Investigation of the mechanism of action of IGF-II binding to the IGF-1R and IR-A and how signaling pathways are preferentially activated

Author: Andrew Blyth

Blyth, Andrew, 2023 Investigation of the mechanism of action of IGF-II binding to the IGF-1R and IR-A and how signaling pathways are preferentially activated, Flinders University, College of Medicine and Public Health

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Abstract

The insulin like growth factor (IGF) system is essential for regulating metabolic and mitogenic signaling in mammalian cells. This system is particularly important during fetal development and postnatal growth. The IGF system is regulated by three ligands IGF-II, IGF-I and insulin all of which share a high degree of sequence and structural similarity. We are particularly interested in IGF-II which is capable of binding to the type 1 insulin like growth factor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. IGF-II expression is commonly upregulated in cancer, as is the IR-A, resulting in cancer cell survival.

While insulin has been heavily studied for its role in diabetes, in comparison IGF-II has been poorly investigated. Sequence and structural similarities between insulin and IGFII has indirectly advanced our understanding of IGF-II. However, there is a lack of information specific to IGF-II such as unique structural elements that contribute to differences in stability, receptor specificity and signaling outcome.

In mammalian insulins and IGFs there is a high degree of conservation of residues involved in receptor binding. The so-called ‘aromatic FFY/FYF’ motif found in insulin and the IGFs respectively, is an example of this. Removal of this motif in human insulin leads to essentially no receptor binding affinity. In recent years an influx of research into invertebrate insulins has led to the discovery of several insulins in the venom of cone snails which totally lack this aromatic FYF motif yet are still capable of binding and activating the human insulin receptor with high affinity. Structural and biochemical analysis of these cone snail venom insulins has revealed novel mechanisms of binding to the IR. These mechanisms have since been introduced into human insulin analogues lacking the FYF motif and the resultant analogues have been shown to have unique signaling kinetics compared to human insulin.

The overall aim of my thesis was to better understand residues of IGF-II that are responsible for structural integrity and stability. Also, I aimed to further interrogate the mechanism of IGF-II action through understanding how signaling pathways are preferentially activated. To do this IGF-II mutants lacking elements of the FYF motif were produced to investigate the role of the FYF motif in receptor binding by IGF-II. This is the first time an IGF-II analogue lacking all elements of the FYF motif has been produced.

Also introduced into IGF-II were the equivalent mutations found in cone snail venom insulins that are responsible for the alternate mechanism of binding. From these 3 investigations several residues of IGF-II were identified that are important for protein folding, structure and stability. Furthermore, an IGF-II analogue with increased specificity for the IR-A but no change in signaling potency was characterised. Finally, by combining changes in the FYF motif and the equivalent mutations found in cone snail venom

insulins that are responsible for the alternate mechanism of binding I sought to recapitulate the alternate binding mechanism utilised by cone snail insulins for insulin receptor binding. Characterisation of these IGF-II analogues provides valuable information toward developing highly selective small molecule antagonists with low signaling capabilities for the treatment of IGF dependent cancers.

Keywords: Insulin like growth factor II, IGF-II, Insulin like growth factor 1 receptor, IGF-1R, insulin receptor, IR, cone snail venom insulin

Subject: Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Briony Forbes