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Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes.

Rajapaksha H, Forbes BE - Front Endocrinol (Lausanne) (2015)

Bottom Line: The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization.With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential.We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Adelaide , Adelaide, SA , Australia.

ABSTRACT
The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His(4), Tyr(15), Thr(49), Ile(51)) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of AKT (pS473) and MAPK. Serum-starved R− IR-A cells were treated with 10 or 100 nM insulin (INS), IGF-II, qIGF-I, or S597 for 10 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (A) and pMAPK (B). Representative blots are shown in the lower panels. Relative phosphorylation levels are expressed as a fraction of the level detected when cells were stimulated with 10 nM insulin for 10 min. Basal phosphorylation in the presence of serum-free medium (SFM) is shown. Data are normalized to the loading control eIF4E. ***p value < 0.001, **p value 0.001–0.01, *p value 0.01–0.05. R− IR-A cells were also treated with 10 nM insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) over 30 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (C) and pMAPK (D). Relative phosphorylation levels are expressed as a percentage of the level detected when cells were stimulated with 10 nM insulin for 30 min (100%). In each case, the loading control eIF4E was used. Graphs represent the average of three independent experiments ± SEM. Error bars are shown when greater than the size of the symbols.
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Figure 4: Phosphorylation of AKT (pS473) and MAPK. Serum-starved R− IR-A cells were treated with 10 or 100 nM insulin (INS), IGF-II, qIGF-I, or S597 for 10 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (A) and pMAPK (B). Representative blots are shown in the lower panels. Relative phosphorylation levels are expressed as a fraction of the level detected when cells were stimulated with 10 nM insulin for 10 min. Basal phosphorylation in the presence of serum-free medium (SFM) is shown. Data are normalized to the loading control eIF4E. ***p value < 0.001, **p value 0.001–0.01, *p value 0.01–0.05. R− IR-A cells were also treated with 10 nM insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) over 30 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (C) and pMAPK (D). Relative phosphorylation levels are expressed as a percentage of the level detected when cells were stimulated with 10 nM insulin for 30 min (100%). In each case, the loading control eIF4E was used. Graphs represent the average of three independent experiments ± SEM. Error bars are shown when greater than the size of the symbols.

Mentions: gpMAPK stimulated by 10 nM ligand: insulin was denoted 100%, from Figures 2 and 4.


Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes.

Rajapaksha H, Forbes BE - Front Endocrinol (Lausanne) (2015)

Phosphorylation of AKT (pS473) and MAPK. Serum-starved R− IR-A cells were treated with 10 or 100 nM insulin (INS), IGF-II, qIGF-I, or S597 for 10 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (A) and pMAPK (B). Representative blots are shown in the lower panels. Relative phosphorylation levels are expressed as a fraction of the level detected when cells were stimulated with 10 nM insulin for 10 min. Basal phosphorylation in the presence of serum-free medium (SFM) is shown. Data are normalized to the loading control eIF4E. ***p value < 0.001, **p value 0.001–0.01, *p value 0.01–0.05. R− IR-A cells were also treated with 10 nM insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) over 30 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (C) and pMAPK (D). Relative phosphorylation levels are expressed as a percentage of the level detected when cells were stimulated with 10 nM insulin for 30 min (100%). In each case, the loading control eIF4E was used. Graphs represent the average of three independent experiments ± SEM. Error bars are shown when greater than the size of the symbols.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493403&req=5

Figure 4: Phosphorylation of AKT (pS473) and MAPK. Serum-starved R− IR-A cells were treated with 10 or 100 nM insulin (INS), IGF-II, qIGF-I, or S597 for 10 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (A) and pMAPK (B). Representative blots are shown in the lower panels. Relative phosphorylation levels are expressed as a fraction of the level detected when cells were stimulated with 10 nM insulin for 10 min. Basal phosphorylation in the presence of serum-free medium (SFM) is shown. Data are normalized to the loading control eIF4E. ***p value < 0.001, **p value 0.001–0.01, *p value 0.01–0.05. R− IR-A cells were also treated with 10 nM insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) over 30 min. Whole-cell lysates were immunoblotted for phosphorylated pAKT (pS473) (C) and pMAPK (D). Relative phosphorylation levels are expressed as a percentage of the level detected when cells were stimulated with 10 nM insulin for 30 min (100%). In each case, the loading control eIF4E was used. Graphs represent the average of three independent experiments ± SEM. Error bars are shown when greater than the size of the symbols.
Mentions: gpMAPK stimulated by 10 nM ligand: insulin was denoted 100%, from Figures 2 and 4.

Bottom Line: The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization.With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential.We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Adelaide , Adelaide, SA , Australia.

ABSTRACT
The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His(4), Tyr(15), Thr(49), Ile(51)) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

No MeSH data available.


Related in: MedlinePlus