Insulin, IGF-II and a low-affinity insulin analog differentially regulate insulin receptor isoform A trafficking and induce a different balance of metabolic and mitogenic effects
A. Morcavallo1, M. Genua1, A. Palummo1, E. Kletvikova3, J. Jiracek3, A. Brzozowski4, R. Iozzo2, A. Belfiore1 & A. Morrione2
Introduction: The isoform A of the human insulin receptor (IR-A) binds insulin with high affinity, and binds IGF-II with a 310 folds lower affinity. Cells lacking the insulin-like Growth Factor-I receptor (IGF-IR) and overexpressing the human IR-A (R-/IR-A cells) respond to IGF-II with reduced metabolic effects but unaltered or increased mitogenesis as compared to insulin stimulation. We hypothesized that this altered ratio of metabolic-to-mitogenic effects of IGF-II in R-/IR-A cells may depend on the differential regulation of IR-A trafficking. Moreover, we hypothesized that similar different biological effects are features of other low-affinity IR-A ligands.
Methods: We used R-/IR-A cells and evaluated IR-A phosphorylation and trafficking after stimulation with insulin, IGF-II and a synthetic insulin analog (NMeTyrB26-insulin) that binds IR-A with a similar affinity to IGF-II.
Results: We found that NMeTyrB26-insulin has a similar mitogenic activity than IGF-II. Insulin induced strong IR-A phosphorylation, which was instead reduced after IGF-II and NMeTyrB26-insulin stimulation. A clear reduction in cell surface IR-A was strongly induced by insulin, while IGF-II and NMeTyrB26-insulin promoted only modest IR-A internalization. Prolonged cell stimulation with insulin, but not with IGF-II or NMeTyrB26-insulin, targeted the IR-A for proteosomal and lysosomal degradation despite similar levels of IR-A ubiquitination. IRS-1 was also down-regulated by insulin but not by IGF-II or NMeTyrB26-insulin. Clathrin-dependent endocytosis was critical for IR-A-dependent Akt activation, while clathrin-independent IR-A endocytosis regulated ERKs activation. p70S6K activation was instead slightly increased by inhibiting IR-A internalization.
Conclusions: The lower IR-A phosphorylation elicited by IGF-II or NMeTyrB26-insulin, as compared to insulin, may protect IR-A and IRS-1 from negative feed-back down-regulation mechanisms, thus sustaining potent mitogenic stimuli in spite of decreased metabolic activity. These data may have profound implications in the design of insulin analogs and in our understanding of the role of IGF-II in cancer.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.