Author: Steven Thomas Henderson
Henderson, Steven Thomas, 2011 The Generation and Characterisation of Human Insulin-like Growth Factor Mutants, Flinders University, School of Biological Sciences
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Human insulin-like growth factor-I and -II (IGF-I and -II) are structurally homologous ligands with differential binding affinities for the insulin/IGF family of cell surface receptors. High affinity binding of IGF-I and IGF-II ligands to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms stimulates receptor tyrosine kinase activity regulating numerous biological responses such as cell cycle progression, proliferation and cell survival. Deregulation of the IGF-IR and IR-A signalling axis is common in cancer. Both IGF-I and IGF-II have been implicated in cancer initiation and progression due to ligand overexpression and autocrine ligand production. The focus of this study was to diversify human IGF-I as part of the ongoing development of an in vivo gene diversification system associated with the filamentous fungus Neurospora crassa (Catcheside et al., 2003). The aim of this project was to generate, express, secrete and screen novel IGF-I variants to identify IGF-IR antagonists as potential cancer therapeutics. Mutant IGF-I DNA sequences were generated with high efficiency using the Neurospora Repeat-Induced Point Mutation (RIP) & HotSpot-Mediated Recombination (HSMR) gene diversification platforms. Multiple IGF-I expression vectors were constructed and the novel secretion of human IGF-I protein in Neurospora was achieved with the fusion of the IGF-I protein to the endogenous glucoamylase (gla-1) protein. However, the abundance of mature IGF-I protein in culture medium was relatively low due, at least in part, to extracellular proteolytic degradation. In addition, the IGF-IR binding assay was found to be unsuitable for reliable screening of secreted IGF-I protein in Neurospora culture medium. Resolution of these issues were not pursued in this project as the generation of protease deficient Neurospora expression strains and development of a more robust screening methodology were already being undertaken by Neugenesis Corporation and were unlikely to be resolved within the duration of this project. Consequently, the project direction was diverted to the characterisation of IGF residues involved in receptor binding and activation through site-directed mutagenesis. The IGF-I and IGF-II ligands share a common domain structure and exhibit differential receptor binding and activation which is primarily due to the IGF C-domain. Understanding the role of specific residues within the C-domains could make a significant contribution to our fundamental understanding of ligand-receptor interactions in the insulin/IGF system and enable the rational design of IGF-IR and IR-A antagonists for use as anti-cancer agents. As such, a second aspect of this study aimed to delineate the C-domain residues conferring differential receptor binding affinity and activation by generating and characterising three IGF-II analogues. Mutants C1 and C2 contained partial IGF-I C-domain substitutions (IGF-I residues underlined): GYGSSSRRSR & SRVSRRAPQT, respectively. Mutant C3 contained a truncated IGF-I C-domain (GSSSRRAT) which approximated the size of the IGF-II C-domain. The three IGF-II analogues were characterized by competitive receptor binding assay, receptor phosphorylation assay and cell survival assay using the IR-A, IR-B and IGF-IR receptors. The C1 analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with only moderate affinity suggesting a negative interaction between the C1 C-domain and the exon-11 encoded peptide in the IR-B. The IR-A, IR-B and IGF-IR activation and cell survival responses were generally proportional to the relative binding affinity of C1. In contrast, C2 acted as a partial agonist against the IR-A, IR-B and IGF-IR with high receptor binding affinities but substantially lower activation and cell survival responses in all three receptors. These data indicated both flanks of the IGF C-domain play important roles in receptor binding, activation and cell survival. The C3 analogue had generally poor IR-A, IR-B and IGF-IR binding affinities, activation and cell survival responses which indicated this analogue lacked critical C-domain residues required for high affinity receptor binding and activation.
Keywords: IGF-I,IGF-II,Neurospora,directed evolution,gene diversification,mutagenesis,analogues
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Professor David Catcheside