Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Abstract

Background: Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.

Methodology/principal findings: In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.

Conclusions/significance: This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

Figure 1. The amino acid sequence alignment of IGF-II, IGF-I, and insulin.

Insulin residues important for IR binding and defined as site 1 residues (ValB12, TyrB16, GlyB23, PheB24, Phe B25, TyrB26, GlyA1, IleA2, ValA3, GlnA5, TyrA19, AsnA21) are shown in bold type and those defined as site 2 residues (HisB10, GluB13, LeuB17, SerA12, LA13, Glu17) are underlined and in italics . IGF-I and IGF-II site 2 residues are underlined and in italics. Conserved residues are boxed in light gray, residues conserved between IGF-II and IGF-I are boxed in dark gray and the domain structure is below. Residue Glu 12 of IGF-II mutated in this study is highlighted with an asterisk.

Figure 2. Competitive binding of IGF-II and Glu12 mutants to IGF-1R and IR-A either solubilised or on intact cells.

Immunocaptured solubilised IGF-1R and IR-A (A and B) or P6 IGF-1R and R⁻IR-A cells (C and D) were incubated with Eu-IGF-II in the presence or absence of increasing concentrations of IGF-II (•), Glu12Asp IGF-II (▴), Glu12Ala IGF-II (▪), Glu12Gln IGF-II (▵), Glu12His IGF-II (○), Glu12Arg IGF-II (▿) or Glu12Lys IGF-II (□). Results are expressed as a percentage of binding in the absence of competing ligand (%B/Bo), and the data points are the mean±SEM of at least three separate experiments with each point performed in triplicate. Error bars are shown when greater than the size of the symbols.

Figure 3. Dose-response curves for negative cooperativity.

Accelerated dissociation of prebound Eu-IGF-II in the presence of increasing concentrations of (A) IGF-II (•), Glu12Asp IGF-II (▴),Glu12Gln IGF-II (▵), Glu12His IGF-II (○) and (B) IGF-II (•), Glu12Ala IGF-II (▪), Glu12Arg IGF-II (▿) or Glu12Lys IGF-II (□) from the IGF-IR on P6 IGF-1R cells. Results are expressed as a percentage of binding in the absence of competing ligand (%B/Bo) after 30 min, and the data points are the mean±SEM of three assays with each concentration measured in triplicate. Error bars are shown when greater than the size of the symbols. Curves are separated into two graphs for clarity.

Figure 4. Activation of the IGF-1R and IR-A by IGF-II and Glu12 mutants.

P6-IGF-1R cells (A) and R⁻IR-A cells (B) were serum starved for 4 h and then incubated with increasing concentrations of IGF-II (•), Glu12Asp IGF-II (▴), Glu12Ala IGF-II (▪), Glu12Gln IGF-II (▵), Glu12His IGF-II (○), Glu12Arg IGF-II (▿) or Glu12Lys IGF-II (□) for 10 min. Solubilised IGF-1R (A) and IR-A (B) were immunocaptured, and phosphorylated tyrosines were detected with Eu-PY20. Receptor phosphorylation is expressed as a percentage of the maximal phosphorylation induced by IGF-II. The data points are means±SEM of three assays with each concentration measured in triplicate. Error bars are shown when greater than the size of the symbols.

Figure 5. Induction of Akt phosphorylation upon IGF-1R and IR-A activation by IGF-II and Glu12 mutants.

Serum-starved P6 IGF-1R (A) and R⁻IR-A cells (B) were treated with IGF-II, Glu12Asp IGF-II, Glu12Ala IGF-II, Glu12Gln IGF-II, Glu12His IGF-II, Glu12Arg IGF-II or Glu12Lys IGF-II at 10 nM (hatched bars) or 100 nM (solid bars) for 10 min. Whole-cell lysates were prepared and subjected to SDS-PAGE and then immunoblotted for phosphorylated Akt (pAkt). Representative blots are shown in the lower panels, and lanes 1–9 are 10–15 are from separate blots in both A and B. Each blot included lanes from cells untreated (basal) and treated with 100 nM IGF-II. In upper panels densitometry of three independent experiments ± SEM are shown as a column graph. Relative pAkt levels are expressed as a fraction of the level detected when cells were stimulated with 100 nM IGF-II. In each case pAkt was expressed as a fraction of the loading control (β-tubulin). a = p value<0.001 , b = p value 0.001 to 0.01, c = p value 0.01 to 0.05 when compared to IGF-II at the same concentration.