Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration

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O'Donoghue, Jordan Christopher
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University College Cork
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The IGF-1R has long been implicated in numerous malignant characteristics of cancers including enhanced proliferation, insensitivity to apoptosis, chemoresistance, increased migratory and invasive capacity, epithelial-to-mesenchymal transition, and metabolic augmentation. IGF-1R, much like other receptor tyrosine kinases, has been characterised as a substrate for gamma-secretase-mediated regulated intramembrane proteolysis (RIP). This proteolytic pathway begins with ectodomain shedding, mediated by sheddases, which produces a soluble fragment in the extracellular space. Following ectodomain shedding, a membrane anchored C-terminal fragment (CTF) is produced which is then cleaved by gamma-secretase at the internal juxtamembrane region to produce a soluble intracellular domain (ICD). Classically, RIP was thought to be a means by which cells would terminate receptor signalling, however contemporary research indicates a more nuanced role. It is now understood that the fragments generated through RIP can retain signalling of the full-form receptors and, in some instances, may acquire novel functionality. Although the RIP of IGF-1R has been outlined, the underlying dynamics of this pathway and the consequences of such remain to be elucidated. With this in mind, we endeavoured to identify the regulatory mechanisms underlying IGF-1R RIP and subsequently aimed to identify potential functions of the fragments produced. Firstly, we generated a GFP-tagged K1003 IGF-1R point mutant, termed a kinase dead mutant, which lacks the ability to catalyse trans-autophosphorylation of the IGF-1R kinase domains thus preventing downstream signal transduction. Trans-autophosphorylation is a critical step in the activation of receptor tyrosine kinases during which the kinase domain of one intracellular domain catalyses the phosphorylation of tyrosine residues within the other intracellular domain. With this tool, we validated the cleavage of IGF-1R and the production of its CTF via transient transfection of HEK293T cells with plasmids expressing either wild-type or K1003R kinase dead mutant forms of IGF-1R C-terminally tagged with GFP. We subsequently identified receptor kinase activation as a potential driver of IGF-1R RIP and determined that ligand stimulation significantly catalyses the accumulation of IGF-1R CTF. This was accomplished via transient transfection of HEK293T cells with plasmids expressing the aforementioned IGF-1R forms. Following this, we elucidated clathrin-mediated endocytosis as a probable regulatory pre-requisite step for IGF-1R RIP by gamma-secretase. Furthermore, our data indicates that ectodomain shedding of IGF-1R is likely catalysed in a metalloprotease dependent fashion, consistent with other receptor tyrosine kinases. We also confirmed that the CTF can undergo nuclear translocation in a clathrin-mediated endocytosis dependent manner, consistent with the translocation of the holoreceptor. Utilising wound healing assays, conducted in Hela cells, our data indicates that gamma-secretase inhibition by DAPT (a small molecule gamma-secretase inhibitor) alone does not antagonise IGF-1-induced cell migration. Similarly, the administration of Pitstop alone does not impinge upon IGF-1 induced cell migration. Interestingly, the administration of batimistat (a broad spectrum metalloprotease inhibitor) significantly suppresses cell migration in the presence of IGF-1. Collectively, our data provides a framework for the construction of the IGF-1R RIP cascade. Following ligand-binding it appears that IGF-1R undergoes metalloprotease-dependent ectodomain shedding leading to loss of the extracellular alpha-chains. The CTF, which remains in the plasma membrane, then undergoes clathrin-mediated endocytosis and packaging into early endosomes. Following internalisation, the CTF is cleaved by gamma-secretase to generate a soluble ICD. Our data also demonstrates that IGF-1R CTF undergoes nuclear translocation in a clathrin-mediated endocytosis dependent fashion. Lastly, data gathered from our wound healing assays indicates that IGF-1R ectodomain shedding is likely a required event for the efficient IGF-1-induced migration of cells, possibly through the proteolytic augmentation of focal adhesion complexes containing IGF-1R.
Gamma-secretase , Proteolysis , Cell migration , Cancer , IGF-1R , IGF-1
O'Donoghue, J. C. 2023. Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration. MRes Thesis, University College Cork.
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