Limits...
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

Time course of IR-A internalization (A) and mitogenic potency measured by [3H] thymidine incorporation (B) induced by insulin, IGF-II, qIGF-I, and S597. (A) Serum-starved R− IR-A cells were treated with 10 nM ligand in a time course of 30 min. The cell surface proteins were biotinylated and IR-A was immunocaptured after cell lysis. The immunocaptured IR-A was probed with Eu-streptavidin and biotinylated IR-A was measured by time-resolved fluorescence. Internalization was measured by comparing the proportion of IR-A on the cell surface (biotinylated) relative to the proportion in the presence of serum-free medium (SFM = 100%, black dotted line). 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. (B) L6 rat muscle myoblasts overexpressing the IR-A were treated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼). The results are illustrated as percentage incorporation of [3H] thymidine above basal (no stimulation). Curves are plotted using the average of three assays ± SEM with each concentration measured in triplicate, and are analyzed using a sigmoidal dose–response curve fit with variable slope (Graphpad Prism V5.04). Error bars are shown when greater than the size of the symbols. The EC50 was determined for insulin (0.8 ± 0.17 nM), IGF-II (4.4 ± 0.4 nM), and qIGF-I (1.15 ± 0.01 nM) but not for S597.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493403&req=5

Figure 5: Time course of IR-A internalization (A) and mitogenic potency measured by [3H] thymidine incorporation (B) induced by insulin, IGF-II, qIGF-I, and S597. (A) Serum-starved R− IR-A cells were treated with 10 nM ligand in a time course of 30 min. The cell surface proteins were biotinylated and IR-A was immunocaptured after cell lysis. The immunocaptured IR-A was probed with Eu-streptavidin and biotinylated IR-A was measured by time-resolved fluorescence. Internalization was measured by comparing the proportion of IR-A on the cell surface (biotinylated) relative to the proportion in the presence of serum-free medium (SFM = 100%, black dotted line). 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. (B) L6 rat muscle myoblasts overexpressing the IR-A were treated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼). The results are illustrated as percentage incorporation of [3H] thymidine above basal (no stimulation). Curves are plotted using the average of three assays ± SEM with each concentration measured in triplicate, and are analyzed using a sigmoidal dose–response curve fit with variable slope (Graphpad Prism V5.04). Error bars are shown when greater than the size of the symbols. The EC50 was determined for insulin (0.8 ± 0.17 nM), IGF-II (4.4 ± 0.4 nM), and qIGF-I (1.15 ± 0.01 nM) but not for S597.

Mentions: eIR internalization at 30 min by 10 nM ligand: surface/total IR insulin = 80%, from Figure 5A.


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)

Time course of IR-A internalization (A) and mitogenic potency measured by [3H] thymidine incorporation (B) induced by insulin, IGF-II, qIGF-I, and S597. (A) Serum-starved R− IR-A cells were treated with 10 nM ligand in a time course of 30 min. The cell surface proteins were biotinylated and IR-A was immunocaptured after cell lysis. The immunocaptured IR-A was probed with Eu-streptavidin and biotinylated IR-A was measured by time-resolved fluorescence. Internalization was measured by comparing the proportion of IR-A on the cell surface (biotinylated) relative to the proportion in the presence of serum-free medium (SFM = 100%, black dotted line). 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. (B) L6 rat muscle myoblasts overexpressing the IR-A were treated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼). The results are illustrated as percentage incorporation of [3H] thymidine above basal (no stimulation). Curves are plotted using the average of three assays ± SEM with each concentration measured in triplicate, and are analyzed using a sigmoidal dose–response curve fit with variable slope (Graphpad Prism V5.04). Error bars are shown when greater than the size of the symbols. The EC50 was determined for insulin (0.8 ± 0.17 nM), IGF-II (4.4 ± 0.4 nM), and qIGF-I (1.15 ± 0.01 nM) but not for S597.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Time course of IR-A internalization (A) and mitogenic potency measured by [3H] thymidine incorporation (B) induced by insulin, IGF-II, qIGF-I, and S597. (A) Serum-starved R− IR-A cells were treated with 10 nM ligand in a time course of 30 min. The cell surface proteins were biotinylated and IR-A was immunocaptured after cell lysis. The immunocaptured IR-A was probed with Eu-streptavidin and biotinylated IR-A was measured by time-resolved fluorescence. Internalization was measured by comparing the proportion of IR-A on the cell surface (biotinylated) relative to the proportion in the presence of serum-free medium (SFM = 100%, black dotted line). 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. (B) L6 rat muscle myoblasts overexpressing the IR-A were treated with increasing concentrations of insulin (●), IGF-II (■), qIGF-I (▲), or S597 (▼). The results are illustrated as percentage incorporation of [3H] thymidine above basal (no stimulation). Curves are plotted using the average of three assays ± SEM with each concentration measured in triplicate, and are analyzed using a sigmoidal dose–response curve fit with variable slope (Graphpad Prism V5.04). Error bars are shown when greater than the size of the symbols. The EC50 was determined for insulin (0.8 ± 0.17 nM), IGF-II (4.4 ± 0.4 nM), and qIGF-I (1.15 ± 0.01 nM) but not for S597.
Mentions: eIR internalization at 30 min by 10 nM ligand: surface/total IR insulin = 80%, from Figure 5A.

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