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Controlled Dimerization of Insulin-Like Growth Factor-1 and Insulin Receptors Reveal Shared and Distinct Activities of Holo and Hybrid Receptors

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Controlled Dimerization of Insulin-Like Growth Factor-1 and Insulin Receptors Reveal Shared and Distinct Activities of Holo and Hybrid Receptors JBC Papers in Press. Published on January 12, 2018 as Manuscript M117.789503 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.789503 Controlled dimerization of insulin-like growth factor-1 and insulin receptors reveal shared and distinct activities of holo and hybrid receptors Jingci Chen* a, b, Alison M. Nagle* b, c, Yu-Fen Wang b, David N. Boone b, e, Adrian V. Lee b, c, d, # aSchool of Medicine, Tsinghua University, Beijing 100084, China bWomen’s Cancer Research Center at the UPMC Hillman Cancer Center, cDepartment of Pharmacology & Chemical Biology, dDepartment of Human Genetics, and eDepartment of Biomedical Informatics, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA *Authors contributed equally to this work. Running title: Hybrid IGF1R/InsR signals and functions similar to holo-IGF1R Downloaded from Keywords: breast cancer, holo-IGF1R, holo-InsR, HybR, homodimer, heterodimer # Corresponding author: http://www.jbc.org/ Adrian V. Lee, Ph.D. Magee Womens Research Institute 204 Craft Avenue, Room A412 at University of Pittsburgh on January 18, 2018 Pittsburgh, PA 15213 1 Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Abstract (IGF2R), insulin receptor-A and B (InsR-A and InsR-B), and IGF-1/insulin hybrid receptor Breast cancer development and progression is (HybR) (2-4) play a crucial role in normal influenced by insulin-like growth factor 1 (IGF1R) mammary gland development and physiology (5). and insulin receptor (InsR) signaling that drive cancer phenotypes such as cell growth, IGF1R and InsR are both tetrameric proteins proliferation, and migration. IGF1R and InsR form composed of two extracellular α subunits IGF1R/InsR hybrid receptors (HybR) consisting of covalently linked to two intracellular β subunits, one molecule of IGF1R and one molecule of InsR. which contain the tyrosine kinase domains (6,7). The specific signaling and functions of HybR are The binding of ligands to IGF1R and InsR leads to largely unknown as HybR is activated by both conformational changes in structure and IGF1 and insulin, and no cellular system expresses transphosphorylation, which triggers the activation HybR in the absence of holo-IGF1R or holo-InsR. of two main cascades: the phosphatidylinositol 3- Here we studied the role of HybR by constructing kinase/AKT kinase (PI3K/AKT) pathway and the inducible chimeric receptors, and compared HybR RAF kinase/mitogen activated protein kinase Downloaded from signaling with that of holo-IGF1R and holo-InsR. (RAF/MAPK) pathway (4). Although IGF1R and We cloned chemically-inducible chimeric IGF1R InsR share common downstream pathways, their and InsR constructs consisting of the extracellular biological roles are not completely identical: domains of the p75 nerve growth factor receptor IGF1R mainly mediates proliferation, migration, fused to the intracellular β subunit of IGF1R or transforming, and anti-apoptotic events, whereas http://www.jbc.org/ InsR, and a dimerization domain. Dimerization InsR mainly controls the metabolism of glucose with the drugs AP20187 or AP21967 allowed (8). Nevertheless, there is crosstalk between specific and independent activation of holo- IGF1R and InsR: IGF-1 can also bind InsR and IGF1R, holo-InsR, or HybR, resulting in insulin can bind IGF1R, but with a lower affinity activation of the PI3K pathway. Holo-IGF1R and compared to their own targeted receptors (9). at University of Pittsburgh on January 18, 2018 HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose Here we focus on the role of three receptors: holo- uptake, and only holo-IGF1R showed anti- IGF1R, holo-InsR, and HybR. Hybrid receptors apoptotic effects. We also found that the three (HybR) consisting of one subunit of IGF1R receptors differentially regulated gene expression: and one subunit of InsR have been reported holo-IGF1R and HybR upregulated EGR3; holo- (10-12). Assays for determination of HybR InsR specifically downregulated JUN and activity have shown that it responds with much BCL2L1; holo-InsR downregulated, but HybR greater potency to IGF1 than insulin (13), and has upregulated HK2; and HybR specifically different binding affinity compared to holo- upregulated FHL2, ITGA6, and PCK2. Our receptors (14). However, as most cells express a findings suggest that when expressed and activated combination of IGF1R, InsR and HybR, and few in mammary epithelial cells, HybR acts in a methods exist to specially examine activated manner similar to IGF1R, and supports further HybR in living cells (15), attributing specific investigation of the role of HybR in breast cancer. effects to HybR has been challenging. HybR content exceeds IGF1R content in >75% breast Introduction cancer specimens (16). Breast cancer is the most common cancer among To directly study the function of HybR, we used a women worldwide, excluding skin cancer, and it method which has been used to demonstrate the remains a significant cause of morbidity and function of ErbB1/ErbB2 heterodimers (17). We mortality (1). The insulin-like growth cloned chimeric holo-IGF1R, holo-InsR, and factor/insulin (IGF/insulin) system, consisting of HybR, which have low affinity to growth factors three ligands, IGF-1, IGF-2, and insulin; six and took advantage of chemically induced system ligand-binding proteins IGFBP1-6; and five to activate them respectively, and compare their receptors, insulin-like growth factor 1 receptor different biological roles in a breast cancer cell (IGF1R), insulin-like growth factor 2 receptor line. Although, we understand that the chimeric 2 system is artificial and highly engineered, it is a cells expressed endogenous IGF1R, but chimeric powerful tool to systematically isolate the IGF1R was only detected in the holo-IGF1R and signaling cascades of each receptor individually, HybR cells as expected. For InsR, endogenous and provides novel insight into the potential levels were low, but were increased in the holo- biological effects of the holo and hybrid chimeric InsR and HybR cells as expected. However, the receptors. Additionally, while we cannot directly chimeric holo-InsR had a slightly higher molecular compare the signaling and biological effects of the weight. The detection of the Glu-Glu tag was chimeric receptors to the native receptors in this challenging due to lack of sensitivity of the study due to factors such as differing potencies of antibody, however, was repeatedly detected in the the dimerizing agent and endogenous ligand(s), HybR cells. Of note, we observed slight increases inability of chimeric receptor to cross-talk with in native receptor expression with the addition of endogenous receptor or other RTK family chimeric receptor expression potentially due to members, and altered molecular conformation of changes in stability of the native receptor, however the chimeric receptor (further detail outlined in we did not test this in this study. In summary, we Discussion), we have included endogenous generated stable cells lines expressing vectors that Downloaded from receptor action to observe if the directional trend allow chemical activation of holo-IGF1R, holo- in response is similar. InsR and HybR. We attempted to obtain clones with equal expression of the respective Results components, but this was not possible. http://www.jbc.org/ Cloning and expression of chimeric dimerizable Cells were serum starved by placing in serum-free holo-IGF1R, holo-InsR, and HybR. media, to maintain a low level of activation of endogenous IGF1R, InsR, and HybR, and then To construct chimeric IGF1R and InsR and allow treated with increasing concentrations of for chemically induced dimerization, 4 parts were dimerizing agent (0 to 2.5 μM) and activation of at University of Pittsburgh on January 18, 2018 cloned into the pBabe retroviral vector: 1) PI3K and Erk1/2 pathway detected by Extracellular domain of the low-affinity nerve immunoblotting (Fig 2A-C, Fig S1-2). In all three growth factor receptor (p75), 2) β subunit of cell model systems (holo-IGF1R (Fig. 2A), holo- IGF1R or InsR, 3) a binding site for a dimerizing InsR (Fig 2B), and HybR (Fig 2C)), as little as agent (one FRB or two FKBP domains), and 4) an 10nM of dimerizer resulted in an increase in p-Akt epitope tag (HA or Glu-Glu) (Fig 1A). The role of with a dose-response increase up to 2.5uM. IGF1 p75 is to replace the ligand binding α subunit of and insulin were used as positive controls for IGF1R or InsR, and prevent chimeric receptors signaling activation, however, insulin had only from being activated by IGF-1 or insulin. p75 has minor effects reflecting the low levels of a low affinity for nerve growth factor (which is not endogenous InsR in these cells. Phosphorylation expressed by breast cancer cells) and thus has no of Erk1/2 was minimally increased by dimerizer ligand and/or activity. AP20187 was used to compounds, but was not as robust as Akt dimerize FKBP domains together to activate holo- activation (Fig 2A-C, Fig S2). Additionally, we IGF1R or holo-InsR, while AP21967 was used to did a time course analysis (30m – 8hr) in holo- dimerize an FRB and an FKBP domain to activate IGF1R and HybR cells treated with 500 nM HybR (Fig 1A). After transient transfection and dimerizing agent (Fig S3). As expected the viral infection, stable cell lines were generated and signaling (p-Akt and p-Erk1/2) declines over time, immunoblotting performed. As shown in Fig 1B, however the holo-IGF1R signaling appears more compared with wild type (WT) MCF7, holo- potent and sustained than the HybR signaling. As IGF1R, holo-InsR, and HybR transfected cells a control, stimulating untransfected wild type expressed endogenous proteins, and chimeric MCF7 with dimerizing agents did not have any receptors were also detected with a larger effect upon p-IGF1R or p-Akt levels (Fig S4) and molecular weight (Fig S1). All three transfected also did not alter cell proliferation (Fig S5) cell lines expressed HA tags as expected; however, different expression levels were noted with holo- Activated chimeric holo-IGF1R does not IGF1R expressing higher levels.
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