UNIVERSITY of CALIFORNIA, SAN DIEGO Intracellular and Extracellular Interactions of the Low Density Lipoprotein Receptor Related
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Intracellular and Extracellular Interactions of the Low Density Lipoprotein Receptor Related Protein (LRP-1) A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry by Miklos Guttman Committee in charge: Professor Elizabeth Komives, Chair Professor Tracy Handel Professor Kim Prather Professor Susan Taylor Professor Hector Viadiu-Ilarraza 2009 Copyright© Miklos Guttman, 2009 All rights reserved. The dissertation of Miklos Guttman is approved, and it is acceptable in quality and form for publication on microfilmand and electronically: ______________________________________________________ ______________________________________________________ ______________________________________________________ ______________________________________________________ ______________________________________________________ Chair University of California, San Diego 2009 iii TABLE OF CONTENTS Signature Page……………………………………………………................... iii Table of Contents……………………………………………………................ iv List of Symbols and Abbreviations……………………………..................... vi List of Figures…………………………………………………………………… ix List of Tables……………………………………..…………………………….. xi Acknowledgements…………………………….…………………….………… xii Vita, Publications, Fields of Study........……………… …………………….. xiv Abstract of the Dissertation…………………….…………………………….. xvi CHAPTER 1………………………………………………………………... 1 Introduction The LDL family of receptors…….………...….....……………………. 2 The LDL-receptor (LDLR)......…………..…… ……………….……… 5 Apolipoprotein E..………………………………… ……………..……. 7 LDL-receptor related protein (LRP-1)………… ………..….…..... 8 Cytoplasmic interactions of LDLRs.………… ……………………… 11 Goals of dissertation..…………………… …………………………… 13 References…………………………………………………….….. …… 14 CHAPTER 2…………………………………………………………………… 22 Expression Refolding and Characterization of CR domains Introduction..………………………………….……………… ………. 23 Materials and Methods…………………………….…..................... 25 Results…….……...…………………………….…...……….……..... 28 Discussion.…………………………….…….................................... 39 References…………....…………………………………….………… 45 iv CHAPTER 3……………………………………………………….................. 49 Properties of Ligand Binding Modules Governing the Interaction with ApoE Introduction...…………………………...…………….…………. …… 50 Materials and Methods………………………................................... 54 Results…………………………………………………….........…....... 59 Discussion…………………………………….....…………….………. 75 References…………………………………………….....……………. 83 CHAPTER 4..……………………………………..…………………… …...... 89 Structure of the Minimum Interface between ApoE and LRP Introduction..……………………...…………………………… ……… 90 Materials and Methods……...……………...………………………… 92 Results…………..…………………………..………………………..... 99 Discussion….……………………………………….……….…..... …… 124 References..................................................................................... 138 CHAPTER 5….……………………………………………………..………… 144 Interactions of the NPXY Microdomains of LRP-1 Introduction.................................................................................... 145 Materials and Methods.................................................................... 147 Results........................................................................................... 155 Discussion……………………………………………………….……. 165 References…………………………………..................................... 178 v LIST OF SYMBOLS AND ABBREVIATIONS LDL Low density lipoprotein VLDL Very low density lipoprotein LDLR LDL- Receptor LRP LDLR-Related Protein sLRP soluble LRP VLDLR VLDL-Receptor CR Complement repeat LA Ligand binding module GST Glutatione-S-Transferase RAP Receptor Associated Protein α2M Alpha-2-macroglobulin ApoE Apolipoprotein E β-VLDL Beta-very low density lipoprotein EDTA Ethylenediaminetetraacetic acid HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid TCEP (tris(2-carboxyethyl)phosphine) HPLC High performance liquid chromatography SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis NMR Nuclear magnetic resonance TOCSY Total correlation spectroscopy NOE Nuclear Overhauser effect NOESY Nuclear Overhauser effect spectroscopy vi COSY Correlation spectroscopy HSQC Heteronuclear single quantum coherence hNOE 1H-15N heteronuclear NOE R1 Longitudinal relaxation rate R2 Transverse relaxation rate ppm parts per million HDX Hydrogen Deuterium exchange RMSD Root mean square deviation MALDI-TOF Matrix assisted laser desorbtion ionization- time of flight AUC Analytical ultracentrifugation CD Circular dichroism kDa kiloDalton SPR Surface plasmon resonance KD Dissociation equilibrium constant NC Negative control Std Standard HMW High molecular weight PCR Polymerase chain reaction APP Amyloid precursor protein CAMKII Calmodulin-dependent kinase II CSK C-src kinase HA Influenza hemagglutinin protein vii LRP1-CT LRP1 cytoplasmic domain (amino acids 4745-4807) MEF Mouse embryonic fibroblasts MUDPIT Multidimensional protein identification technology PDGF Platelet-derived growth factor PI3K Phosphoinositide-3 kinase PTB Phosphotyrosine binding domain Snx17 Sorting nexin 17 viii LIST OF FIGURES Figure 1.1: Mechanism of receptor mediated endocytosis…................... 4 Figure 1.2: Schematic of three members of the LDL receptor family ....... 6 Figure 1.3: Crystal structure of Apolipoprotein E………………………….. 10 Figure 2.1: SDS-PAGE analysis of expression and purification CRs…… 30 Figure 2.2: 1H,15N HSQC spectra of each CR/LA…....................... …….. 35 Figure 2.3: Analytical RP-HPLC of denaturing vs. native conditions……. 36 Figure 2.4: 1H,15N HSQC of LA4 after native HPLC purification…........... 37 Figure 2.5: Calcium binding isotherms of each individual CR/LA ………. 38 Figure 2.6: Calcium binding of LA45, CR16-18 and LA4 at pH 5.2…....... 40 Figure 2.7: Affinity pulldown assays of CR16-18 with ApoE(130-149)….. 42 Figure 3.1: Schematic diagram of LRP, LDL, and VLDL ......................... 52 Figure 3.2: Binding of smaller units of CR16-18 to ApoE(130-149)......... 62 Figure 3.3: NMR titration of Ub-ApoE(130-149) into each CR/LA........... 63 Figure 3.4: NMR titration of Ub-ApoE(130-149) into LA4-5 & CR16-18.. 64 Figure 3.5: Amide perturbations and plots of titrations…………………… 67 Figure 3.6: NMR titration of Ub-RAPD3 into each CR/LA……………….. 70 Figure 3.7: ApoE(130-149) affinity pulldowns with LA/CR constructs…... 72 Figure 3.8: ApoE(1-191)-DMPC and GST-RAP pulldown assays............ 73 Figure 3.9: Sequence alignments of LA4/VLA5 and LA5/VLA6 from various species………………………………… ……………….... …….. 74 Figure 3.10: Proposed models of how LDL receptors might bind ApoE- containing lipoprotein particles ……….……………................ 82 ix Figure 4.1: Perturbations of CR17 from binding various ligands.............. 101 Figure 4.2: Perturbations of ApoE(130-149) from CR17 binding.............. 102 Figure 4.3: Chemical shift index plot for ApoE(130-149)…………………. 103 Figure 4.4: Solution structure of CR17……………...……….................... 106 Figure 4.5: Calcium binding of CR17(K5A) and CR17-ApoE(130-149)… 109 Figure 4.6: HSQC comparison of CR17 to CR17-ApoE(130-149)……… 110 Figure 4.7: Native tryptophan fluorescence of CR17 constructs……….. 113 Figure 4.8: NMR titrations with fusion and CR17(K5A) constructs…….. 114 Figure 4.9: Solution structure of CR17-ApoE(130-149)……..... ……….. 117 Figure 4.10: Structural and dynamic consequences of ligand binding….. 118 Figure 4.11: NMR perturbations within CR17-ApoE(130-149)........... ….. 119 Figure 4.12: Relaxation measurements for CR17 constructs.................... 123 Figure 4.13: A different binding mode of ApoE(130-149) on CR17.…….. 131 Figure 4.14: Comparison of receptor interactions in ApoE and RAP......... 136 Figure 5.1: Anti pTyr blots of LRP-1 immuno-precipitates……...………... 158 Figure 5.2: SDS-PAGE of proteins binding NPXpY 4507………………… 159 Figure 5.3: Analysis of binding proteins to each microdomain................. 161 Figure 5.4: Effect of dephosphorylating NPXpY 4507 on binding………. 162 Figure 5.5: Specific detection of interacting proteins …………………….. 164 Figure 5.6: Detection of interactions with recombinant proteins…..…….. 167 x LIST OF TABLES Table 2.1: MALDI TOF analysis of expressed CR constructs…………….. 31 Table 2.2: Thermodynamics of calcium binding for each CR….………….. 41 Table 3.1: Binding affinities for various ApoE-LA/CR interactions………. 68 Table 4.1: CR17 & CR17-ApoE(130-149) refinement statistics…………. 107 Table 5.1: LRP1 NPXpY4507 interacting proteins……………...…………… 173 Table 5.2: LRP1 NPXpY4507 interacting proteins………………………….. 175 Table 5.3: LRP1 NPXpY4473 interacting proteins…………………………… 175 Table 5.4: LRP1 NPXY4507 interacting proteins……………………………. 176 xi ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Elizabeth Komives for her mentorship and encouragement throughout my graduate career. My fellow lab members, past and present, have all been tremendously helpful along the way. I would like to especially thank former graduate students J. Helena Prieto, Johnny E. Croy, and Gina Betts for paving the way for this project, and also Mela Mulvihill continuing the research in this area. It was a pleasure to work together with Jonathan Ostrem through the toughest parts of this project. Carla Cervantes has been greatly helpful in designing and analyzing NMR experiments. Thank you Simon Bergqvist, from whom I learned everything about calorimetry and ultracentrifugation, and Stephanie Truhlar who helped me with advice along the way. I would like to thank some of the senior scientists that have aided in my scientific