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. 2007 May;27(10):3569-77.
doi: 10.1128/MCB.01447-06. Epub 2007 Feb 26.

Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors

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Free PMC article

Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors

Adam Denley et al. Mol Cell Biol. .
Free PMC article

Erratum in

  • Mol Cell Biol. 2007 Sep;27(17):6264

Abstract

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.

Figures

FIG. 1.
FIG. 1.
Activation of IR-A and IR-B by insulin, IGF-II, and IGF-I. R IR-A and IR-B cells were serum starved overnight and treated with 10 nM ligand for 5 min. (A and B) Lysates were blotted for IR pY960 (A) or IR pY1158/1162/1163 (B), and phospho-IR levels were normalized for IR β-subunit levels in either R IR-A or R IR-B cells by stripping and reprobing with an IR β-subunit antibody. (C and D) IR immunoprecipitates were blotted with anti-phosphotyrosine (PY20) antibody. Total IR phosphorylation was also normalized for IR β-subunit levels by stripping and reprobing with an IR β-subunit antibody. Panel D shows a representative blot of the experiment shown in panel C and also illustrates that the R IR-B cells express ∼50% of the level of IR in the R IR-A cells. The data in panel C are represented as percentages of the maximal (max) level of phosphorylation seen with insulin, since the lack of background signal in the basal PY20 immunoprecipitation samples precluded calculation of the increase over basal values. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to basal levels of phosphorylation or, where indicated, between the bracketed samples. P values are represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. IP, immunoprecipitation; WIB, Western immunoblotting.
FIG. 2.
FIG. 2.
Time course of IRS-1 and IRS-2 activation by insulin, IGF-II, and IGF-I. Cells were treated as described in the legend to Fig. 1, and the lysates were immunoprecipitated (IP) with anti-IRS-1 and IRS-2 antibodies, followed by Western immunoblotting with anti-IRS and PY20 antibodies. (A) IRS-1 activation in R IR-A cells; (B) IRS-1 activation in R IR-B cells; (C) IRS-2 activation in R IR-A cells; (D) IRS-2 activation in R IR-B cells. Representative blots are shown beneath each graph. In each case, the PY20 signal was normalized for IRS-1 or IRS-2 levels as determined by blotting with the respective antibody separately for each sample. Error bars represent SEM of three independent experiments. The bottom portion of the gel image in each case corresponds to a single representative IRS-1 or IRS-2 control blot. max, maximum; pIRS-1, phosphorylated IRS-1; pIRS-2, phosphorylated IRS-2.
FIG. 3.
FIG. 3.
Graphical summary of the 5-min activation of IRS-1 and IRS-2 from Fig. 2. (A) IRS-1 activation in R IR-A cells; (B) IRS-1 activation in R IR-B cells; (C) IRS-2 activation in R IR-A cells; (D) IRS-2 activation in R IR-B cells. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to basal levels of phosphorylation or, where indicated, between the bracketed samples. P values are represented as follows: **, P < 0.01; ***, P < 0.001. NS, not significant; max, maximum.
FIG. 4.
FIG. 4.
IRS-1 and IRS-2 activation by insulin, IGF-II, and IGF-I in NIH 3T3/IR-A cells. Cells were treated and analyzed as described in the legend to Fig. 2. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to basal levels of phosphorylation or, where indicated, between the bracketed samples. P values are represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant; IP, immunoprecipitation; WB, Western blotting; max, maximum.
FIG. 5.
FIG. 5.
Effect of Long R3 IGF-I on IRS-2 activation in (A) R IR-A cells and (B) NIH 3T3/IR-A cells. Cells were treated and analyzed as described in the legend to Fig. 2. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to basal levels of phosphorylation or, where indicated, between the bracketed samples. P values are represented as follows: **, P < 0.01; ***, P < 0.001. NS, not significant; max, maximum; IP, immunoprecipitation; WB, Western blotting.
FIG. 6.
FIG. 6.
Effect of IR transfection on IGF-I and Long R3 IGF-I activation of IRS-2. Control R cells and RI R-A and R IRB cells were treated with 10 nM IGF-I (open bars) or Long R3 IGF-I (closed bars) and analyzed as described in the legend to Fig. 2. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to levels of phosphorylation in IGF-I-treated R cells or, where indicated, between the bracketed samples. P values are represented as follows: **, P < 0.01; ***, P < 0.001. NS, not significant; max, maximum; IP, immunoprecipitation; WB, Western blotting.
FIG. 7.
FIG. 7.
Time course of Akt activation by insulin and IGF-I in R IR-B cells. Cells were treated with 10 nM ligand over 60 min. (A) Graphical representation of three experiments. Results are expressed as a percentage of the maximal (max) Akt phosphorylation seen with insulin. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to basal levels of Akt phosphorylation. P values are represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Representative Western immunoblot. pAKT, phosphorylated Akt.
FIG. 8.
FIG. 8.
Effect of 10 nM insulin, IGF-II, and IGF-I on R IR-A (A) and R IR-B (B) cell survival from butyrate-induced apoptosis relative to serum-free and 5 mM butyrate controls. Results are expressed as a percentage of the maximal survival response stimulated by 200 nM insulin. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to butyrate-treated control. P values are represented as follows: *, P < 0.05; ***, P < 0.001.
FIG. 9.
FIG. 9.
Proliferation/viability of R IR-B cells after IRS-2 knockdown with siRNA transfection. (A) Western immunoblot of IRS-2 and IRS-1 after 48-h transfection of negative-control and IRS-2 siRNAs. (B) WST assay with negative-control siRNA Smartpool. (C) WST assay with IRS-2 siRNA Smartpool. Error bars represent SEM of three independent experiments. Statistical significance shown is in relation to vehicle control at each time point. P values are represented as follows: **, P < 0.01; ***, P < 0.001.

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