THE ROLE OF THE hGRBl4 ADAPTOR PROTEIN BPS DOMAIN IN INSULIN SIGNALING BY SHERzuE KELLY A Thesis Submitted to the Faculty of Graduate Studies In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Biochemistry and Medical Genetics University of Manitoba Winnipeg, Manitoba @ Sherrie Kelly, September 2002 NationatLibrarY Bibliothèque nationale l*I du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 W€ll¡nglon Stre€t 395. rue Wellington OttawaON K1A0|\,|4 Ottawa ON KIA 0N4 Canacla Canada Your 6ls Votro rélèmæ Our frlâ Itotre rèlérûæ The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loar't, distibute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/filn, de reproduction sur paprer ou sur format électronique. The ar¡thor retains ownership of the L'auteur conserve la propriété du copyrieht in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial exhacts from it Ni la thèse ni des extraits substantiels may be printed or othenyise de celle-ci ne doivent être imprimés reproduced without the author,s ou autrement reproduits sâns son permission. autorisation. 0-612-76780-9 Canad'ä THE TINIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES x**** COPYRIGHT PERMISSION PAGE The Role of the hGrbl4 Adaptor protein Bps Domain in Insulin signaling BY Sherrie Kelly A Thesis/Practicum submitted to the Faculty of Graduate Studies of The Universitv of Manitoba in partial fulfittment of the requirements of the degree of MASTER OF SCIENCE SHERRIE KELLY @2002 Permission has been granted to the Library of The University of Manitoba to lend or sell copies this of thesisþracticum, to the National Library of Canada io microfihn this thesis and to lend or sell copies of the film, and to University Microfilm Inc. to publish an abstract of this thesis/practicum. The author reserves other publication rights, and neither this thesisþracticum nor extensive extracts from it may be printed or otherwise reproduced without the author's written permission. TABLE OF CONTENTS TABLE OF CONTENTS.. LIST OF FIGURES. LIST OF TABLES. IX LIST OF ABBREVIATIONS X ACKNOWLEDGMENTS ... xii ABSTRACT..... 13 I. INTRODUCTION t4 1.1. Diabetes mellitus t4 1.2. Insulin 15 1.3. lnsulin receptor. 15 1.4. Insulin signaling pathway. T7 1.5. Proteins interacting with the IR. 20 1.6. GrbT family of adaptor proteins. 2T 1.6.1. Growth factor receptor bound-7 (Grb7) 2l 1.6.2. Growth factor receptor bound-10 (Grb10). 23 1.6.3. Growth factor receptor bound-14 (Grb14). 24 1.7. Objective... 26 1.8. Hypothesis... 26 1.9. Aims. 26 2. MATERIALS AND METHODS 27 2.1. Reagents.... 27 2.1.1. Antibodies 27 2.1.2. Yeast strains. 28 2.1.3. Bacterial strains. 28 2.1.4. Oligonucleotides. 29 2.2. Transformation of E. coli. 30 2.2.I. Preparation of electrocompetent cells. 30 2.2.2. Electroporation. .. 30 2.3. Preparation of plasmid DNA 31 2.4. Polymerase chain reaction (PCR). 31 2.4.I. PCR-based mutation detection. 32 2.5. DNA cloning techniques. 34 2.5.I. Treatment of DNA fragments with restriction endonuclease, Klenow, and phosphatase. 34 2.5.2. Isolation of DNA from gels. 34 2.5.3. Ligations. 35 2.5.4. Manual DNA sequencing. 35 2.5.5. Automated Sequencing 36 2.6. Bioinformatic tools and methods. 36 2.7. Yeast two-hybrid system. 37 2.7.1. Yeast two-hybrid plasmids. JI 2.7 .2. Transformation of S. cerevisiae 42 2.7.3. B-Galactosidase filter lift assay. 42 2.7 .4. Liquid B-galactosidase.. 43 2.7.5. hGrbl4 protein expression in yeast. 44 2.8. Mammalian cell culture methods. 45 2.8.1. Mammalian cell lines. 45 2.8.2. Cell transfection. 45 2.8.3. Insulin stimulation 47 2.8.4. Cell lysis. 47 2.9. Bradford protein assay. 47 2.10. Immunoblotting 48 2.1 1. Immunoprecipitation. 48 2.I2. Transgenic experiments...... 49 2.12.1. Animals. 49 2.12.2. Transgenic construct preparation 50 2.12.3. Microinjection of mouse embryos 50 2.12.4. Preparation of mouse-tail DNA. 50 lll 2.12.5. Founder screening. 52 2.12.5.I. PCR reactions. 52 2.12.5.2. Southern blot analysis..... 52 2.I2.5.3. Protein expression analysis. 54 3. RESULTS AND DISCUSSION... 55 3.1. Analysis of the interaction of hGrbl4-IR in the yeast two-hybrid system. 55 3. 1 . 1 . Analysis of the interaction of full-length hGrb 1 4 with the IR. .. 55 3.1.2. Confirmation that hGrbl4 interacts with the IR in a kinase dependent manner. 57 3.1.3. Analysis of the role of the SH2 domain in the hGrbl4-IR interaction 60 3.I.4. Identification of a second hGrbl4 domain involved in IR bindins. 63 3.1.5. Bioinformatic analysis of the BPS domain..... 64 3.1.6. Analysis of the role of the BPS domain in hGrb14-IR interaction.. 69 3.1.7. Analysis of the interaction of the BPS+SH2 domain fusion protein with the IR.... 78 3.1.8. Identifying akey interacting residue in the BPS domain............. 82 3.I.9. Analysis of BPS-mutant hGrbl4 fusion protein interaction with the IR. 84 3.1.10. Analysis of the hGrbl4 protein expression levels in yeast. 87 3.2. hGrbl4-IR interaction in mammalian cells and effect on downstream insulin signaling. 88 3.2.1. Transfection of hGrbl4 into CHO-IR cells.. 91 3.2.2. Interaction of hGrbl4 wild-type and mutant forms with the IR..... 95 3.2.3. Tyrosine phosphorylation of proteins in CHO-IR cells overexpressing wild-type hGrbl4. 99 3.3. hGrbl4 transgenic mice. 102 3.3.1. Strategy for creating hGrbl4 transgenic mice. 102 3.3.2. hGrbl4 protein expression in mouse tissues. 106 4. CONCLUSION. 109 5. FUTURE DIRECTIONS... II2 6. APPENDIX. .. 1 13 6.1. MutationAnalysis..... 113 6.1.1. Electropherogram of pGAD424::hGrb14¡ R387Q using the GAL4AD reverse primer. 113 1V 6.1.2. Mutation analysis of pGAD424::hGrb14¡ R387Q R466Q using the GAL4AD reverse primer r1,4 6.2. Peptide mass analysis. 114 6.3. Media 115 6.3.r. Bacterial media. 115 6.3.1.1. Luria Bertani (LB) medium 115 6.3.I.2. Low Salt LB medium 115 6.3.1.3. SOC medium..... 115 6.3.2. Yeast media. 115 6.3.2.1. YPAD media. 115 6.3.2.2. SC media. rt6 6.3.3. Mammalian cell culture media. rt6 6.3.3.1. Ham's F12 media 116 6.4. Buffers t17 6.4.1. Cell lysis buffer 117 6.4.2. Immunoprecipitation lysis buffer rt7 6.4.3. PBS buffer. 117 6.4.4. 4x SDS loadins buffer r17 6.4.5. STE buffer tt7 6.4.6. Tissue lysis buffer IT7 6.4.7. TAE buffer 117 6.4.8. TBE buffer tT7 6.4.9. TBS-T buffer. 118 6.4.10. TCA buffer. 118 6.4.11. TCA Laemmli loading buffer. 118 6.4.12. Zbuffer. 118 6.4.13. Zbufferlþ-ME/X-gal. 118 6.5. Sequencing gel.... 118 6.6. Southem blotting analysis solutions. 118 6.6.I. Denaturation solution. 118 6.6.2. Neutralization solution tt9 6.6.3. 20 x Standard Saline Citrate (SSC). 119 6.6.4. Hybridization buffer. 119 6.6.5. Wash #1.... 119 6.6.6. Wash #2.... rt9 6.6.1 . V/ash #3.... 119 REFERENCES t20 VI LIST OF FIGT]RES Figure 1: Diagram of the structure of the IR... t6 Figure 2: Overview of the insulin signaling pathway. 18 Figure 3: Diagram of the structure of the GrbT protein family. 22 Figure 4: Diagram depicting the PCR-based site directed mutagenesis and PCR- aa based mutation detection methods. JJ Figure 5: Diagram of the yeast-two hybrid system 38 Figure 6: Diagram of the yeast two-hybrid plasmids.... 40 Figure 7:Diagram of the pRcCMV::hGrbl4e vector. 46 Figure 8: Diagram of hGrbl4 MfeUBamHI DNA fragment used for microinjection to create transgenic mice. 51 Figure 9: Diagram of the pGAD424::hGrb14r vector. s6 Figure 10: Analysis of hGrbl4-IR interactions using B-galactosidase filter lift assays. 58 Figure 11: Interaction of hGrbl4 full-length and sub-domain GAL4 AD fusion proteins with the IRK in yeast. 59 Figure 12: Diagramof the pGAD424::SH2 vector. .. 6r Figure 13: Multiple sequence alignment of GrbTll01r4 proteinsequences. 65 Figure 14: Dendrogram ofthe GrbTlI\ll4 protein sequences. 67 Figure 15: BPS domain BLAST query output. 68 Figure 16: Multiple sequence alignment of the GrbTll0/r4 BpS domain............ 70 Figure 17 : Diagram of the pG AD424::BPS vector. 7l Figure 18: Diagram of the pGBTS::BPS vector. 73 Figure 19: Diagram of the pGAD424::PH+BPS vector. 75 Figure 20:Diagramof the pGAD424.:1/2 PH+BPS vector. 76 Figure 2l:Diagrarnof the pGAD424::BPS dropout vector. 77 Fi gure 22 : Diagr am o f the pG AD 424 :: BP S+ SH2 vector. 79 Figure 23:lnteraction of the hGrbl4 BPS+SH2 GAL4AD reqion with the IRK in yeast. .......... 81 Figure 24: Detection of hGrb14 substitution mutants by PCR-based methods.. ... 85 Figure 25: Interaction of mutant GAL4AD::hGrbl4 fusion proteins with the IRK in yeast. 86 Figure 26: Western blot analysis of GAL4AD::hGrbl4e and GAL4AD: :BPS+SH2F fusion proteins. 89 vil Figure 27: V/estern blot analysis of GAL4AD::SH2, BPS dropout, and BPS fusion proteins.