DISSERTATION Titel der Dissertation Comparative Studies of the Avian and the Mammalian LDLR angestrebter akademischer Grad Doktor/in der Naturwissenschaften (Dr. rer. nat.) Verfasserin / Verfasser: Mag. Melanie Schiff Matrikel-Nummer: 9801745 Dissertationsgebiet (lt. Mikrobiologie und Genetik Studienblatt): Betreuerin / Betreuer: O. Univ. Prof. DI. Dr. Wolfgang J. Schneider Wien, im August 2009 Directory Directory Directory ...................................................................................................................... i Abbreviations ..............................................................................................................iv List of figures and tables............................................................................................vii Abstract....................................................................................................................... 9 Zusammenfassung ................................................................................................... 11 Introduction............................................................................................................... 13 Amnionless............................................................................................................ 13 Amnionless in human disease............................................................................... 14 The interaction of Amnionless and Cubilin ............................................................ 15 The role of cubam and LPR2 during embryonic development and nutrition.......... 17 The LDL receptor gene family ............................................................................... 18 The LDL receptor .................................................................................................. 19 Functions of the LDLR: The LDLR pathway .......................................................... 21 Other members of the LDL receptor gene family................................................... 23 VLDL receptor (VLDLR) ........................................................................................ 25 LR8, the avian VLDLR homologue........................................................................ 26 The mutant restricted ovulator (R/O) chicken strain .............................................. 27 Apolipoprotein E receptor 2 (apoER2)................................................................... 28 LR7/8B, the chicken homologue of apoER2.......................................................... 28 Mammalian and chicken LRP1/ α 2-macroglobulin receptor................................... 28 LR11/SorLA and its chicken counterpart............................................................... 29 LRP2, previously named gp330 and megalin........................................................ 30 LRP1B/LRP-DIT.................................................................................................... 31 Interaction of the LDLR with apolipoproteins......................................................... 31 Apolipoprotein B (apoB)..................................................................................... 31 Apolipoprotein E (apoE)..................................................................................... 33 Binding of apoE to the LDL receptor.................................................................. 36 Adaptor proteins in endocytosis ............................................................................ 40 The adaptor protein Numb................................................................................. 42 Aims of the thesis...................................................................................................... 45 Materials and Methods.............................................................................................. 46 Animals ................................................................................................................. 46 -i - Directory Preparation of membrane protein extracts ............................................................ 46 SDS-PAGE and Western blot analysis.................................................................. 47 Ligand blotting....................................................................................................... 47 Site-directed mutagenesis of the ligand binding domain (LA7) of the chicken LDLR .............................................................................................................................. 48 Expression, purification, and refolding of ggLA7 wild type and ggLA7 mutants .... 49 Co-Immunoprecipitation ........................................................................................ 49 Antibodies ............................................................................................................. 49 Cell culture and ApoE preparation......................................................................... 50 Cloning of chicken Numb isoforms........................................................................ 50 Prokaryotic expression of recombinant His-tagged chicken Numb isoforms......... 51 Cloning of the chicken Numb PRR insert and prokaryotic expression................... 51 GST-tagged fusion proteins .................................................................................. 52 His-tagged fusion proteins..................................................................................... 52 Results...................................................................................................................... 53 LDL receptor in human and chicken...................................................................... 53 Characterization of the chicken LDLR ligand binding domain ............................... 55 Site-directed mutagenesis of the ligand binding domain.................................... 55 Expression, purification, and refolding of the LDLR ligand binding domains ..... 57 Analysis of the ligand binding ability of the recombinant LA7 fusion proteins.... 59 Characterization of the protein interaction properties of the intracellular binding domain of LDLR family members .......................................................................... 60 Interaction of adaptor proteins with the cytoplasmic tail of the LDLR................. 60 Chicken Numb ................................................................................................... 61 Genomic organization of chicken Numb according to the Ensembl chicken database ........................................................................................................ 61 Chicken Numb protein.................................................................................... 61 Chicken Numb isoforms ................................................................................. 64 Cloning of the chicken Numb isoform1 .............................................................. 65 Bacterial expression of recombinant chicken Numb isoform1 ........................... 66 Cloning of the chicken Numb isoform2 .............................................................. 67 Bacterial expression of recombinant chicken Numb isoform2 ........................... 68 Cloning of the chicken Numb isoform3 .............................................................. 69 Bacterial expression of recombinant chicken Numb isoform3 ........................... 70 - ii - Directory Binding of chicken Numb isoforms to the cytoplasmic tails of chicken and human LDLR ................................................................................................................. 71 Cloning of the chicken Numb PRR insert........................................................... 74 Bacterial expression of the recombinant chicken Numb PRR insert.................. 74 Interaction of the chicken Numb PRR insert with cytoplasmic domains of the chicken and human LDLR ................................................................................. 76 Binding of chicken LR8 and LR8B to chicken Numb.......................................... 77 Interaction of the chicken Amnionless cytoplasmic tail with adaptor proteins.... 80 Discussion ................................................................................................................ 85 Characterization of the chicken LDLR ligand binding domain ............................... 85 Protein interaction properties of the intracellular domains of LDLR family members .............................................................................................................................. 88 References ............................................................................................................... 93 Acknowledgements................................................................................................. 105 Addendum .............................................................................................................. 107 -iii - Abbreviations Abbreviations A aa amino acid Aβ amyloid β -peptide ACAT acyl-CoA:cholesterol acyltransferase AD Alzheimer’s disease α2M α2-macroglobulin α2MR α2-macroglobulin receptor Amn Amnionless AP adaptor protein apo apolipoprotein ApoER2 Apolipoprotein E receptor type 2 APP amyloid precursor protein ARH Autosomal Recessive Hypercholesterolemia B bp base pair BMP bone morphogenetic protein BSA bovine serum albumin C CCP clathrin-coated pits CCV clathrin-coated vesicles CLASP clathrin-associated sorting proteins CM chylomicrons CME clathrin-mediated endocytosis