Details

Autor(en) / Beteiligte

• Rezgui, Dellel

Titel

Functional evaluation of the igf2-igf2r interaction

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2008

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The aim of this work was to further our understanding of the molecular basis of the IGF2-IGF2R interaction by studying natural and acquired mutations of domain 11. Firstly, I evaluated the evolutionary development of the IGF2-IGF2R interaction from fish to humans. Following species specific recombinant protein expression and site directed mutagenesis (SDM), I quantified the acquisition and optimisation of the binding interaction using real time surface plasmon resonance (SPR). The acquisition of an active IGF2 binding site predated Igf2 and Igf2r genomic imprinting and was associated with molecular evolution of the CD loop conformation from birds to monotremes. Following establishment of the hydrophobic CD core, subsequent evolutionary mutations were in second sphere residues in the FG and AB loops. Secondly, I evaluated the G1619R IGF2R (5002A>G) non-synonymous single nucleotide polymorphism (SNP) of the IGF2 specific binding domain, which had been correlated with impaired childhood growth and is in stable Hardy Weinberg equilibrium. Functional investigation of the effects of this polymorphism utilised SDM, SPR analysis, quantitative PCR, 35S pulse chase and flow cytometry. Comparison between proteins with polymorphism at 1619 had no direct effect on the receptor binding kinetics, half-life and distribution. Other potential SNPs in linkage disequilibrium may have accounted for the correlation with childhood growth. Finally, I developed directed evolution of domain 11 for enhanced IGF2 affinity using a Pichia Pastoris surface display. This approach aimed to evolve domain 11-IGF2 binding in the laboratory, in order to discovery whether it could acquire a higher affinity for IGF2. This technique also has application to the therapeutic treatment of cancer.