-like Growth Factor-I (IGF-I) in Mouse Lung Cancer Cells.

Insulin-like growth factor-I (IGF-I) is an important mitogen in many types of malignancies. The purpose of this study was to evaluate the role of the IGF system on cell proliferation and cell death in mouse lung cancer cell lines (3LL). Northern analysis was performed in 3LL cells. We evaluated the phosphorylation of IGF-I receptor (IGF-IR) with IGF-I stimulation. MTT assay was performed after treating 3LL cells with IGF-I and the treatment effect on cell death in the presence of anticancer drug was investigated. Northern analysis revealed the presence of IGF-I and IGF-IR mRNA expression in 3LL cells. IGF-I increased cellular proliferation in serum free media. IGF-I also stimulated the tyrosine phosphorylation of two proteins: one, with a molecular mass of 95 kDa, was the beta-subunit of IGF-IR; the other, with an approximate molecular mass of 185 kDa, was originally identified as the insulin receptor substrate-I (IRS-I). IGF-I at a low concentration inhibited the cell death induced by adriamycin. IGF-I, a mitogen through the phosphorylation of the IGF-IR beta-subunit, acts as a survival factor to inhibit cell death. Therefore, these findings suggest that IGF-I and IGF-IR are involved in both the cell proliferation and cell death associated with cancer cell growth.

Aurintricarboxylic acid induces a distinct activation of the IGF-I receptor signaling within MDA-231 cells.

Aurintricarboxylic acid (ATA), a polymeric carboxylated triphenylmethane derivate, prevents apoptotic death in a variety of cell systems. Recently, we have shown that the survival promoting effect of ATA is transduced via activation of the IGF-I receptor (IGF-IR) signaling pathway. In breast cancer MDA-231 cells exposed either to the protein synthesis inhibitors cycloheximide or ricin or to the anticancer drug adriamycin, we have found that ATA, but not IGF-1, is a powerful antiapoptotic agent. The purpose of this study was to compare the ability of ATA and IGF-I to activate the IGF-IR signaling cascade and to correlate this ability to their survival potency. MDA-231 cells were exposed to ATA or IGF-I, up to 7 h, and the dynamics of activation of the IGF-IR signaling cascade was evaluated. Our results show that: 1) The amount of tyrosine phosphorylated IGF-IR proteins was greater after exposure to ATA, compared with IGF-I. 2) Two phosphorylated IGF-IR beta-subunits (a 95-kDa and a 75-kDa) were induced after exposure to ATA, whereas IGF-1 induced only the 95-kDa form. Immunoprecipitation of both receptor forms by antibodies against the alpha-subunit and against the carboxy terminus of the beta-subunit of the IGF-IR suggests that the 75-kDa form could be the beta-chain truncated at the amino terminus above the alpha-beta disulphide bridges. 3) The ATA-activated IGF-IR forms underwent slow dephosphorylation, compared with a rapid dephosphorylation of the IGF-I activated receptor. 4) The insulin receptor substrate-1/2-associated PI3K, Shc proteins, and the kinases Akt and Erk1/2, downstream mediators of the antiapoptotic signaling by IGF-IR, were activated to a higher extent and for a longer time period by ATA, compared with IGF-I. Taken together, the sustained activation of the IGF-IR signaling pathway by ATA may explain its stronger antiapoptotic effect. We suggest that this enhanced activity, and the different susceptibility of the IGF-IR to certain proteases and phosphatases, may indicate a distinct conformation of the ATA-activated IGF-IR.

Differential effects of insulin-like growth factor (IGF)-binding protein-3 and its proteolytic fragments on ligand binding, cell surface association, and IGF-I receptor signaling.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), the predominant IGF carrier protein in circulation, is posttranslationally modified in vivo by IGFBP-3 protease(s) into a number of fragments. Based on the ascertained and predicted recognition sites for known IGFBP-3 proteases, FLAG-epitope tagged intact IGFBP-3, NH2-terminal (1-97), intermediate fragment (88-148), and COOH-terminal fragments (98-264) and (184-264) were generated in a baculovirus and/or Escherichia coli expression system and examined, by Western ligand blot and affinity cross-linking assays, for their ability to bind IGF and insulin. The NH2- and COOH-terminal fragments bound both IGF and insulin specifically (albeit with significantly reduced affinity) for IGF but higher affinity for insulin, when compared with intact IGFBP-3. The effect of IGFBP-3 and the fragments on IGF-I receptor (IGFIR) signaling pathways was studied by testing IGF-I-induced receptor autophosphorylation in IGFIR-overexpressing NIH-3T3 cells. IGFBP-3 showed a dose-dependent inhibition of autophosphorylation of the beta-subunit of IGFIR. The (1-97)NH2-terminal fragment inhibited IGFIR autophosphorylation at high concentrations, and this effect seems largely attributable to sequestration of IGF-I. In contrast, no inhibition of IGF-I-induced IGFIR autophosphorylation was detectable with the (98-264) and (184-264) COOH-terminal fragments, despite their ability to bind IGF. However, unlike the (1-97)NH2-terminal fragment, the COOH-terminal fragments of IGFBP-3 retained their ability to associate with the cell surface, and this binding was competed by heparin, similar to intact IGFBP-3.

Double-stranded ribonucleic acid decreases C6 rat glioma cell numbers: effects on insulin-like growth factor I gene expression and action.

Poly(IC), a synthetic double-stranded RNA copolymer of inosinic and cytidilic acids, decreases the growth of normal and tumorigenic cells. We tested the hypothesis that Poly(IC) decreases C6 glioma cell growth by disrupting an autocrine insulin-like growth factor I (IGF-I) growth loop. Addition of Poly(IC) decreased C6 cell number in confluent and sparse cultures in a dose-dependent manner. Addition of exogenous IGF-I partially compensated for the decrease in cell number caused by Poly(IC) in confluent and subconfluent cultures of C6 cells, suggesting that one mechanism of Poly(IC) action is through down-regulation of IGF-I gene expression and/or action. Treatment of confluent C6 cells with 10 and 200 microg/ml Poly(IC) for 24 h decreased IGF-I messenger RNA (mRNA) levels to 50% and 25% of the control value, respectively. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGF-I receptor mRNA levels to 50% of the control level. IGF-binding protein-1 (IGFBP-1), -2, and -6 mRNAs were not expressed in the C6 cells used in this study. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGFBP-4 mRNA and IGFBP-5 mRNA levels to 26% and 29% of the control level, respectively. There was no significant change in IGFBP-3, insulin receptor, or actin mRNA levels with Poly(IC) treatment. Treatment of confluent C6 cells with 200 microg/ml Poly(IC) for 24 h decreased levels of immunoreactive IGF-I in conditioned medium (CM) to 55% of the control value, decreased IGF-I receptor beta-subunit levels to 28% of the control value, and decreased levels of IGFBP-3, IGFBP-4, and IGFBP-5 protein in CM to 45%, 50%, and 30% of the control values, respectively. There was no significant change in actin and tubulin protein levels with Poly(IC) treatment. These results suggest that IGF-I gene expression is down-regulated by Poly(IC) treatment and that IGF-I bioavailability and action in C6 cells are also altered due to decreases in IGF-I receptor and binding protein levels.

Stimulation of inorganic phosphate transport by insulin-like growth factor I and vanadate in opossum kidney cells is mediated by distinct protein tyrosine phosphorylation processes.

Insulin-like growth factor I (IGF-I) stimulates sodium-dependent inorganic phosphate (Pi) transport across the apical plasma membrane of confluent opossum kidney (OK) cells. Previous studies indicated that vanadate, at doses known to inhibit protein tyrosine phosphatases, mimicked the effect of IGF-I and suggested the involvement of tyrosine phosphorylation processes in Pi transport regulation. In this study, protein tyrosine phosphorylation and activation of several cellular signaling pathways were investigated in confluent OK cells in response to IGF-I and vanadate. We report that IGF-I and vanadate induced tyrosine phosphorylation of distinct proteins. Tyrosine phosphorylation of p95 (IGF-I receptor beta-subunit) was rapidly and dose dependently increased in response to IGF-I. Associated with phosphorylation of the receptor, the increase in tyrosine phosphorylation of a protein of 50 kDa was observed. Vanadate did not mimic the effect of IGF-I, but increased phosphorylation of seven major proteins of 170, 140, 100, 83, 70-82, 60, and 35 kDa. Among the different tyrosine kinase inhibitors tested, only staurosporine affected Pi transport up-regulation by IGF-I and vanadate, attenuating the effect of IGF-I and completely blocking the response to vanadate. Staurosporine decreased tyrosine phosphorylation of several constitutively phosphorylated proteins and interfered with the increase in tyrosine phosphorylation induced by vanadate. Phosphorylation of p95 in response to IGF-I was not affected. Staurosporine also markedly decreased constitutive association of the adapter protein Nck with tyrosine-phosphorylated proteins and attenuated increases in phosphotyrosine-associated Nck induced by IGF-I and vanadate. In contrast, signaling to other downstream effectors common to IGF-I and vanadate, such as mitogen-activated protein kinase and phosphatidylinositol-3-kinase, was not affected by staurosporine. In conclusion, our results suggest that although IGF-I and vanadate induce distinct protein tyrosine phosphorylation in OK cells, they activate an overlapping set of signaling molecules, among which Nck appears as an interesting candidate to link activation of tyrosine kinases to the stimulation of Pi transport.

Increased basal and induced tyrosine phosphorylation of the insulin- like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera

We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum- free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

Increased Basal and Induced Tyrosine Phosphorylation of the Insulin-Like Growth Factor I Receptor β Subunit in Circulating Mononuclear Cells of Patients With Polycythemia Vera

We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum-free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

The IGF-I receptor sub-membrane domain is intact in GH-secreting pituitary tumours.

Clinical acromegaly is characterized by dysregulation of somatotroph GH secretion in the presence of high circulating serum IGF-I levels. Physiologically, IGF-I exerts a negative feedback on GH secretion at both the hypothalamic and the pituitary levels. We have previously shown that the 943 and 950 tyrosine residues in the IGF-I receptor beta-subunit are required for ligand signalling to the GH gene, as substitution of these residues abrogates IGF-I signal transduction. To determine whether a mutation within the IGF-I receptor submembrane domain may be involved in the pathogenesis of GH secreting tumours, we studied this region in these tumours. Exon 15 of the IGF-I receptor containing both the 943 and 950 tyrosines was analysed in 19 GH-secreting tumours by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products. Tumour DNA and patients' lymphocyte DNA, which served as normal controls, were analysed. All samples exhibited normal migration patterns in the SSCP analysis which was further confirmed by direct DNA sequencing. We conclude that mutations in the IGF-I receptor sub-membrane domain which disrupt the negative feedback loop are not involved in the pathogenesis of acromegaly.

Insulin-like growth factor-I (IGF-I) dependent phosphorylation of the IGF-I receptor in MG-63 cells

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63. by acting through the IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of the IGF-I receptor in this cell line. Serum starved MG-63 cells were metabolically labeled with [32P]orthophosphoric acid and the cells were treated with IGF-I. Phosphotyrosine containing proteins were immunoprecipitated from the cell lysates with antiphosphotyrosine-Agarose and eluted with phenyl phosphate. Further immunoprecipitation with IGF-I receptor monoclonal antibodies (alpha IR-3, 18E9) and analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography demonstrated IGF-I dependent autophosphorylation of the IGF-I receptor. Phosphoamino acid analysis of the IGF-I receptor beta subunit and the observation that antiphosphotyrosine-Agarose did not immunoprecipitate [35S]methionine-labeled receptor from unstimulated cells, demonstrated that in the absence of IGF-I, the receptor was not phosphorylated on tyrosine residues. Western blotting of cell lysates with a monoclonal phosphotyrosine antibody did not identify the IGF-I receptor or pp185 but demonstrated IGF-I dependent phosphorylation on tyrosine residues in three other proteins, p110, p70 and p40.

A developmentally regulated form of insulin-like growth factor receptor beta-subunit in C2 myoblasts exhibiting altered requirements for differentiation.

Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of the C2 myoblast cell line. Permissive myoblasts differentiate spontaneously, whereas myoblasts of the inducible subclone require exogenous IGFs to undergo terminal differentiation. Permissive myoblasts contain beta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against tyrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP5) domains of the insulin receptor and insulin receptor monoclonal antibody 29B4. The tryptic phosphopeptide map of the 95-kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 101-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because it forms a hybrid complex with the 95-kDa protein, but it does not react directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 101-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit. Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its immunoreactivity and phosphopeptide map are virtually identical to those of the 101-kDa IGF-I receptor beta-subunit from permissive cells. However, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. These data demonstrate 1) an alpha 2 beta 2 IGF receptor that has high sensitivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodium dodecyl sulfate-polyacrylamide gels from either of those found in permissive cells; and 3) expression of this beta-subunit is developmentally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha 2 beta 2 IGF receptor subtype that functions as an IGF-II receptor.

Structure-function of the human insulin-like growth factor-I receptor: a discordance of somatotroph internalization and signaling.

Insulin-like growth factor-I (IGF-I), a GH-dependent growth factor, suppresses GH secretion by pituitary cells. To clarify the role of ligand-mediated receptor internalization for IGF-I signaling to GH, human IGF-I receptor (IGF-IR) cDNAs mutated in the beta-subunit were stably transfected into GC rat pituitary cells. Overexpression of wild-type IGF-IR markedly enhanced IGF-I suppression of GH 4-fold (P < 0.005) compared to that of untransfected cells. A mutant IGF-IR with a 943Tyr-->Ala substitution in the IGF-IR submembrane domain only partially suppressed GH (73% of IGF-IR wild type), while replacement of 957Tyr-->Ala or 940Gly-->Ala produced IGF-IRs that retained enhanced IGF-I signaling to GH. Substitution of 950Tyr-->Ala or 1003Lys-->Ala in the human IGF-IR beta-subunit failed to enhance IGF-I signaling to GH above that of untransfected cells. Intracellular phosphorylation of insulin-responsive substrate-I by these mutant IGF-IRs paralleled the observed IGF-I suppression of GH, with no phosphorylation of IRS-I by 950Tyr-->Ala. Ligand-mediated receptor internalization, however, was not reduced by substitution of either 943Tyr-->Ala or 950Tyr-->Ala. In contrast, substitution of 957Tyr-->Ala reduced the internalization of labeled IGF-I to 35% that of wild-type IGF-IR. Substitution of 1003Lys-->Ala abolished IGF-IR internalization, as expected. These results demonstrate that both 950Tyr and 943Tyr are important for IGF-I signaling to GH and that IGF-IR internalization is discordant for IGF-I signaling to the GH gene.

Human insulin-like growth factor I receptor 950tyrosine is required for somatotroph growth factor signal transduction.

Insulin-like growth factor I (IGF-I), a growth hormone (GH)-dependent growth factor exerts feedback regulation of GH by inhibiting GH gene expression. IGF-I inhibition of GH secretion is enhanced 3-5-fold in GC rat pituitary cells overexpressing the wild type 950Tyr human IGF-I receptor which autophosphorylates appropriately. To determine the critical amino acid sequence responsible for IGF-I signaling, insertion, deletion, and site-directed mutants were constructed to substitute for 950Tyr in exon 16 of the human IGF-I receptor beta-subunit transmembrane domain. All mutant transfectants bound IGF-I with a similar Kd to untransfected cells but had markedly increased (7-34-fold) IGF-I-binding sites. GH responsiveness to IGF-I was tested in mutant transfectants. Overexpressed site-directed and insertion mutant IGF-I receptors exhibited a modest suppressive effect on GH in response to the IGF-I ligand, similar to that observed in untransfected cells. Deletion mutant (IG-FIR delta 22) (amino acid 944-965) did not transduce the IGF-I signal to the GH gene. Site-directed and insertion mutants therefore did not enhance the IGF-I response of the endogenous rat receptor, unlike the 950Tyr wild type transfectants which enhanced the IGF-I signal. All mutant transfectants, except the deletion mutant, internalized radioactive ligand similarly to 950Tyr wild type transfectants. 950Tyr of the human IGF-I receptor is therefore required for IGF-I signal transduction in the pituitary somatotroph, but not for IGF-I-mediated internalization.

Neuronal protein tyrosine kinases associated with synaptosomal glycoproteins.

Protein tyrosine kinase (PTK) activity associated with synaptosomal membrane glycoprotein (SMGP) fractions of rat brain was examined. The synthetic substrate poly(Glu4-Tyr) was phosphorylated by SMGP in the presence of Mg2+ and Mn2+, whose stimulatory effects were additive. In contrast, endogenous tyrosine phosphorylation in SMGPs was strictly dependent on Mn2+. Anti-phosphotyrosine antibodies (PY20) immunoprecipitated two polypeptides in SMGPs of Mr 170K and 60K. Upon preincubation with IGF-I, 97/90K polypeptides were phosphorylated, corresponding to the IGF-I receptor beta-subunits, and were immunoprecipitated with both PY20 and anti-IGF-I-receptor antibodies. Immunoblot analysis using anti-src antibody revealed that there was src protein associated with the glycoprotein fractions of solubilized synaptosomal membranes. Additional experiments revealed that the 60K tyrosine-phosphorylated polypeptide present in the PY20 precipitates was indeed pp60c-src. This was confirmed by subjecting the PY20 immunoprecipitates to immunoblotting using anti-src antibodies. In addition, src protein was directly immunoprecipitated by anti-src antibodies from the SMGP preparations. Hence, IGF-I receptors and glycoprotein-associated PTKs including pp60c-src may play an important role in synaptic transmembrane signalling, plasticity, and neuronal survival.

Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor.

Monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor I (IGF-I) receptors were prepared and characterized. Three IgG mAbs were specific for the human IGF-I receptor and displayed negligible crossreactivity with the human insulin receptor. They stimulated 125I-labeled IGF-I (125I-IGF-I) or 125I-IGF-II binding to purified human placental IGF-I receptors and to IGF-I receptors expressed in NIH 3T3 cells in contrast to the well-studied mAb alpha IR-3, which inhibits 125I-IGF-I or 125I-IGF-II binding to both forms of IGF-I receptors. The mAbs introduced in this study stimulated DNA synthesis in NIH 3T3 cells expressing human IGF-I receptors approximately 1.5-fold above the basal level and the IGF-I- or IGF-II-stimulated level. In contrast, alpha IR-3 inhibited both basal and IGF-I or IGF-II-stimulated DNA synthesis by approximately 30%. Inhibition of IGF-II-stimulated DNA synthesis by alpha IR-3 was as potent as its inhibition of IGF-I-stimulated DNA synthesis, although IGF-II binding to the IGF-I receptors was not inhibited by IGF-II as potently as was IGF-I. With the purified IGF-I receptors, both inhibitory and stimulatory mAbs were shown to activate autophosphorylation of the IGF-I receptor beta subunit and to induce microaggregation of the receptors. These results suggest that conformational changes resulting from receptor dimerization in the presence of either type of mAb may affect the signal-transducing function of the IGF-I receptor differently. These additional mAbs and alpha IR-3 immunoprecipitated nearly 90% of IGF-I binding activity from Triton X-100-solubilized human placental membranes, indicating that IGF-I receptor reactive with these mAbs is the major form of the IGF-I receptor in human placenta.

Insulin like growth factor-I induces limited association of phosphatidylinositol 3-kinase to its receptor.

Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell line that overexpresses human IGF-I receptors, led to an increase in the amount of a phosphatidylinositol kinase that could be immunoprecipitated by anti-IGF-I receptor or anti-phosphotyrosine antibodies. The identity of the lipid produced in phosphatidylinositol kinase assays of anti-IGF-I receptor or anti-phosphotyrosine immunoprecipitates indicated that IGF-I selectively increased the amount of immunoprecipitated phosphatidylinositol 3-kinase activity. The amount of immunoprecipitated phosphatidylinositol 3-kinase activity that was increased by IGF-I followed a time course that paralleled the stimulation of IGF-I receptor beta-subunit autophosphorylation. The amount of phosphatidylinositol 3-kinase activity detected in anti-IGF-I receptor immunoprecipitates represented only 2% of that which was immunoprecipitated by anti-phosphotyrosine antibody. Furthermore, phosphatidylinositol 3-kinase activity which was recovered with anti-phosphotyrosine antibody was present in both cytosol and particulate cell fractions at approximately similar levels. Taken together, these results suggest that the stimulation of the IGF-I receptor tyrosine kinase leads to an increase in the amount of phosphatidyl inositol 3-kinase activity immunoprecipitated by antiphosphotyrosine and anti-IGF-I receptor antibodies and to a limited association with the IGF-I receptor itself, even though these cells express very high levels of IGF-I receptors. That the majority of phosphatidylinositol 3-kinase activity does not tightly associate with the IGF-I receptor after IGF-I stimulation suggests that it may be associated with other tyrosine phosphorylated proteins. Alternatively, the kinase itself may become phosphorylated on tyrosine and dissociate from the IGF-I receptor. In this manner, an increase of phosphatidylinositol 3-kinase activity by IGF-I deviates from the activation of phosphatidylinositol 3-kinase by platelet-derived growth factor receptor in that a tight association with the receptor is not produced after stimulation.

Gene expression and receptor binding of insulin-like growth factor-II in pig choroid plexus epithelial cells.

To elucidate the function of insulin-like growth factor-II (IGF-II) in the choroid plexus, the gene expression and receptor binding of IGF-II were studied in isolated epithelial cells from the porcine choroid plexus. The choroid plexus expressed multiple IGF-II transcripts of 1.2, 1.6, 2.4, and 4.4 kb, at levels higher than those found in porcine liver and kidney. These data suggest that IGF-II is synthesized by the choroid plexus. Choroid plexus epithelial cells contained high levels of IGF-I receptors on the cell surface whereas very low levels of receptor binding were found for 125I-IGF-II and 125I-insulin. Solubilization of epithelial cells showed that a large proportion of the IGF-I receptors were present in the detergent-insoluble fraction whereas IGF-II receptors and insulin receptors were concentrated in the detergent-soluble fraction. These results suggest that IGF-I receptors are located in clathrin-coated pits of the plasma membrane whereas IGF-II receptors and insulin receptors are present in endosomal vesicles. The tyrosine kinase activity of the IGF-I receptor beta-subunit was stimulated by IGF-I, IGF-II, and insulin, in order of potency, suggesting that these peptides exert a regulatory function in the choroid plexus epithelium. In conclusion, we propose that the IGF-I receptor tyrosine kinase on the surface of the epithelial cells in the pig choroid plexus mediates effects of IGF-I and IGF-II, whereas IGF-II receptors are down-regulated due to the synthesis and secretion of IGF-II in these cells.