INVESTIGATION INTO THE MOLECULAR SPECIFICITY OF THE IGFs: A CHIMERIC APPROACH

Transcription

1 INVESTIGATION INTO THE MOLECULAR SPECIFICITY OF THE IGFs: A CHIMERIC APPROACH By Adam Denley, BBiotecb (Hons.) A Thesis submitted to the University of Adelaide, South Australia in fulfilment of the requirements for the degree of Doctor of Philosophy THE UNIVERSITY OF ADELAIDE AUSTRALIA School of Molecular and Biomedical Science The University of Adelaide Adelaide, South Australia. December, 2004

2 ABSTRACT Insulin-like growth factors (lof)-1 and -II induce a variety of cellular outcomes including cell proliferation, diffentiation, cell migration and protection from apoptosis. IOF-I an IOF-II share a high degree of structural similarity reflecting their high level of sequence similarty. Despite this both IOP-l and IOF-II exhibit different binding affinities to almost all proteins with which they both interact. To date few studies have investigated the structural basis whereby IOP-l and lop-ii bind with different affmities to the type-j IOF receptor (lof-ir), insulin receptor (IR) isoforms, type-2 lop receptor (IOP-2R) and IOF binding proteins (lopbps). The major objective of this thesis is to investigate the molecular basis for the unique rector and binding protein binding properties of IOF-I and lof-ii To address the question of what parts ofiof-1 and IOF-II confer their unique receptor and binding protein binding affnities, chimeras of IOF-I and IOF-II were enginee where their C and D domains were exchanged either singly or together. " These are the first ever whole domain chimeras of IOP-I and IOF-II. Rectly the altertively spliced isoform of the insulin receptor lacking 12 amino acids encoded by exon 11 (IR-A) has been shown to be overexpressed in foetal and cancer tissues and bind lop-ii but not IOF-l with high affnity. Binding analysis of the engineered chimeras to the lr-a showed that the C and D domains of IOF-I1 allow high affnity binding whereas the same domains of IOF-l preclude high affinity binding to the IR-A. The C and D domains also regulate the differential interaction of IOF-l and IOF-I1 with the insulin receptor isoform contag the amino acds encoded by exon 11 (IR-B). The C domain of IOF-II, in the background of IOF-II or IOF-I, also allows potent induction of phosphorylation of various tyrosine residues in the intracellular 13

3 subunits of the lr-a and lr-b, whereas the same domain of lgf-i in the background of lgf-ii did not. In addition this same domain also is the molecular basis for the differential ability of IGF-II and IGF-I to phosphorylate IRS- l and activate PKBI Akt. Furthennore, the differential ability of IGF-II and lgf-l to induce cell survival and promote cell migration via the IR-A is due to their C domains. The lgf-ir binds lgf-i with a 3-5 fold higher affnity than IGF-II. Here in this thesis the differential affinity of lgf-i and IGF-II for the lgf-1 R is shown to be in a large part due to their C and D domains. The lgf-2r is a receptor that does not contain any intrinsic signaling activity and in the context of the IGF system appears to regulate the concentration of IGF-II. lgf-2r binds lgf-ii with high affnity but binds lgf-i with very low affity. Reported here is the novel identification of the IGF-II C domain as an important contributor for wildtype IGF-2R binding. lgfbps act to both potentiate and inhibit the cellular actions of the lgfs. Almost all lgfbps bind lgf-i and lgf-ii with different affinities, particularly IGFBP-6 which has a fold higher affinity for lgf-ii compared to lgf-i. Binding analysis of IGF chimeras to IGFBP-l, -2, -3 revealed that the C and D domains of the IGFs do not regulate any IGFBP affnity difference,, however the IGF-II C domain may play a small role in binding to IGFBP-6. Recently a patient exhibiting mental retardation, short stature and gondal dysfunction was shown to be homozygous for a mutation in his IGF-l gene resulting in a protein with a methionine instead of a valine at position 44. Here in this thesis the biochemical characterization of this mutant is reported. This mutation causes almost a 90-fold reduction in IGF-IR binding affnity, while completely abrogating binding to either lr isoform. Interestingly binding to IGFBP-2, 3 and -{ is unaffected. In addition, NMR analysis showed only small perturbations in the structure localized around the site of the mutation.

4 In conclusion this thesis has contributed to the understanding of ligand receptor interactions of the insulinllgf system.

5 TABLE OF CONTENTS ABSTRA CT n U... n.d... U n STATEMENT OF ORIGINALITY... IV ACKNO'\\'LEDGEMENTS... 'u... n... nu V TABLE OF CONTENTS... VII LIST OF PUBLICATIONS... XIV ABBREVIATIONS u... u u XVII CHAPTER 1: INTRODUCTION INTRODUCTION Introduction to the IGFaxis.. "... "... " IGF EXPRESSION AND TRANSCRIPTIONAL REGULATION IGF Structure and Function ".""... "." INSULIN-LIKE GROWTH FACTOR I RECEPTOR (IGF-IR) -STRUCTURE AND FtmCTIO... " IGF-1R signalling... "" Role of the IGF-IR in cancer... " IGF residues involved in IGF-1R binding SummaryofIGF residues involved in IGF-IR binding Mechanism ofigf binding to theigf-lr INSULIN RECEPTOR Insulin receptor (IR) -structure and fonction Insulin receptor isoforms Insulin receptor isoform tissue distribution The role of the insulin receptor isoforms in cancer The role of insulin receptor isoforms in type 2 diabetes Residues of IGF that are involved in IR binding Summary ofigf residues involved in 1R binding Mechanism of insulin binding to the [R... " HYBRID IRlIGF-IR THE INSULIN-LIKE GROVlTII FACTOR TYE 2 RECEPTOR (IGF-2R)... 48

6 1.6.1 IGF residues involved in IGF-2R binding Summary of IGF residues involved in IGF-2R binding Mechanism of IGF-II binding to the IGF-2R INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS (IGFBPs)-STRUCTURE AND FUNCTION IGF residues involved in IGFBP binding Summary of 1GF residues involved in 1GFBP binding Mechanism of IGF-I and IGF-II binding to IGFBPs COMPARISON OF THE RECEPTOR AND BINDING PROTEIN BINDING SITES ON THE IGFs AIMS AND EXPERIMENTAL STRATEGy CHAPTER 2: CONSTRUCTION OF VECTORS FOR IGF CHIMERA EXPRESSION u... u... u u INTRODUCTION GENERAL MATERIALS Bacterial media and buffers Bacterial Strains MATERIALS FOR MOLECULAR BIOLOGy Molecular Biology Solutions Kits For Molecular Biology Expression Vector Oligonucleotides METHODS Agarose Gel Electrophoresis Of DNA Restriction Enzyme Digestion Of DNA Purification Of DNA By Denaturing Polyacrylamide Gel Electrophoresis Polymerase Chain Reaction (PCR) Purifcation Of PCR Products From Agarose Gels TA Cloning Of PCR Fragments Dye Terminator DNA Sequencing Removal Of 5 '-Phosphate From Vector DNA Ligation Preparation Of Competent Cells Bacterial Transformations... 81

7 2.5 RESULTS AND DISCUSSION Codon Optimisation Of Sequences For Expression Strategy to ConstnLCt Coding Sequence for IGF chimeras Summary CHAPTER 3: EXPRESSION AND PURIFICATION OF HUMAN IGF-I, IGF-II AND NOVEL IGF CHIMERAS INTRODUCTION MATERIALS Expression and Purification buffrs Protein Electrophoresis bu fers BIAcore bu fers 3.3 MErnODs Analytical HPLC Exression Of Proteins I n E. coli SDS Polyacrylamide Gel Electrophoresis Lysis OfE. coli B y French Press Gel Filtration Refolding Of Fusion Proteins I on Exchange Chromatography Hydroxylamine Cleavage a-lytic Protease Cleavage Preparative Reverse Phase HPLC Freeze Drying Quantitation Of Puriied Protein Mass Spectrometry And N-Terminal Sequencing JOO BIAcore Analyses RESULTS IGF-I IGF-II IGF-I Dll IGF-I Cll IGF-I ClIDII IGF-II Dl IGF-II Cl

8 3.4.8IGF-/I CIDl BIAcore Analyses DISCUSSION CHAYfER 4: BINDING AND ACTIVATION OF IR-A AND IR-B BY INSULIN, IGF-II, IGF-I AND IGF CHIMERAS INTRODUCTION MATERIALS MEllIODS Construction O/Cells exressing The Human IR-A And IR-B Binding Analyses O/Chimeras To Insulin Receptor Is% rms Insulin Receptor Phosphorylation Assays RESULTS Binding analyses 0/ insulin, IGF-II, /GF-I and IGF chimeras to the IR-A Binding analyses o/insulin, IGF-II, IGF-I and IGF chimeras to the IR-B Phosphorylation o/the IR-A and IR-B by stimulation with chimeric IGFs DISCUSSION CHAPTER 5: INSULIN, IGF-II, IGF-I AND IGF CIDMERA SIGNALING THROUGH THE IR-A AND IR-B INTRODUCTION , 5.2 MATERIALS MEllIODS Construction O/Cells Expressing The IR Is% rms IR-A And IR-B Phosphorylation And Activation O/Intra-Cellular Signalling Molecules In Response To Insulin. IGF-I, IGF-/I And IGF Chimeras Western Blot Analysis Migration Assays Cell Survival Assays RESULTS A timecourse o/phosphorylation 0/Y960 on the IR-A stimulated by insulin. IGF-II, IGF-I and IGF C domain chimeras Induction 0/ autophosphorylation o/tyrosines in the activation loop o/the IR-A and IR-B by insulin, 1GF-II, IGF-I, and IGF chimeras

9 5.4.4 Activation ofy960 on IR-A and Y972 on IR-B by insulin, IGF-II. IGF-L and IGF chimeras...,...,...,...,..., ,4.5 Phosphorylation of IRS-l by IR-A and IR-B activated by insulin. IGF-L IGF-IL and IGF chimeras..."..."".." Phosphorylation ofirs-2 by IR-A and IR-B activated by insulin, IGF-L IGF-II and IGF chimeras Activation of AktlPKB by insulin, IGF-IL IGF-I. and IGF chimeras in KIR- A and KIR-B cells ,8 Activation oferk1l2 by insulin, IGF-IL IGF-L and IGF chimeras in KIR-A and K1R-B cells...,..,...,...,..,...,..,..,...,.., KIR-A cell protection from sodium butyrate-induced apoptosis by Insulin, IGF-IL IGF-I, and IG chimeras Insulin, IGF-L IGF-IL and IGF chimera stimulated chemotaxis of RJR-A cells...,...,...,..,...,...,..,...,..., DISCUSSION..., CHAPTER 6: BINDING AND ACTIVATION OF THE HUMAN IGF-IR BY INSULIN, IGF-II, IGF-I AND IGF CHIMERAS INTRODUCTION..., MATERlALS METHODS... " RESULTS Binding of insulin. IGF-IL IGF-I and IGF chimeras to the IGF-1R Phosphorylation of the IGF- IR by stimulation with chimeric IGFs DISCUSSION CHAPTER 7: BINDING OF IGF-I, IGF-II AND IGF CHIMERAS TO THE IGF-2R AND IGFBPS INTRODUCTION Type II IGF receptor (IGF-2R} Insulin-like growth factor binding proteins (IGFBPs) MATERIALS METHODS Production Of IGF-2R Fragments..." BIAcore Analyses Of IGF-2R Interactions

10 " BIAcore Analyses Of IGFBP Interactions RESULTS...,..., ,1 Kinetic analysis of IGF-L IGF-ll and IGF chimeras binding to IGF-2R fragments...,...,..., Kinetic analysis of IGF-L IGF-ll and IGF chimeras binding to IGFBP-1, -2, -3 and -6...,...,...,...,..., ,5 DISCUSSION...,...,...".,...,.,..." CHAPTER 8: STRUCTURAL AND FUNCTIONAL ANALYSIS OF 8.1 INTRODUCTION.. VAL44 IET IGF-I "...,..."...,', MATERIALS.. " "......,... "..., METHODS Construction Of Expression Plasmids Encoding Human 1GF-I And Val44Met IGF-l....., Recombinant IGF-I And Val44Met IGF-I Production Binding Analysis OfVal44Met IGF-I To The IGF-1R And IR Isoforms IR And IGF-1 R Phosphorylation Assays BIAcore Analyses Of IGFBP Binding RESULTS Purifcation ofval44met IGF-l , IGF-1R binding and activation IR binding and activation Receptor signalling and biological activity in fibroblasts IGFBP binding...,..., Structural Analysis ofval44met IGF-1 by NMR DISCUSSION CHAPTER 9: FINAL DISCUSSION DISCUSSION SUMMARY OF FINDINGS EVOLUTION OF THE IGFS DIVERGENCE OF THE IOF-I AND IOF-II C AND D DOMAINS WHICH C AND D DOMAIN RESIDUES CONFER SPECIFICITY AND WHY? FUTURE DIRECTIONS

11 9.7 DESIGNING INHIBITORS OF IGF ACTION RATHER THAN OF IGF-IR ACTION.,." ,8 CONCLUSION"...,... " "..,... ",.. " ,... "... ". "..,....., ',... "...".,..,",."" REFERENCES... u... ".nu"... uu... "... u 240,