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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 IR-A in response to insulin, IGF-II, qIGF-I, and S597. R− IR-A cells were incubated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) for 10 min (A) or 30 min (B) following 4 h serum starvation. Solubilized IR-A was immunocaptured, and phosphorylated tyrosines were detected with Eu-PY20. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at 10−6 M. Basal phosphorylation in serum-starved cells was 2% (not shown). In addition, R− IR-A cells were incubated with 10 nM (C) or 100 nM (D) ligand for increasing times up to 30 min. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at t = 30 min. 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.
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Figure 1: Phosphorylation of IR-A in response to insulin, IGF-II, qIGF-I, and S597. R− IR-A cells were incubated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) for 10 min (A) or 30 min (B) following 4 h serum starvation. Solubilized IR-A was immunocaptured, and phosphorylated tyrosines were detected with Eu-PY20. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at 10−6 M. Basal phosphorylation in serum-starved cells was 2% (not shown). In addition, R− IR-A cells were incubated with 10 nM (C) or 100 nM (D) ligand for increasing times up to 30 min. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at t = 30 min. 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.

Mentions: In order to measure the ability of the ligands to activate IR-A phosphorylation upon binding R− IR-A cells were stimulated with a series of concentrations (0.3–1000 nM) of insulin, IGF-II, qIGF-I, or S597 and the potency of each ligand to phosphorylate IR-A was measured using a kinase receptor activation (KIRA) assay. The highest level of total tyrosine phosphorylation induced by insulin was achieved at 1000 nM after 10 min stimulation, although phosphorylation had not reached a maximum at this concentration (Figure 1A). IGF-II, which had a 10-fold lower affinity for IR-A, was less potent than insulin in stimulating IR-A phosphorylation. There was a rightward shift in EC50 and the highest level of total tyrosine phosphorylation induced by IGF-II was achieved at 1000 nM but maximal phosphorylation was not reached (Figure 1A; Table 2). Unexpectedly, at 10 min stimulation, qIGF-I, which had a threefold lower affinity for IR-A than insulin, was the most potent ligand in stimulating total phosphorylation, with maximal tyrosine phosphorylation achieved at 300 nM and higher concentrations resulted in less than maximal phosphorylation. S597, which had a twofold higher affinity for IR-A, induced a similar total tyrosine phosphorylation to insulin after 10 min stimulation but maximal IR-A tyrosine phosphorylation was achieved at 300 nM. Generally, the pattern of phosphorylation induced by insulin, IGF-II, and qIGF-I after 30 min stimulation (Figure 1B; Table 2) was similar to that seen after 10 min stimulation (Figure 1A). Interestingly, however, S597 was the most potent ligand at 30 min stimulation with a lower EC50 and a greater maximal response than qIGF-I, insulin, or IGF-II.


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 IR-A in response to insulin, IGF-II, qIGF-I, and S597. R− IR-A cells were incubated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) for 10 min (A) or 30 min (B) following 4 h serum starvation. Solubilized IR-A was immunocaptured, and phosphorylated tyrosines were detected with Eu-PY20. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at 10−6 M. Basal phosphorylation in serum-starved cells was 2% (not shown). In addition, R− IR-A cells were incubated with 10 nM (C) or 100 nM (D) ligand for increasing times up to 30 min. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at t = 30 min. 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Phosphorylation of IR-A in response to insulin, IGF-II, qIGF-I, and S597. R− IR-A cells were incubated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼) for 10 min (A) or 30 min (B) following 4 h serum starvation. Solubilized IR-A was immunocaptured, and phosphorylated tyrosines were detected with Eu-PY20. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at 10−6 M. Basal phosphorylation in serum-starved cells was 2% (not shown). In addition, R− IR-A cells were incubated with 10 nM (C) or 100 nM (D) ligand for increasing times up to 30 min. The receptor phosphorylation is expressed as a percentage of the phosphorylation induced by insulin at t = 30 min. 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.
Mentions: In order to measure the ability of the ligands to activate IR-A phosphorylation upon binding R− IR-A cells were stimulated with a series of concentrations (0.3–1000 nM) of insulin, IGF-II, qIGF-I, or S597 and the potency of each ligand to phosphorylate IR-A was measured using a kinase receptor activation (KIRA) assay. The highest level of total tyrosine phosphorylation induced by insulin was achieved at 1000 nM after 10 min stimulation, although phosphorylation had not reached a maximum at this concentration (Figure 1A). IGF-II, which had a 10-fold lower affinity for IR-A, was less potent than insulin in stimulating IR-A phosphorylation. There was a rightward shift in EC50 and the highest level of total tyrosine phosphorylation induced by IGF-II was achieved at 1000 nM but maximal phosphorylation was not reached (Figure 1A; Table 2). Unexpectedly, at 10 min stimulation, qIGF-I, which had a threefold lower affinity for IR-A than insulin, was the most potent ligand in stimulating total phosphorylation, with maximal tyrosine phosphorylation achieved at 300 nM and higher concentrations resulted in less than maximal phosphorylation. S597, which had a twofold higher affinity for IR-A, induced a similar total tyrosine phosphorylation to insulin after 10 min stimulation but maximal IR-A tyrosine phosphorylation was achieved at 300 nM. Generally, the pattern of phosphorylation induced by insulin, IGF-II, and qIGF-I after 30 min stimulation (Figure 1B; Table 2) was similar to that seen after 10 min stimulation (Figure 1A). Interestingly, however, S597 was the most potent ligand at 30 min stimulation with a lower EC50 and a greater maximal response than qIGF-I, insulin, or IGF-II.

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