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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 [(His4, Tyr15, Thr49, Ile51) 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.
Highlights
The insulin and insulin-like growth factor (IGF) system comprises the three highly similar ligands
Insulin bound to the insulin receptor (IR)-A with an EC50 of 1.57 nM, whereas insulin-like growth factor-II (IGF-II) bound with an IC50 of 15.21 nM (Table 1), a 10-fold lower affinity than insulin. qIGF-I bound IR-A with a threefold lower affinity than insulin, and of all the ligands, S597 had the highest affinity for the IRA with an IC50 of 0.75 nM
IR-A Tyrosine Phosphorylation IR-A Total Tyrosine Phosphorylation Induced by Different Ligand Concentrations 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
Summary
The insulin and insulin-like growth factor (IGF) system comprises the three highly similar ligands (insulin, IGF-I, and IGF-II). Insulin binds with high affinity to the IR to promote metabolic signaling and IGF-I and IGF-II act via the IGF-1R and hybrid receptors to promote mitogenic signaling, such as cell survival, growth, and proliferation [2, 3]. Whereas the IR-B includes the 12 amino acids encoded by exon 11 [5] and its activation by insulin leads to metabolic actions, the IR-A binds both IGF-II and insulin with high affinity to promote mitogenic outcomes [6,7,8]. Cancer cells can use this pathway as an additional or alternate mitogenic pathway to signaling via the type-1 IGF receptor (IGF-1R), and can provide a mechanism by which cancer cells can become resistant to treatments targeting the IGF-1R [4, 12]. An understanding of how mitogenic processes are activated downstream of the IR-A will lead to improved strategies to inhibit this signaling pathway and will potentially provide novel cancer treatments
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