molecular events during reelin signaling

FH-Studiengang Biotechnologie

Vienna Biocenter

1030 Wien

Molecular events during Reelin signaling

Molekulare Mechanismen im Reelin Signalweg

Sarah Duit

Diplomarbeit zur Erlangung des akademischen GradesDipl. Ing. (FH)

Eingereicht am 25. Oktober 2006

Abstract

The Reelin signaling pathway plays a crucial role in the development of the mam-malian central nervous system where it is responsible for the correct positioningof neurons in laminated brain structures. The pathway is thought to influencemigration of neurons, modulate cell-cell interactions, and induce cytoskeletal re-arrangements. Disruption of the pathway in mice results in ataxia, tremors, im-balance and a reeling gait. The large glycoprotein Reelin is secreted during braindevelopment by specialized cells, the Cajal-Retzius cells, in the neocortex andexternal granule cells in the developing cerebellum. The key events triggering thesignaling pathway are binding of Reelin to Apolipoprotein E receptor 2 (ApoER2)and very low density lipoprotein receptor (VLDLR) expressed by target neurons,and subsequent phosphorylation of the intracellular adaptor protein disabled 1(Dab1).

Although the first steps in Reelin signaling are well understood, the knowledgeon the modulation and regulation of the initial signal is still scarce. Here, it couldbe demonstrated that both receptors and Reelin are subjected to regulation bydifferent mechanisms independently from the primary signaling event. Reelin isinternalized and degraded at different rates by cells expressing either ApoER2or VLDLR, leading to prolonged membrane association or rapid clearance fromthe cell surface, respectively. While VLDLR recycles to the plasma membraneafter degradation of the ligand, ApoER2 is degraded via the lysosomal pathway.Furthermore, extra- and intracellular fragments of ApoER2 are produced fromsecretase-mediated cleavage. These fragments could also exhibit important ef-fects in the modulation of the Reelin signaling pathway.

Zusammenfassung

Der Reelin Signalweg spielt eine entscheidende Rolle in der Entwicklung desZentralnervensystems von Sugetieren. Er gewhrleistet die korrekte Position-ierung von Neuronen in mehrschichtigen Strukturen im Gehirn, indem er Migra-tion, Zell-Zell-Interaktionen und Umstrukturierungen des Zytoskeletts von Neuro-nen kontrolliert. Eine Strung dieses Signalwegs fhrt im Maus-Modell zu Koor-dinationsstrungen, Gleichgewichtsstrungen und einem taumelnden Gang. DasGlycoprotein Reelin wird whrend der embryonalen Hirnentwicklung von spezial-isierten Zellen, den Cajal-Retzius Zellen, im Neocortex und im entstehendenCerebellum sezerniert. Die Schlsselereignisse, die den Reelin Signalweg ein-leiten, sind die Bindung von Reelin an Apolipoprotein E Rezeptor 2 (ApoER2) undvery low density Lipoprotein Rezeptor (VLDLR) und die anschlieende Phos-phorilierung des intrazellulren Adapterproteins Disabled 1 (Dab1).

Auch wenn die ersten Schritte im Reelin Signalweg seht gut untersucht sind,so bleiben doch Fragen bezglich Modulation und Regulierung des Signals of-fen. In der vorliegenden Arbeit konnte gezeigt weren, dass beide Rezeptorenund auch Reelin einer Regulation durch verschiedene Mechanismen unterworfensind, die unabhngig von der Signalweitergabe agieren. Reelin wird von ApoER2-und VLDLR-exprimierenden Zellen in unterschiedlicher Geschwindigkeit inter-nalisiert und abgebaut, was entweder zu einer lnger anhaltenden Membran-Assoziation oder zu einer schnellen Beseitigung von Reelin von der Zellober-flche fhrt. Whrend VLDLR nach dem Abbau des Liganden an die Zellmem-bran zurckgefhrt wird, wird ApoER2 ber das Lysosom abgebaut. Darberhinaus wird ApoER2 einer spezifischen Secretase-vermittelten Spaltung unter-zogen, wodurch extra- und intrazellulre Rezeptor-Fragmente entstehen. Auchdiese Fragmente knnten sich als wichtige Faktoren fr die Modulation des ReelinSignalwegs herausstelen.

Contents

List of Figures v

List of Tables vii

1 Introduction 11.1 The low density lipoprotein receptor family . . . . . . . . . . . . . . 1

1.1.1 Structural organization of the LDLR family members . . . . 11.1.2 The low density lipoprotein receptor . . . . . . . . . . . . . 41.1.3 The very low density lipoprotein receptor . . . . . . . . . . 51.1.4 The apolipoprotein E receptor 2 . . . . . . . . . . . . . . . 61.1.5 The LDL receptor related protein . . . . . . . . . . . . . . . 71.1.6 LRP1b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.1.7 Megalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.1.8 MEGF7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2 The Reelin signaling pathway . . . . . . . . . . . . . . . . . . . . . 91.2.1 Reelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2.2 The Reelin signaling cascade . . . . . . . . . . . . . . . . . 101.2.3 Reelin signaling in brain development . . . . . . . . . . . . 121.2.4 Reelin signaling in the adult brain . . . . . . . . . . . . . . . 14

1.3 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.3.1 Clathrin-mediated endocytosis . . . . . . . . . . . . . . . . 161.3.2 Lipid rafts and caveolae . . . . . . . . . . . . . . . . . . . . 17

2 Aims of this study 19

3 Material and Methods 223.1 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223.2 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.3 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.3.1 Cells and cell lines . . . . . . . . . . . . . . . . . . . . . . . 233.3.2 Freezing of cells . . . . . . . . . . . . . . . . . . . . . . . . 25

Contents iv

3.3.3 Thawing of cells . . . . . . . . . . . . . . . . . . . . . . . . 253.3.4 Transient transfection . . . . . . . . . . . . . . . . . . . . . 253.3.5 Conditioned media . . . . . . . . . . . . . . . . . . . . . . . 26

3.4 Protein extraction and analysis . . . . . . . . . . . . . . . . . . . . 263.4.1 Preparation of cell extracts (Hunt buffer) . . . . . . . . . . . 263.4.2 Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 263.4.3 Western blotting . . . . . . . . . . . . . . . . . . . . . . . . 27

3.5 Western blot based assays . . . . . . . . . . . . . . . . . . . . . . 283.5.1 Membrane localization assay . . . . . . . . . . . . . . . . . 283.5.2 Dab1 phosphorylation assay . . . . . . . . . . . . . . . . . 293.5.3 Reelin uptake and degradation assay . . . . . . . . . . . . 293.5.4 Reelin depletion assay . . . . . . . . . . . . . . . . . . . . . 303.5.5 Surface biotinylation assay . . . . . . . . . . . . . . . . . . 303.5.6 Receptor degradation assay . . . . . . . . . . . . . . . . . 313.5.7 Receptor fragmentation assay . . . . . . . . . . . . . . . . 313.5.8 Membrane binding assay . . . . . . . . . . . . . . . . . . . 32

3.6 Immunofluorescence Microscopy . . . . . . . . . . . . . . . . . . . 323.6.1 Subcellular receptor distribution . . . . . . . . . . . . . . . . 323.6.2 Reelin internalization . . . . . . . . . . . . . . . . . . . . . . 333.6.3 Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . 33

4 Results 354.1 Localization of ApoER2 and VLDLR within the plasma membrane 354.2 Reelin internalization by ApoER2 and VLDLR . . . . . . . . . . . . 384.3 Surface expression of ApoER2 and VLDLR . . . . . . . . . . . . . 424.4 Reelin-induced degradation of ApoER2 . . . . . . . . . . . . . . . 474.5 Reelin-induced fragmentation of ApoER2 . . . . . . . . . . . . . . 50

5 Discussion 555.1 Membrane localization of the Reelin receptors . . . . . . . . . . . 565.2 Reelin endocytosis and degradation . . . . . . . . . . . . . . . . . 575.3 Surface expression of the Reelin receptors . . . . . . . . . . . . . 615.4 Reelin-induced degradation and specific cleavage of the receptors 62

References 67

List of Figures

1.1 Structural organization of the LDLR family members . . . . . . . . 21.2 Structure of the Reelin protein . . . . . . . . . . . . . . . . . . . . . 101.3 The Reelin signaling pathway . . . . . . . . . . . . . . . . . . . . . 111.4 Neocortical layer formation by radial migration . . . . . . . . . . . . 121.5 Defective neocortical layer formation in reeler mouse mutants . . . 141.6 Multiple mechanisms of endocytosis. . . . . . . . . . . . . . . . . . 161.7 Clathrin-mediated endocytosis . . . . . . . . . . . . . . . . . . . . 171.8 Membrane organization of lipid rafts and caveolae . . . . . . . . . 18

4.1 Membrane distribution of ApoER2. . . . . . . . . . . . . . . . . . . 364.2 Reelin-induced phosphorylation of Dab1 is independent of receptor

localization to rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . 374.3 Different Reelin internalization rates . . . . . . . . . . . . . . . . . 384.4 Receptor-mediated Reelin depletion from the medium . . . . . . . 394.5 Reelin fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . 404.6 Chimeric receptors of ApoER2 and VLDLR . . . . . . . . . . . . . 414.7 Reelin internalization by chimeric receptors . . . . . . . . . . . . . 424.8 Reelin-induced phosphorylation of Dab1 is independent of endo-

cytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.9 Subcellular distribution of ApoER2 and VLDLR . . . . . . . . . . . 434.10 Surface expression of ApoER2 and VLDLR . . . . . . . . . . . . . 444.11 Trafficking of ApoER2 upon Reelin stimulation . . . . . . . . . . . . 454.12 Trafficking of ApoER2 in the presence of Dab1 . . . . . . . . . . . 464.13 Multivalent ligands induce degradation of ApoER2 . . . . . . . . . 474.14 Multivalent ligands induce degradation of ApoER2 in neurons . . . 484.15 Reelin stimulation induces lysosomal degradation of ApoER2 . . . 494.16 Raft-disrupting agents do not interfere with ApoER2 degradation . 494.17 Multivalent ligands induce fragmentation of ApoER2 . . . . . . . . 504.18 The -secretase inhibitor DAPT causes accumulation of the mem-

brane-bound intracellular fragments of ApoER2 and VLDLR . . . . 51

List of Figures vi

4.19 The intracellular fragment of ApoER2 produced by -secretase isbound to the plasma membrane . . . . . . . . . . . . . . . . . . . 52

4.20 Chloroquine causes accumulation of the membrane-bound intra-cellular fragment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.21 Methyl--cyclodextrin inhibits secretase-mediated fragmentation ofApoER2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.22 Presence of Dab1 enhances secretase-mediated ApoER2 frag-mentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.23 Fragmentation of ApoER2 does not occur at 4C . . . . . . . . . . 54

5.1 Reelin fragments produced by metalloproteinase cleavage . . . . . 60

List of Tables

3.1 Composition of SDS polyacrylamide gels . . . . . . . . . . . . . . 273.2 Antibodies used in Western blotting . . . . . . . . . . . . . . . . . . 283.3 Antibodies used for immunofluorescence . . . . . . . . . . . . . . 34

1 Introduction

1.1 The low density lipoprotein receptor family

The low density lipoprotein receptor (LDLR) gene family includes several struc-turally similar transmembrane receptors in mammals which are involved in molec-ular transport and signal transduction. The best known member of this gene fam-ily is the low density lipoprotein (LDL) receptor which plays an important rolein cholesterol homeostasis. Other core-members of the LDLR family are theapolipoprotein E receptor 2 (apoER2), the very low density lipoprotein recep-tor (VLDLR), the LDL receptor related protein (LRP, also referred to as LRP1),LRP1b, megalin (also known as LRP2 or gp330) and the less well characterizedMEGF7 (also known as LRP4). More distantly related receptors include LRP5,LRP6 and SorLA (also known as LR11) [Herz and Bock , 2002; May et al., 2005].

Traditionally, the physiologic role of membrane proteins was believed to be en-docytosis of macromolecules and delivery of ligands to the lysosome for degra-dation. More recent findings however connected these receptors to signal trans-duction pathways, including G-protein mediated protein kinase activation [Goret-zki and Mueller , 1998], modulation of the cytoskeletal organization via cytosolicadaptor proteins [Trommsdorff et al., 1998], and neuronal migration in brain de-velopment [Herz et al., 2000].

1.1.1 Structural organization of the LDLR family members

All core members of the LDL receptor family show a modular organization com-prising four distinct domains [Gent and Braakman, 2004; Hussain et al., 1999]:the N-terminal ligand-binding domain, the epidermal growth factor (EGF) precur-sor homology domain, a single transmembrane domain and a cytoplasmic tail. Afifth region, termed the O-linked sugar domain, can only be found in LDL receptor,VLDL receptor and ApoER2 and is located between the EGF precursor homology

1 Introduction 2

region and the transmembrane domain. Combinations of these domains build upthe core members of the LDLR family (figure 1.1), ranging in molecular massesfrom about 130 kDa (LDLR, VLDLR, ApoER2) up to approximately 600 kDa (LRP,LRP1b, megalin).

Figure 1.1 Structural organization of the LDL receptor family core members. The receptorsof this family contain various numbers of LA-repeats at the amino-terminus, followed by the EGFprecursor homology domain, the O-linked sugar domain and the transmembrane domain. Thecytoplasmic tails of the core members contain at least one NPxY motif. ApoER2 and VLDLRare depicted including differentially spliced regions (marked with *): several LA-repeats and theO-linked sugar domain in both receptors as well as the furin cleavage site and the proline richinsert in ApoER2. In contrast to LDLR, ApoER2, and VLDLR, MEGF7 has four EGF precursorhomology domains. LRP, LRP1b and megalin are much larger than the other core members of theLDLR family and contain multiple clusters of LA-repeats and EGF precursor homology domains.Modified from Nykjaer and Willnow [2002].

The ligand-binding domains are made up of head-to-tail arranged LDL receptortype A repeats, known as LA-repeats. Each of these repeats consists of about

1 Introduction 3

40 amino acids including six conserved cysteine residues forming three disulfidebonds. In the endoplasmic reticulum (ER), the receptor associated protein (RAP)binds to the LA-repeats to prevent premature interaction of other ligands with thereceptors [Bu, 2001]. Furthermore, RAP acts as a chaperone to assist in receptorfolding [Bu, 2001]. Since RAP binds to all receptors of the LDLR family, it can beused as a unique tool to study ligand-receptor interactions.

The EGF precursor homology domains consist of cysteine-rich growth factor re-peats of about 40 amino acids separated by YWTD repeats. The growth fac-tor repeats (EGF-like repeats) contain six conserved cysteine residues, differingfrom those of the ligand-binding repeats in their pattern of disulfide bond forma-tion. Located in between are regions termed YWTD repeats, containing the con-sensus tetrapeptide Tyr-Trp-Thr-Asp (YWTD), which form a -propeller structure[Jeon et al., 2001; Springer , 1998]. The EGF precursor homology domains in-cluding the -propeller structures are necessary for the pH-dependent disso-ciation of ligands from the receptor in endosomes [Jeon and Blacklow , 2005;Rudenko et al., 2002].

Adjacent to the EGF precursor homology domain of LDL receptor, VLDL recep-tor, and ApoER2, is the so called O-linked sugar domain, a short region rich inserine and threonine residues that undergo O-linked glycosylation. For ApoER2and VLDLR, distinct splice variants containing or lacking this glycosylation re-gion are expressed in different tissues [Korschineck et al., 2001; Nakamura et al.,1998]. Although it is not exactly clear which biological functions are regulated byits presence or absence, it is known that the O-linked sugar domain contributes toreceptor stability and has a critical influence on the rate of proteolytic processingof the receptors [Magran et al., 1999; May et al., 2003].

A single transmembrane domain anchoring the receptors into the membrane via astretch of hydrophobic residues connects the extracellular regions to the cytoplas-mic tail. The relatively short cytoplasmic region includes at least one NPxY (Asp-Pro-Xxx-Tyr, where Xxx denotes any amino acid) motif which can interact withadaptor proteins and is important for the localization of the receptors to clathrin-coated pits and their internalization [Chen et al., 1990].

In the more distant relatives such as LRP5, LRP6, and SorLA, not all of the men-tioned elements can be found. LRP5 and LRP6 are closely related to each other.Compared to the LDLR family core members, they show an inverted order of the

1 Introduction 4

ligand binding and the EGF precursor domain. Furthermore, their cytoplasmictails do not contain an NPxY motif. The structure of SorLA differs even more fromthe basic structure of the LDLR family members because it contains a domain withhomology to a yeast receptor for vacuolar protein sorting (VPS10) and fibronectintype III repeats which can also be found in neuronal adhesion molecules [Jacob-sen et al., 1996].

1.1.2 The low density lipoprotein receptor

The LDL receptor is the name giving and best studied member of the LDLRfamily. The N-terminal ligand binding domain of the mature receptor consistsof seven LA-repeats, arranged in a head-to-tail fashion. The EGF precursor ho-mology domain contains three EGF-like repeats and one YWTD region, two of thegrowth factor repeats being located N-terminal and one C-terminal of the YWTDsequence. These modules are followed by an O-linked sugar domain, a trans-membrane segment and a short intracellular tail containing an NPxY motif [Jeonand Blacklow , 2005].

The physiological role of the LDL receptor in mammals is to maintain cholesterolhomeostasis by cellular uptake and catabolism of plasma cholesterol. In the cir-culatory system, cholesterol and other lipids are transported as a part of lipopro-teins. These particles consist of a lipid core composed of esterified cholesteroland triglycerides, surrounded by a monolayer of phospholipids and unesterifiedcholesterol with associated apolipoproteins. Depending on their triglyceride pro-portion and their buoyant density, the lipoprotein particles are divided into differentsubcategories: chylomicrons, very low density lipoprotein (VLDL), intermediatedensity lipoprotein (IDL), low density lipoprotein (LDL), and high density lipopro-tein (HDL). Dietary fat is transported from the intestinal mucosa to the liver in formof chylomicrons. In the circulation, triglycerides are extracted from the chylomi-crons and degraded by lipoprotein lipases to free fatty acids which are taken upfrom cells as energy source or stored by adipose tissue. The chylomicron rem-nants are taken up by the liver via receptor mediated endocytosis. There, VLDL issynthesized from triglycerides and cholesterol delivered by the chylomicron rem-nants and released to the blood stream. In the circulation, the VLDL particles areconverted to IDL and later LDL by further extraction of triglycerides by lipopro-tein lipases. LDL finally carries cholesterol to other tissues where it is recognized

1 Introduction 5

and internalized by LDL receptor. HDL collects cholesterol from the tissues anddelivers it back to the liver.

The binding of lipoprotein particles to lipoprotein receptors is mediated by apo-lipoproteins. Natural ligands for the LDL receptor are apolipoprotein E (apoE) andapolipoprotein B-100 (apoB-100), which mediates binding of VLDL, IDL and LDL.The internalization of LDL particles after binding to LDLR is carried out by clathrin-mediated endocytosis (originally referred to as receptor-mediated endocytosis)[Goldstein et al., 1985]. This process is dependent on the localization of thereceptors to clathrin-coated pits via the NPxY motifs in their cytoplasmic tail [Chenet al., 1990]. The coated pit pinches off the cell membrane to form a coatedvesicle containing the receptor-lipoprotein-complex. After shedding the clathrincoat, the vesicle fuses with a late endosome where the receptor is exposed toa slightly acidic pH environment. This causes conformational changes in thereceptor leading to the release of the LDL particle. Subsequently, the receptorand the lipoprotein are separated for further processing. The LDL receptor caneither be degraded or delivered back to the cell surface by receptor recycling. Thelipoprotein particle is degraded in the lysosome, liberating cholesterol which canbe stored in the cell in the form of cholesterol ester or used as a component ofplasma membranes or for the synthesis of bile acids or steroid hormones. Severalfeedback mechanisms ensure the maintenance of a constant level of cholesterolin the cell and protect it from cholesterol overaccumulation [Brown and Goldstein,1986]. On the one hand, LDL receptor expression is downregulated, preventingfurther uptake of LDL and cholesterol. On the other hand, the level of cellularcholesterol biosynthesis is reduced in the presence of intracellular cholesterolby suppression of the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and HMG-CoA reductase. Furthermore, free cholesterol in thecell activates acyl-CoA-cholesterol:acyltransferase (ACAT) which facilitates thestorage of excess cholesterol as cholesterol esters.

Apart from delivering cholesterol to the cells in an appropriate amount, receptor-mediated uptake of LDL also guarantees the clearance of lipoproteins from thecirculation. A genetic disorder affecting the functionality of the LDL receptorleads to the disease familial hypercholesterolemia (FH) which is characterized bylipoprotein accumulation in the blood stream due to the defective cellular uptakemechanism. Symptoms caused by elevated plasma LDL levels are prematureatherosclerosis and heart attacks.

1 Introduction 6

1.1.3 The very low density lipoprotein receptor

The structure of the very low density lipoprotein receptor is almost identical tothat of the LDL receptor except for an additional LA-repeat in the ligand bindingdomain. The receptor recognizes triglyceride-rich lipoproteins containing apoEwhich includes VLDL, IDL and chylomicrons but not LDL. VLDLR is expressedat high levels on the capillary endothelium of skeletal muscle, heart and adiposetissue [Wyne et al., 1996] where it occurs in splice variants containing or lackingthe O-linked sugar domain. The variant including this domain seems to be morestable at the cell surface [Magran et al., 1999]. In the brain, a third splice variantlacking one LA-repeat can be found [Takahashi et al., 2004].

Since VLDLR is only expressed on the endothelium of tissues involved in theperipheral metabolism of triglyceride-rich lipoproteins, it was suggested that itsphysiological role is the delivery of VLDL to peripheral tissues [Wyne et al., 1996].Another possibility is that the main function of VLDLR is the binding of triglyceride-rich lipoproteins to the surface of endothelial cells, facilitating the hydrolysis oftriglycerides by lipoprotein lipase (LPL) resulting in the delivery of free fatty acidsto the adjacent tissue. This view is supported by the findings that the presence ofLPL enhances the binding of lipoproteins to VLDLR [Takahashi et al., 1995] andthat VLDLR enhances the LPL mediated triglyceride hydrolysis [Goudriaan et al.,2004]. Moreover, it was shown that the VLDL receptor is involved in the transcy-tosis of LPL across endothelial cells [Obunike et al., 2001]. However, lipoproteinprofiles of VLDLR deficient mice do not differ from those of animals expressingthe receptor [Frykman et al., 1995]. Nevertheless, the knockout mice are partiallyresistant to diet-induced obesity [Goudriaan et al., 2001] and tend to be leanerdue to a reduction in adipose tissue mass [Frykman et al., 1995] indicating re-duced fatty acid transport into adipocytes. Since mice are high density lipoproteinanimals, LDLR/VLDLR double knockout mice which have increased LDL and de-creased HDL levels resemble the human lipoprotein profile better. Keeping theseanimals under a high fat diet, a significant increase in serum triglyceride levelscould be observed [Tacken et al., 2000]. Together, these results clearly show arole for the VLDL receptor in peripheral triglyceride uptake. Beyond this function,VLDLR also plays a role in signal transduction, angiogenesis and tumor growth.Its ligands include urokinase plasminogen activator (uPA)/plasminogen activatorinhibitor 1 complex [Argraves et al., 1995], thrombospondin [Mikhailenko et al.,

1 Introduction 7

1997], tissue factor pathway inhibitor (TFPI) [Hembrough et al., 2001] and Reelin[Hiesberger et al., 1999].

1.1.4 The apolipoprotein E receptor 2

ApoER2 is almost exclusively expressed in brain, testis and placenta. Alterna-tive splicing generates distinct variants of ApoER2 which differ in the number ofLA-repeats and the presence or absence of the O-linked sugar domain, a furincleavage site, and a 59 amino acid proline-rich insertion in the cytoplasmic tail.

In analogy to the VLDL receptor, the apoER2 variant lacking the O-linked sugardomain is more susceptible to proteolytic processing [May et al., 2003]. Depend-ing on the species and organs expressing the receptor, apoER2 can contain eitherthree, four, five, seven or eight LA-repeats [Brandes et al., 2001; Kim et al., 1997;Sun and Soutar , 1999]. In man and mouse but not chicken, an additional exonat the carboxy-terminus of the ligand binding domain constitutes a furin consen-sus cleavage site. Cleavage by the calcium-dependent serine endoprotease furin[Thomas, 2002] at this site produces a soluble receptor fragment containing theentire ligand binding domain consisting of four LA-repeats. The secreted frag-ment is able to inhibit Reelin signaling in primary neurons [Koch et al., 2002]. Inthe cytoplasmic domain of the human and murine (but not the avian) receptor,differential splicing of a single exon leads to a longer version of the tail contain-ing a proline-rich insertion of 59 amino acids [Brandes et al., 1997]. This insertand features in or near the transmembrane domain exclude ApoER2 from carry-ing out clathrin-mediated endocytosis [Sun and Soutar , 2003]. Furthermore, thisregion is capable of binding JIP-1 (c-jun N-terminal kinase interaction protein 1)and JIP-2 [Stockinger et al., 2000]. JIP was originally described as a scaffold pro-tein being involved in a MAP kinase pathway. The main protein of this pathwayis c-jun N-terminal kinase (JNK). Once activated, it translocates to the nucleuswhere it modulates target gene expression. Another adaptor protein binding tothe proline-rich insert is the postsynaptic density protein PSD-95 which interactswith both ApoER2 and the N-methyl-D-aspartate (NMDA) type gutamate receptorsimultaneously in the presence of Reelin. This interaction plays a role in synapticplasticity and memory in the adult brain by regulating synaptic calcium influx viathe NMDA receptor [DArcangelo, 2005].

1 Introduction 8

1.1.5 The LDL receptor related protein

The LDLR related protein (LRP, LRP1) is a multifunctional receptor which is ex-pressed in a broad range of cell types, including hepatocytes, neurons, vascularsmooth muscle cells, macrophages and embryonic tissues and interacts with awide variety of ligands. This multifunctionality is likely the reason for the earlyembryonic lethality of mouse embryos in which the LRP gene has been disruptedin all cells by conventional gene targeting [Herz et al., 1992].

In the liver, LRP mediates the uptake of circulating chylomicron remnants[Rohlmann et al., 1998]. In the brain, LRP plays an important role in the balancebetween synthesis and clearance of the -amyloid peptide A. A is a majorfactor in the pathogenesis of Alzheimers disease and is generated by proteolyticprocessing of the -amyloid precursor protein (APP). Furthermore, LRP has aninfluence on synaptic plasticity due to PSD-95 mediated interaction with NMDAreceptors [Qiu et al., 2002] induced by binding of activated 2-macroglobulin (2-M) to LRP. Other ligands for LRP include apolipoprotein E, lipoprotein lipase,platelet derived growth factor (PDGF), transforming growth factor (TGF), andurokinase plasminogen activator (uPA):plasminogen activator inhibitor-1 (PAI-1)complex.

1.1.6 LRP1b

LRP1b is highly homologous to LRP except for an additional LA-repeat and analternatively spliced unique exon within its cytoplasmic tail coding for 33 aminoacids. In mice, the primary site of LRP1b expression is the central nervous sys-tem but it was recently shown that expression in humans is more widespread [Liet al., 2005]. LRP1b was originally discovered as a putative tumor suppressor[Liu et al., 2000]. Further findings linking homozygous deletions, DNA methyla-tion or abnormal transcripts of the LRP1b gene to different types of cancer havestrengthened this theory [Hirai et al., 2004; Langbein et al., 2002; Pineau et al.,2003; Sonoda et al., 2004].

1 Introduction 9

1.1.7 Megalin

Megalin is highly expressed in the renal proximal tubules, where it plays an im-portant role in renal resorption. Megalin was first discribed as pathogenic antigenfor Heymann nephritis, an autoimmune disease in rats [Kerjaschki and Farquhar ,1982]. Under physiological conditions, megalin mediates transepithelial trans-port of various macromolecules including transcobalamin/vitamin B12 complex[Moestrup et al., 1996], myoglobin [Gburek et al., 2003], retinol binding protein(RBP) in complex with retinol (vitamin A) [Marin et al., 2001], insulin [Orlandoet al., 1998], and sonic hedgehog [Morales et al., 2006].

1.1.8 MEGF7

MEGF7 (also known as LRP4) was discovered in 1998 during a screen for pro-teins containing multiple EGF-like domains in human brain [Nakayama et al.,1998]. Recently, it was revealed that MEGF7 deficient mice exhibited a severemalfunction of digit differentiation. A role of MEGF7 as a modulator of cellu-lar signaling pathways including wingless/int proteins (Wnt), bone morphogenicproteins (Bmp), fibroblast growth factors (Fgf) and sonic hedgehog (Shh) wassuggested, since the interaction of these pathways is required for proper limbpatterning [Johnson et al., 2005].

1.2 The Reelin signaling pathway

The Reelin signaling pathway plays a crucial role in the development of the cen-tral nervous system (CNS) where it is responsible for the correct positioning ofneurons in laminated brain structures. Disruption of the pathway results in ataxia,tremors, imbalance and a reeling gait that becomes apparent two weeks afterbirth [DArcangelo and Curran, 1998]. Although the specific mechanisms affectedby Reelin signaling are not fully understood, Reelin is thought to influence migra-tion of neurons, modulate cell-cell interaction and induce cytoskeletal rearrange-ments.

Analysis of mutant mice has led to the identification and characterization of genesinvolved in this pathway. Naturally occurring mutants showing this phenotype

1 Introduction 10

include reeler, scrambler and yotari mice. While the causative mutation of reelermice is in Reelin, scrambler and yotari mice arise from mutations in Disabled 1,a cytoplasmic adaptor protein [Sheldon et al., 1997]. The reeler phenotype canalso be observed in ApoER2 and VLDLR double knock out mice [Trommsdorffet al., 1999].

1.2.1 Reelin

Reelin is a large secreted glycoprotein of 3461 amino acids with a molecular massof approximately 430 kDa. Mouse and human Reelin are 94.2% identical [DeSilvaet al., 1997]. Reelin contains a cleavable signal peptide at the N-terminus fol-lowed by eight consecutive repeats, each containing two subdomains separatedby EGF-like motifs [DArcangelo et al., 1995]. It harbors two protease cleavagesites located between repeats two and three and repeats six and seven (figure1.2). In vivo, Reelin is processed by a metalloproteinase giving rise to two frag-ments of approximately 300 kDa and 180 kDa which can be detected in addition tothe full length protein [DArcangelo et al., 1999; Lambert de Rouvroit et al., 1999].

Figure 1.2 Structure of the Reelin protein. Reelin contains a cleavable signal peptide atthe N-terminus (segment S) followed by an F-spondin homology domain (segment SP) and aunique region (segment H) with no sequence homology. The main body of the protein consistsof eight repeats (segments 1-8), each containing an EGF-like motif at the center, flanked by twosubdomains (A and B). The C-terminal region of the protein is rich in basic residues and is requiredfor secretion [DArcangelo et al., 1997]. The two arrows point to the cleavage sites responsible forReelin processing in vivo. Modified from Tissir and Goffinet [2003].

In murine embryos, Reelin is first detected at embryonic day 11.5 (E11.5), in-creases up to birth and remains high until postnatal day 11 (P11). It plays animportant role in neuronal migration during development of laminar structures ofthe mammalian brain including the cerebral cortex and cerebellum. The highestexpression of Reelin can be detected in the marginal zone of the developing cere-bral cortex, where it is secreted by Cajal-Retzius neurons as well as in externalgranule cells of the developing cerebellum [DArcangelo et al., 1995]. Besidesthe brain, Reelin is also present at low levels in peripheral organs such as adult

1 Introduction 11

blood, liver, and kidney [Smalheiser et al., 2000]. The function of Reelin signalingin non-neuronal tissues however is still not clear. Besides its signaling proper-ties, Reelin acts as a serine protease on adhesion molecules of the extracellularmatrix [Quattrocchi et al., 2002], thereby possibly facilitating cellular migration.

1.2.2 The Reelin signaling cascade

Reelin molecules form disulfide-linked homodimers [Kubo et al., 2002] which arecapable of interacting with ApoER2 as well as with VLDLR [DArcangelo et al.,1999]. Binding of Reelin dimers to the ligand binding domains of ApoER2 and/orVLDLR leads to dimerization or oligomerization of the corresponding receptors(figure 1.3). When clustered, the receptors are able to induce tyrosine phospho-rylation of Dab1 [Strasser et al., 2004] on residues 198 and 220 [Benhayon et al.,2003; Keshvara et al., 2001]. Interaction of Dab1 with the receptors occurs be-tween the unphosphorylated NPxY motif in the cytoplasmic tails of ApoER2 andVLDLR and the phosphotyrosine binding (PTB) domain of Dab1 [Howell et al.,1999; Trommsdorff et al., 1999]. The kinase responsible for Dab1 phosphoryla-tion is a member of the Src family kinases (SFK), with Fyn being the main Dab1 ki-nase in vivo, followed by Src and, to a lesser extent, by Yes [Arnaud et al., 2003b;Kuo et al., 2005]. Abl does not play a significant role in Dab1 phosphorylation[Arnaud et al., 2003b]. To be a substrate for tyrosine phosphorylation, Dab1 hasto be serine/threonine phosphorylated. Cyclin-dependent kinase 5 (Cdk5) hasbeen shown to be responsible for this phosphorylation which occurs indepen-dently of the presence of Reelin [Keshvara et al., 2002]. Tyrosine phosphorylatedDab1 interacts with phosphoinositide-3-kinase (PI3-K) which leads to the acti-vation of Akt/protein kinase B (PKB) and inhibition of glycogen synthase kinase3 (GSK3) [Beffert et al., 2002; Bock et al., 2003]. Thereby, Reelin stimulationleads to a reduction of the phosphorylation state of the microtubule-associatedprotein tau, a known GSK3 substrate [Beffert et al., 2002; Ohkubo et al., 2003].Furthermore, Reelin eventually has effects on other downstream targets of thePI3-K pathway which is involved in diverse processes, including cell migration.

In addition to PI3-K, other proteins have been shown to interact with Dab1 in aReelin-dependent and phosphorylation state-dependent fashion. These includelissencephaly 1 (Lis1) [Assadi et al., 2003], Nck (noncatalytic region of tyrosinekinase) adapter protein (Nck) [Pramatarova et al., 2003], Crk family members

1 Introduction 12

[Ballif et al., 2004; Chen et al., 2004; Huang et al., 2004], and neuronal Wiskott-Aldrich syndrome protein (N-WASP) [Suetsugu et al., 2004], all of them beinginvolved in cell migration.

Figure 1.3 The Reelin signaling pathway. Reelin dimers bind to both ApoER2 and VLDLRwhich results in dimerization or oligomerization of the receptors. Disabled 1 (Dab1) is ser-ine/threonine phosphorylated by cyclin dependent kinase 5 (Cdk5) which makes it a sub-strate for tyrosine phosphorylation by Src family kinases (SFK). While Cdk5 acts independentlyfrom the Reelin signal, tyrosine phosphorylation is induced by Reelin dependent clustering ofApoER2 and VLDLR. The Src family kinases themselves can be further activated by phospho-rylated Dab1 [Bock and Herz, 2003]. Furthermore, tyrosine phosphorylated Dab1 interacts withphosphoinositide-3-kinase (PI3-K) which leads to activation of Akt/PKB and inhibition of GSK3.These kinases are part of a pathway involved in cell migration which controls neuronal positioningin brain development.

After Reelin stimulated tyrosine phosphorylation, Dab1 is ubiquitinated and de-graded by proteasomes [Arnaud et al., 2003a] which might be a mechanism toensure a transient response to the Reelin signal. Non tyrosine phosphorylatedDab1 is stable with a half-life time of 12 h, while tyrosine phosphorylation de-creases the half-life of Dab1 to 3 h.

1.2.3 Reelin signaling in brain development

For the development of the brain, Reelin signaling is crucial for proper migrationof neuronal progenitors from defined proliferative zones to their final location in

1 Introduction 13

the cerebral cortex and the cerebellum.

The six neuronal layers of the cerebral cortex are formed during embryonic de-velopment by migration of neurons from the subventricular zone (SVZ) outwardsalong radial glial fibers to the pial surface of the brain (figure 1.4). This radialmigration occurs in the mouse between embryonic days 11 and 18.

Figure 1.4 Neocortical layer formation by radial migration. At embryonic day 11 (E11), thepreplate (PP) is established by a wave of postmitotic neurons that has migrated from the ventric-ular zone (VZ) to the pial surface (PS). By E13, a second postmitotic neuronal wave has migratedthrough the intermediate zone (IZ) and split the PP into the marginal zone (MZ) and subplate (SP),creating the cortical plate (CP). During E14-E18, subsequent waves of neurons expand the CP inan inside-out fashion, as each wave of neurons passes its predecessors to settle underneath theMZ. Neuronal positioning occurs mainly by migration along radial glial fibers (shown as verticalbars). In adulthood, the SP degenerates, leaving behind a six-layered neocortex. Modified fromGupta et al. [2002].

The first wave of postmitotic neurons leaving the SVZ establishes a neuronal layerknown as the preplate which is split by the following neuronal wave, resulting in asubplate and a superficial marginal zone. The subplate is located proximal to theventricular zone, while the marginal zone which harbors Reelin secreting Cajal-Retzius cells [Frotscher , 1998] is positioned just underneath the pial surface ofthe brain. The new layer between the subplate and the marginal zone is referredto as the cortical plate. Subsequent waves of migrating neurons traverse the sub-plate and the preexisting neuronal layers of the cortical plate until they encounterReelin. There, they cease migration, detach from the radial glial fibers and formnew defined layers underneath the marginal zone. According to this mechanism,

1 Introduction 14

cortical layers form in an inside-out manner, with the latest-born neurons occupy-ing the outermost layers of the cortical plate. After the cortical plate has been fullyestablished, the subplate degenerates, leaving a cortex consisting of six neuronallayers that persists throughout adulthood.

In mouse mutants exhibiting the reeler phenotype, the Reelin pathway is dis-rupted. As demonstrated in figure 1.5, migrating neurons are unable to bypasspre-existing layers which results in an unsplit preplate (called the superplate) andinverted, disorganized cortical layers [Gupta et al., 2002]. Several hypotheseshave been proposed to explain the function of Reelin in neuronal layer formation:Reelin may act as an attractant molecule for the migrating neurons; it may act as astop signal when the neuron has reached its destination in the cortical plate; or itmay interrupt the association between migrating neurons and radial glia. Anothertheory [Luque et al., 2003] is based upon the finding that neocortical neurons arederived from asymmetrical cell division of radial glia and the observation that theyinherit the glia process, already attached to the pial basement membrane [Miy-ata et al., 2001; Tamamaki et al., 2001]. So, many newborn neurons might usetheir own basement membrane-attached radial processes to reach their targetposition by somatic translocation [Nadarajah et al., 2001]. It was suggested thatReelin signaling is required for the maintenance of the radial glia cytoskeletal phe-notype and that it acts on the inherited radial processes of neocortical neuronsto induce cytoskeletal rearrangements required for somatic translocation [Luqueet al., 2003]. In the absence of Reelin, like in the reeler mouse, somatic translo-cation is impaired, which may explain the incapacity of reeler neurons to passthrough pre-existing layers.

During early postnatal development of the cerebellum, postmitotic Purkinje cellsmigrate outwards from the ventricular zone to the pial surface. When the cellsencounter Reelin which is secreted by granule cells, they align to form a well-defined cortical layer [Miyata et al., 1997]. Progenitors of granule cells in theexternal granule cell layer migrate inwards, passing the Purkinje cell layer, andform the internal granule cell layer, giving rise to the multilayered structure of thecerebellum. In mutant mice with disrupted Reelin signaling, neither the Purkinjecell layer nor the external granule cell layer is formed [Porcionatto, 2006].

1 Introduction 15

Figure 1.5 Defective neocortical layer formation in reeler mouse mutants. In mice withmutations in Reelin, Dab1, or both VLDLR and ApoER2, the preplate (PP) does not split andforms a structure called the superplate (SPP). The cortical plate (CP) forms under the SPP andis inverted, indicating that late-migrating neurons are unable to migrate past their predecessors.Early-migrating and late-migrating neurons are positioned in the superficial and deep layers of theCP, respectively, and layering is disorganized. Modified from Gupta et al. [2002].

1.2.4 Reelin signaling in the adult brain

In the mature brain, synaptic plasticity and long-term potentiation in the hip-pocampus can be modulated by Reelin signaling through ApoER2. In the post-synaptic densities of excitatory synapses, ApoER2 forms a functional complexwith N-methyl-D-aspartate (NMDA) receptors. ApoER2 receptors within this com-plexes contain the proline-rich insert in the cytoplasmic tail which is required forthe interaction of the two receptors mediated by the postsynaptic density proteinPSD-95. In the presence of Reelin, the glutamate-stimulated NMDA receptor-mediated calcium influx is increased through tyrosine phosphorylation of NMDAsubunits [Beffert et al., 2005]. Thereby, Reelin signaling contributes to the mod-ulation of postsynaptic responsiveness, which is a key event in learning andmemory. Thus, disruption of Reelin-dependent synaptic functions may lead tocognitive impairment. Numerous reports implicate involvement of Reelin signal-ing malfunctions in human neurodevelopmental disorders such as schizophrenia[Grayson et al., 2006], autism [Fatemi et al., 2005; Skaar et al., 2005] and au-tosomal recessive lissencephaly [Hong et al., 2000]. Another effect of disruptedReelin signaling was proposed for the development of the neurodegenerative dis-

1 Introduction 16

order Alzheimers disease (AD). AD is characterized by the presence of neu-rofibrillary tangles and amyloid plaques in the brain. The tangles are intracellu-lar aggregates of paired helical filaments, mainly composed of the microtubule-associated protein tau, in a hyperphosphorylated state. Senile plaques areformed by extracellular deposits of -amyloid peptide (A) which is generated bycleavage of the transmembrane glycoprotein -amyloid precursor protein (APP)by the -secretase Presenilin-1 (PS1). The proposal of an involvement of Reelinsignaling in the development of AD is based upon several findings. First, Reelinbinds to ApoE receptors, and some ApoE gene polymorphisms are consideredrisk factors for AD. Moreover, the lack of Reelin is associated with hyperphos-phorylation of tau [Hiesberger et al., 1999], which leads to neurofibrillary tangles.Additionally, APP contains an NPxY motif similar to that found in LDLR familymembers, and is known to bind Dab1 [Howell et al., 1999]. Recent data also indi-cate that Reelin is present in amyloid plaques in a transgenic mouse model of AD[Wirths et al., 2001] and that Reelin levels are increased in brains of AD patients[Botella-Lpez et al., 2006].

1.3 Endocytosis

The plasma membrane separates the cytoplasm from the extracellular environ-ment and regulates the entry and exit of molecules. Essential small molecules,such as amino acids, sugars, and ions, can traverse the plasma membranethrough integral membrane protein pumps or channels. Macromolecules mustbe carried into the cell by endocytosis. Various endocytic pathways are employedfor the internalization of different particles and macromolecules. Phagocytosis,the uptake of large particles is typically restricted to specialized mammalian cells,whereas pinocytosis, the uptake of fluid and solutes, occurs in all cells. Pinocy-tosis includes four basic mechanisms: macropinocytosis, clathrin-mediated en-docytosis (CME), caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis (figure 1.6).

1 Introduction 17

Figure 1.6 Multiple mechanisms of endocytosis The endocytic pathways differ with regard tothe size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and themechanism of vesicle formation. Modified from Conner and Schmid [2003].

1.3.1 Clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME, figure 1.7) represents a constitutive mech-anism in all mammalian cells involved in the continuous uptake of essential nutri-ents, such as LDL particles and transferrin (Tfn) when bound to their correspond-ing receptors [Brodsky et al., 2001; Schmid , 1997]. CME also modulates signaltransduction, both by controlling the levels of surface signaling receptors, and bymediating the rapid clearance and downregulation of activated signaling recep-tors. A prerequisite for CME is the concentration of transmembrane receptorsand their bound ligands into coated pits on the plasma membrane. These mem-brane regions are formed mainly by the cytosolic coat protein clathrin. Clathrinoccurs as a complex of light and heavy chains, building up a threelegged struc-ture, the so-called triskelion [Brodsky et al., 2001; Kirchhausen, 2000]. Triske-lions are capable of forming polygonal cages with the help of assembly proteins.For the formation of clathrin cages at the plasma membrane, the adaptor proteincomplex AP2 is required. AP2 consists of four subunits [Collins et al., 2002]. The-adaptin subunit targets AP2 complexes to the plasma membrane; the -subunitinteracts with clathrin to trigger its assembly; the 2-subunit binds tyrosine-basedinternalization motifs on the cytoplasmic domains of endocytic receptors to con-centrate them into coated pits; and the 2-subunit has a structural role in stabi-lizing the core domain. Besides being recognized by the 2-subunit, the NPxYmotif present in the cytoplasmic domain of the LDL receptor family also interactswith Disabled 2 (Dab2), which can bind to AP2, giving rise to an alternative as-sembly mechanism [Mishra et al., 2002;Morris and Cooper , 2001]. Moreover, the

1 Introduction 18

invagination of coated pits and the formation of clathrin coated vesicles (CCVs)depends on the large GTPase dynamin. Dynamin is thought to self-assemble intoa collar at the neck of the deeply invaginated coated pit and act as a mechano-chemical enzyme to drive membrane fission [Hinshaw and Schmid , 1995; Stowellet al., 1999; Sweitzer and Hinshaw , 1998]. An alternative model suggests that dy-namin recruits or activates downstream effectors [Sever et al., 1999].

Figure 1.7 Clathrin-mediated endocytosis. Clathrin triskelions assemble into a polygonal lat-tice, which helps to deform the overlying plasma membrane into a coated pit. Adaptor protein 2complexes, targeted to the plasma membrane, mediate this assembly. To form a clathrin coatedvesicle (CCV), the GTPase dynamin is recruited to the necks of coated pits where it assemblesto a collar to facilitate membrane fission. The CCV is released into the cytoplasm. Modified fromConner and Schmid [2003].

1.3.2 Lipid rafts and caveolae

Lipid rafts are small (40-60 nm diameter), highly ordered lipid microdomains ofthe plasma membrane. They are rich in free cholesterol and sphingolipids andmove freely within the cell surface [Brown and London, 2000]. Lipid rafts canbe internalized by different endocytic mechanisms, which are either caveolae-dependent or -independent [Kirkham and Parton, 2005]. Caveolae are uncoatedflask-shaped invaginations of the plasma membrane. They are believed to con-stitute a subdomain or a special type of lipid rafts [Anderson, 1998; Kurzchaliaand Parton, 1999]. Like rafts, they have a composition high in free cholesteroland sphingolipids but are additionally defined by the presence of the structuralprotein caveolin. The formation of homo- and hetero-oligomers of caveolins [Sar-giacomo et al., 1995; Scherer et al., 1997] and their interaction with cholesterol[Murata et al., 1995] and glycophospholipids [Fra et al., 1995] is supposed to de-termine the shape of caveolae [Sargiacomo et al., 1995]. Caveolae are abundant

1 Introduction 19

in some cell types (adipocytes, endothelia, muscle) but undetectable in others(lymphocytes, many neuronal cells). Various considerations lead to the conclu-sion that the role of caveolae-mediated endocytosis lies in the regulation of sig-naling processes rather than in bulk fluid-phase uptake [Anderson, 1998; Razaniet al., 2002]. On the one hand, caveolae are only slowly internalized (with a half-time of more than 20 minutes) and carry little volume, and on the other hand,many signaling molecules and membrane transporters are concentrated in thesemicrodomains. Unlike LDLR and VLDLR, ApoER2 is found mostly in caveolae[Mayer et al., 2006; Riddell et al., 2001]. Evidence for caveolae-independentmechanisms of lipid raft internalization was supplied by reports on lipid raft inter-nalization in caveolin-deficient cells [Fra et al., 1994; Simons and Toomre, 2000].Moreover, caveolae-independent lipid raft endocytosis also occurs in cells withabundant caveolae [Kirkham et al., 2005].

Figure 1.8 Membrane organization of lipid rafts and caveolae. Lipid rafts are highly orderedmicrodomains of the plasma membrane. In contrast to the bulk lipid bilayer which is composedmainly of phospholipids, rafts are enriched in sphingolipids and cholesterol. Caveolae are gen-erally considered to be a type of lipid rafts. Due to an enrichment of caveolins, their assemblyto oligomers (shown as dimers for simplicity), and their interaction with cholesterol, raft domainsform the flask-shaped invaginations of caveolae. Modified from Galbiati et al. [2001].

Although the internalization of caveolae and rafts was considered to occur viadifferent mechanisms, another theory has been proposed, suggesting a commonpathway for both and a regulatory role for caveolin [Nabi and Le, 2003]. This the-ory takes into account that reduction of caveolin levels has in some cases eitherno effect on, or even accelerate raft-mediated uptake of molecules. This suggeststhat caveolin is not an essential part of the uptake mechanism; it may even actas a negative regulator of raft-mediated endocytosis. The presence of caveolinin endocytic raft domains may stabilize these structures at the plasma membraneand retard the budding of raft-derived vesicles. This would also be consistent

1 Introduction 20

with the finding that caveolae are largely static. The inhibitory effect could only beovercome by specific signaling events. This proposal is supported by the findingthat phosphatase inhibitors are able to induce the budding of surface-connectedcaveolae.

2 Aims of this study

As outlined in the introduction, two members of the LDLR family, namelyApoER2 and VLDLR are known to be involved in transmitting the Reelin signalacross the plasma membrane of neuroblasts [Trommsdorff et al., 1999]. Dur-ing brain development, the Reelin signaling pathway is activated upon bindingof Reelin to ApoER2 and VLDLR, leading to tyrosine phosphorylation of Dab1.These events seem to constitute a linear pathway, since disruption of either theReelin or Dab1 gene or inactivation of both ApoER2 and VLDLR genes cause thesame phenotype in mice. Obviously, tyrosine phosphorylation of Dab1 is triggeredby Reelin-induced receptor clustering [Strasser et al., 2004] and is mediated bySrc family kinases [Arnaud et al., 2003b]. Still, the knowledge on the modulationof the initial signal and on downstream events guiding migration and positioningof neurons is scarce. Therefore, the aim of this study was to further investigatedifferent aspects of the receptor mediated Reelin signal transduction, includingendocytosis and clearance of Reelin from the cell surface and regulation of theReelin receptors by membrane localization, intracellular trafficking, degradation,and specific cleavage. For this purpose, two different cell systems should beused: first, a NIH 3T3 cell model mimicking the Reelin signaling pathway [Mayeret al., 2006] should be used for most of the experiments since this system of-fers easier availability and greater amounts of cell-material which is necessary formost biochemical analyses. Second, selected experiments should be carried outusing primary rat neurons to verify the results in a more physiological situation.

It was recently shown that ApoER2 and VLDLR reside in distinct subdomainsof the plasma membrane [Mayer et al., 2006; Riddell et al., 2001]: ApoER2 issorted into caveolae/lipid rafts, whereas VLDLR is strictly excluded from these mi-crodomains. Two findings suggested that the differential sorting of the receptorsdoes not influence Reelin signaling: first, knockout of either ApoER2 or VLDLRresulted in only subtle abnormalities in mouse brain architecture, suggesting thatboth receptors can at least partially compensate for each other [Trommsdorffet al., 1999], and second, it was shown that both receptors are equally potent


to phosphorylate Dab1 [Strasser et al., 2004]. Thus, the different localizationof the receptors within the plasma membrane is likely not important for the sig-naling activity of the receptors. To verify this hypothesis, the effectiveness ofDab1 phosphorylation in the presence and absence of functional lipid rafts shouldbe studied. This task should be addressed by the use of methyl--cyclodextrinand nystatin. Both agents disrupt rafts/caveolae by depletion of cholesterol fromthe membrane but have only little effect on other membrane structures such asclathrin-coated pits.

Assuming that differential sorting of the receptors within the plasma membranehas no influence on Dab1 phosphorylation, the question on possible other ef-fects of the distinct membrane localization of both receptors arises. Differen-tial sorting might have an influence on endocytosis and degradation of Reelinand/or on trafficking of the Reelin receptors. Differences in endocytosis ratesof LDLR family members have already been reported [Li et al., 2001]. In agree-ment with this hypothesis, recent results from our laboratory showed that ApoER2and VLDLR exhibit different rates of Reelin endocytosis and degradation [Mayer ,2005]. These results were based on an assay comparing the amounts of cellbound Reelin after different incubation times with Reelin conditioned medium.Here, these results should be verified by an alternative approach based on anal-ysis of Reelin amounts in Reelin conditioned medium during incubation of cellsexpressing either ApoER2 or VLDLR. Since it is not clear to date whether andhow endocytosis of the receptors and Reelin modulates the signaling events andthe cellular response, the potency of endocytosis-inhibited cells to induce Dab1phosphorylation upon Reelin stimulation should be examined, . Moreover, thereceptor domain responsible for the differential sorting of the receptors shouldbe determined. For these experiments, chimeric receptors expressing variouscombinations of the different domains of ApoER2 and VLDLR should be used.

To examine whether the different endocytosis rates of ApoER2 and VLDLR arelinked to differential subcellular distribution, the surface expression of the recep-tors should be analysed by immunofluorescence and biotinylation of surface re-ceptors. Furthermore, a possible influence of Reelin stimulation on the subcellulardistribution of the receptors should be studied.

Regarding receptor degradation, it was observed that levels of ApoER2 but notVLDLR were dramatically decreased upon Reelin stimulation [Mayer et al., 2006].For further investigation of this event, it should be found out if ApoER2 degrada-


tion is initiated by receptor clustering which is also responsible for Reelin-inducedDab1 phosphorylation. This question will be addressed by analysis of cell re-sponse to stimulation with multivalent or monovalent ligands. Furthermore, treat-ment with lysosome or proteasome inhibitors should determine the pathway ofApoER2 degradation under these circumstances.

Besides degradation, the Reelin receptors are substrates for specific, ligand in-duced cleavage, which might add another level of modulating the Reelin signal. Itwas reported recently, that by the action of -secretase, an extracellular solublereceptor fragment is produced [Hoe and Rebeck , 2005]. Subsequent cleavageby -secretase leads to liberation of an intracellular fragment [May et al., 2003].Here, this fragmentation should be further examined in terms of membrane- andraft-association of the intracellular fragment and the influence of Dab1 on thefragmentation process.

The results concerning endocytosis and degradation of Reelin and distribution,degradation, and fragmentation of the receptors are expected to lead to a betterunderstanding of molecular events during Reelin signaling and their modulatoryeffects on the signal produced.

3 Material and Methods

3.1 Antibodies

For the detection of ApoER2, three polyclonal antibodies were used: rabbit 186directed against the entire ligand binding domain (LA-repeats 1-3, 7, 8) of murineApoER2 [Strasser et al., 2004], rabbit 19 directed against the cytoplasmic tail ofApoER2 lacking the proline-rich insert, and rabbit 20 directed against the cyto-plasmic tail of ApoER2 containing the proline-rich insert [Stockinger et al., 1998].For detection of VLDLR, rabbit 187 directed against the entire ligand binding do-main of murine VLDLR was used [Strasser et al., 2004]. Furthermore, HA-taggedreceptors were detected using mouse HA (Covance). Monoclonal antibody D4was used for detection of murine Dab1 in Western blotting. Phosphorylated Dab1was detected by the phosphotyrosine-recognizing mouse antibody PY99 (SantaCruz). For immunoprecipitation of Dab1, polyclonal antibodies were used, i.e.rabbit 54, directed against the first 180 amino acids of the short splice variant ofmurine Dab1 and rabbit 48, directed against the short splice variant of murineDab1. Mouse GFP was purchased from Santa Cruz. Reelin was detected usingthe monoclonal antibody G10. D4 and G10 are a kind gift of Andre Goffinet(University of Louvain, Brussels, Belgium). For detection of endogenous proteinsas loading controls, monoclonal Lis1 antibody and rabbit cav1 (transduction lab-oratories), recognizing caveolin-1 were used. Lis1 is a kind gift of Orly Reiner(Weizmann Institute of Science, Rehovot, Israel).

Secondary antibodies used in Western blotting were HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies (Jackson Immuno Research). Alexa Flour488 goat anti-mouse and Alexa Flour 594 goat anti-rabbit antibodies (MolecularProbes) were used for immunofluorescence microscopy.

3 Material and Methods 25

3.2 Plasmids

Eukaryotic expression plasmids used for the production of stable NIH 3T3 celllines [Mayer et al., 2006] and for transient transfection were based on the vec-tors pMSCVpuro and pfMSCV-IRES-GFP. The constructed plasmids comprisepMSCVpuro-ApoER2(+/-) and pMSCVpuro-VLDLR, coding for murine ApoER2harboring LA repeats 1 to 3, 7, and 8, containing (+) or lacking (-) the proline-richcytoplasmic insert or murine VLDLR lacking the O-linked sugar domain, respec-tively [Mayer et al., 2006]. Furthermore, plasmids coding for chimeric receptorswere used. Chimeric receptors were constructed by swapping intracellular, trans-membrane and extracellular domains between ApoER2 and VLDLR. For con-structs comprising the intracellular domain of ApoER2, the variant containing theproline-rich insert (+) was used. The resulting receptors comprise AeAtmVi (de-rived from ApoER2 by replacing its cytoplasmic tail with that of VLDLR), AeVtmVi(derived from VLDLR by replacing its extracellular domain with the correspond-ing ApoER2 domain), and vice versa, VeVtmAi(+) and VeAtmAi(+) [Mayer , 2005].Plasmids used coding for murine Dab1 were pfMSCV-IRES-GFP-Dab1 andpMSCVpuro-Dab1 [Mayer et al., 2006]. Furthermore, the plasmids pcDNAflux3-ApoER2 and pcDNAflux3-VLDLR [Hoe and Rebeck , 2005] were used for expres-sion of fusion proteins of ApoER2 or VLDLR with a C-terminal hemagglutinin tag,i.e. ApoER2-HA or VLDLR-HA, respectively.

3.3 Cell culture

3.3.1 Cells and cell lines

HEK 293

Human embryonic kidney cell line, ATCC Number CRL-1573

Cells were grown at 37C in a humidified environment of 7.5% CO2 in Dulbeccosmodified Eagle medium (DMEM; Gibco), supplemented with 10% heat-inactivatedfetal calf serum (FCS), 100U/ml penicillin and 100U/ml streptomycin sulphate.Cells were split at confluency every 2-3 days.


NIH 3T3

Mouse embryonic fibroblasts, ATCC Number CRL-1658

Cells were grown at 37C in a humidified environment of 7.5% CO2 in DMEM,supplemented with 10% heat-inactivated FCS, 100U/ml penicillin and 100U/mlstreptomycin sulphate. Cells were split at confluency every 2-3 days.

Primary neuronal cells

Preparation of primary neurons was done by Nuno Andrade and Sophia Blake(Dept. Medical Biochemistry, Vienna Biocenter). Primary neurons were obtainedfrom 16.5 day old (E16.5) rat embryonic brains. Dissected brains were washedtwice with Hanks Balanced Salt Solution (HBSS, Gibco) by centrifugation for 4minat 1,300 rpm and suspended in Dulbeccos modified Eagle medium F-12 (Gibco)containing B27 supplement (Gibco), 2mM L-glutamine, 100U/ml penicillin and100U/ml streptomycin sulphate. The suspension was triturated 30 times with afire polished glass pasteur pipette and neurons were plated on 10 cm cell culturedishes coated with 150 g/ml poly-L-ornithine. Cells were grown at 37C in ahumidified environment of 5% CO2 in DMEM F-12 containing all supplements for72 h before use.

Reelin expressing cell line

The cell line was produced by Harald Mayer and is based on HEK 293 cells[Mayer , 2005]. Cells were grown at 37C in a humidified environment of 7.5%CO2 in DMEM, supplemented with 10% heat-inactivated FCS, 100U/ml penicillin,100U/ml streptomycin sulphate, and 400g/ml G418.

NIH 3T3 cell model for Reelin signaling

As a model cell system for studying Reelin signaling, NIH 3T3 cells stably ex-pressing components of the pathway were used [Mayer et al., 2006]. Cell linesstably expressing murine ApoER2 with ligand-binding repeats 1-3, 7 and 8, con-taining or lacking the proline-rich cytoplasmic insert (3T3 A+ or 3T3 A-, respec-


tively) or murine VLDLR lacking the O-linked sugar domain (3T3 V-) were usedto study receptor mediated endocytosis, receptor degradation, and receptor frag-mentation. Cells co-expressing one of the receptors and murine Dab1 (3T3 A+/D,3T3 A-/D, 3T3 V-/D) were used to study Dab1 phosphorylation and Dab1 influ-ence on molecular events during Reelin signaling. Cell lines expressing chimericreceptors of ApoER2 and VLDLR comprise 3T3 AeAtmVi, AeVtmVi, VeVtmAi(+), andVeAtmAi(+) where e marks the extracellular domain of ApoER2 (A) or VLDLR (V),tm stands for the transmembrane, and i for the intracellular domain. For con-structs comprising the intracellular domain of ApoER2, the splice variant contain-ing the proline-rich insert i(+) was used.

Cells were grown at 37C in a humidified environment of 7.5% CO2 in DMEM,supplemented with 10% heat-inactivated FCS, 100U/ml penicillin, 100U/ml strep-tomycin sulphate, and 500g/ml puromycine. Cells were split at confluency every2-3 days.

3.3.2 Freezing of cells

Cells were washed once with prewarmed PBS, trypsinized, resuspended in growthmedium and pelleted by centrifugation at 500g for 5min. The supernatant wasremoved and the cell pellet was resuspended in FCS containing 10% DMSO andaliquoted to cryotubes (Nunc). Cells were stored at -80C for several days andsubsequently transfered to liquid nitrogen.

3.3.3 Thawing of cells

Cells were rapidly thawed in a 37C waterbath. The cell suspension was graduallydiluted with growth medium and centrifuged for 5min at 500g. The cell pelletwas resuspended in growth medium and transfered to a cell culture dish.

3.3.4 Transient transfection

HEK 293 cells were transiently transfected using PolyFect transfection reagent(Qiagen). Transient transfection of NIH 3T3 cells was performed using Lipofec-


tamine 2000 transfection reagent (Invitrogen). The cells were transfected accord-ing to the manufacturers protocol and used 24-48 h after transfection.

3.3.5 Conditioned media

For the production of Reelin conditioned medium (RCM), HEK 293 cells stablyexpressing Reelin (see 3.3.1) were used. Cells were cultivated in 15 cm cell cul-ture dishes using standard growth medium containing 400 g/ml G418 sulphateto near confluency. The growth medium was replaced by 20ml OptiMEM (Gibco)and cells were incubated at 37C and 7.5% CO2 for 24-48 hours. The super-natant was collected from the cells, the cell debris was removed by centrifugation(2000g, 5 min, RT) and the samples were stored at -20C for further use. Analiquot was used for checking expression of Reelin by Western blotting using theReelin antibody G10. Mock conditioned medium (MCM) was prepared fromuntransfected HEK 293 cells following the procedures described for Reelin con-ditioned medium.

3.4 Protein extraction and analysis

3.4.1 Preparation of cell extracts (Hunt buffer)

Cells were washed 3 times with PBS (137mM NaCl, 0.27mM KCl, 10mMNa2HPO4, 1.7mM KH2PO4, pH7.5) and subsequently scraped into ice-cold Huntbuffer (20mM Tris/HCl pH8.0, 100mM NaCl, 0.5% NP40, 1mM EDTA, Com-plete proteinase inhibitor cocktail (Roche)). Crude extracts were kept on ice for30min and centrifuged at 4C for 15min at 20,000g. Pelleted cell debris wasdiscarded and extracts were stored at -20C or used immediately.

3.4.2 Electrophoresis

Protein samples were resolved by reducing SDS polyacrylamid electrophoresis(SDS-PAGE). Samples were incubated in sample buffer (50mM Tris/HCl pH6.8,2% SDS, 10% glycerol, 0.01% bromphenolblue) containing 1.25% (vol/vol) -mercaptoethanol at 95C for 5 min. Gels were mixed from the components sum-


marized in table 3.1, cast, and polymerized. Gels were run in SDS runningbuffer (25mM Tris, 192mM glycine, 0.1% SDS) at 25-30mA/gel. A prestainedbroad range SDS-PAGE standard (Bio-Rad or Fermentas) was used as a molec-ular weight marker. Full length receptors (120-130 kDa) and Dab1 (80 kDa) wereanaylzed using 8% gels, intracellular receptor fragments (20 kDa) using 15%, andReelin (400 kDa) using 4% gels.

separating gel (5ml) stacking gel (1ml)4% 8% 15% 5%

dH2O (ml) 3 2.3 1.2 0.6830% acrylamid/bisacrylamid 29:1 (ml) 0.65 1.3 2.5 0.171.5M Tris/HCl pH8.8 (ml) 1.3 1.3 1.3 1M Tris/HCl pH 6.8 (ml) 0.1310% SDS (ml) 0.05 0.05 0.05 0.0110% APS (ml) 0.05 0.05 0.05 0.01TEMED (ml) 0.003 0.003 0.003 0.001

Table 3.1 Composition of SDS polyacrylamide gels. Volumes are calculated for one gel (5mlrunning gel and 1ml stacking gel). SDS, sodium dodecyl sulphate; APS, ammonium persulphate;TEMED, tetramethylethylendiamine.

3.4.3 Western blotting

SDS-PAGE gels were transferred to nitrocellulose membranes (Hybond-C ex-tra, Amersham) by semi-dry blotting using semi-dry transfer buffer (25mM Tris,192mM glycine, 0.05% SDS). Blotting was performed at 175mV/cm2 for 1 h atroom temperature. Membranes were subsequently blocked using blocking buffer(PBS containing 0.1% Tween 20 and either 5% non-fat dry milk or 5% BSA) for 1 hat room temperature. Incubation with dilutions of primary antibodies in blockingbuffer was done o/n at 4C. Dilutions and blocking buffers used with the differentantibodies are summarized in table 3.2. Membranes were washed 5-6 times withPBST (PBS containing 0.1% Tween 20) and subsequently incubated with a dilu-tion of the appropriate HRP-conjugated secondary antibody in PBST for one hourat room temperature. After excessive washing, the membranes were incubatedfor 5 min with enhanced chemiluminiscence (ECL) solution (SuperSignal West,Pierce) according to the manufacturers protocol. Excess solution was removedand membranes were exposed to X-ray film (CL-XPosure, Pierce). Exposuretimes varied from 1 s to 20min.


detection of antibody source dilution blocking buffer

Reelin G10 mouse 1: 10,000 BSAApoER2(+) 20 rabbit 1: 7,000 milkApoER2(-) 19 rabbit 1: 1,000 milkApoER2-HA HA mouse 1: 2,000 BSAVLDLR-HA HA mouse 1: 2,000 BSAPhosphorylated Dab1 PY mouse 1: 1,000 BSADab1 D4 mouse 1: 3,000 BSAGFP GFP mouse 1: 500 BSALis1 Lis1 mouse 1: 10,000 BSACaveolin 1 Cav1 rabbit 1: 15,000 milk

primary mouse antibodies ms-HRP goat 1: 20,000 primary rabbit antibodies rb-HRP goat 1: 20,000

Table 3.2 Antibodies used in Western blotting. Source, dilution and blocking buffers for pri-mary and secondary antibodies in Western blotting. Blocking buffers are BSA (5% BSA in PBST)or milk (5% non-fat dry milk in PBST).

3.5 Western blot based assays

3.5.1 Membrane localization assay

NIH 3T3 cells expressing ApoER2 containing the proline-rich insert (3T3 A+) wereseeded in 10 cm cell culture dishes (0.8106 cells/dish) using standard fibroblastgrowth medium. After 24 h, cells were incubated for 1 h with OptiMEM containingor lacking raft disrupting agents (see figure legend) at 37C in a humidified envi-ronment of 7.5% CO2. All subsequent steps were carried out at 4C. Cells werewashed twice with ice-cold TBS and scraped into 1ml TBS. Subsequently, cellswere pelleted by centrifugation for 5min at 1400g and 4C. For lysis, the cell pel-let was resuspended in 1 ml TBS containing 2% Brij 78P (Fluka) and Complete

proteinase inhibitor cocktail. Cells were solubilized by passing 10 times througha 23-gauge needle and incubated on ice for 15min. Cell debris was removed bycentrifugation (10min, 20,000g). 0.6ml of the cell homogenate was mixed with0.6ml of 90% (wt/vol) sucrose in MBS (25mM 2-(4-morpholino)ethanesulfonicacid, 150mM NaCl, pH6.5) and transferred to a SW60-Ti ultracentrifuge tube. Adiscontinuous sucrose gradient was formed above the homogenate by layeringof 2.5ml of 35% (wt/vol) sucrose in MBS, followed by 0.6ml 5% (wt/vol) sucrosein MBS. After centrifugation at 160,000g for 20 h in a Beckman SW60Ti rotorat 4C, 9 fractions of 0.44ml each were collected from the top of the tube. Frac-


tions 2 and 3 at the interface between the 5% and 35% sucrose boundaries weredesignated the caveolae-rich light membrane (CLM) fraction whereas the non-caveolae membrane fraction (NCM) is restricted to the lower fractions (8 and 9).Fractions were resolved by reducing SDS-PAGE and analyzed by immunoblottingusing the ApoER2 specific antibody 20.

3.5.2 Dab1 phosphorylation assay

NIH 3T3 cells expressing Dab1 and either ApoER2 (3T3 A+/D) or VLDLR (3T3V-/D) were seeded in 6 cm or 10 cm cell culture dishes (0.8106 cells/6 cm dish,(1.6106 cells/10 cm dish) using standard fibroblast growth medium. After 24 h,cells were starved for 1 h in serum-free medium (DMEM without supplements)and subsequently incubated with Reelin or mock conditioned medium contain-ing or lacking additional reagents for 1 h at 37C in a humidified environmentof 7.5% CO2. Afterwards, cells were washed with ice-cold PBS and scrapedinto 250 l (for 6 cm dishes) or 750 l (for 10 cm dishes) Hunt buffer containingComplete proteinase inhibitor cocktail and phosphatase inhibitors (50mM NaF,2mM Na3VO4). Cells were lysed on ice for 30min and lysates were centrifugedat 20,000g for 15min at 4C. Extracts were immediately subjected to immuno-precipitation by addition of 3 l 48 and 3 l 54 and incubated o/n at 4C rotat-ing end over end. 40 l of 50% Protein A-Sepharose CL-4B (Amersham) slurry(prepared according to the manufacturers protocol) were added and samples in-cubated for an additional hour at 4C rotating end over end. Protein A-Sepharosebeads were collected by centrifugation at 500g for 1min at 4C, washed threetimes with cold Hunt buffer and subjected to reducing SDS-PAGE. Western blot-ting was done using D4 and phosphotyrosine PY99 for detection of Dab1 andtyrosine phosphorylated Dab1, respectively.

3.5.3 Reelin uptake and degradation assay

Reelin uptake assays were performed with NIH 3T3 cells expressing either of theReelin receptors (3T3 A+, V-) or the chimeric receptors (3T3 AeAtmVi, AeVtmVi,VeVtmAi(+), VeAtmAi(+)). The cells were grown in 6-well plates (0.35106 cells/well)using standard fibroblast growth medium. After 24 hours, cells were starved for1 h in serum-free medium and incubated on ice for 30min. All further steps were


carried out on ice. Cells were washed twice with ice-cold PBS and subsequentlyincubated with Reelin or mock conditioned medium for 1 h. Cells were washed 3times with ice-cold PBS and either Hunt extracts were prepared immediately orcells were overlaid with OptiMEM and incubated in a 37C waterbath for differenttime periods before shifting cells back to ice and lysis in Hunt buffer. Extractswere subjected to SDS-PAGE and immunoblotting using G10 for detection ofReelin.

3.5.4 Reelin depletion assay

NIH 3T3 cells were seeded in 6-well plates (0.35106 cells/well) and transientlytransfected after 24 h using pcDNAflux3-ApoER2 or pcDNAflux3-VLDLR. 48 hafter transfection, cells were incubated for 32 h with 3ml of Reelin conditionedmedium diluted in OptiMEM (1:3) at 37C in a humidified environment of 7.5%CO2. Medium samples of 60 l each were taken after 8, 24, and 32 h and cellswere lysed in Hunt buffer after 32 h. All samples were subjected to reducingSDS-PAGE and Western blotting using the monoclonal Reelin antibody G10 forsamples of the medium or HA to detect ApoER2-HA or VLDLR-HA in the cellextracts. For quantification, integrated optical density (IOD) values of Reelin andreceptor bands were calculated using Gel-Pro Analyzer.

3.5.5 Surface biotinylation assay

NIH 3T3 cells were grown in 6 cm cell culture dishes (0.8106 cells/dish) usingstandard fibroblast growth medium and transiently transfected using pcDNAflux3-ApoER2 or pcDNAflux3-VLDLR. 48 h after transfection, cells were cooled for15min at 4C and subsequently incubated for 2 h with 0.5mg/ml EZ-Link Sulfo-NHSS-S-Biotin (Pierce) in PBS++ (PBS containing 1mM CaCl2 and 1mM MgCl2)at 4C or 37C. Cells were washed once in cold NTC buffer (20mM Tris pH8.8,150mM NaCl, 0.1mM CaCl2, 0.2% BSA) and once in cold PBS++. For the prepa-ration of cell extracts, cells were scraped in 600 l cold TNE buffer (25mM TrispH7.5, 150mM NaCl, 5mM EDTA, 0.1% SDS, 0.1% Triton X-100) containingComplete proteinase inhibitor cocktail. Cells were mechanically disrupted bypassaging 10 times through a 23-gauge needle. Extracts were incubated on icefor 30min and cell debris was pelleted by centrifugation for 15min at 4C and


20,000g. Cleared extracts were incubated with Neutravidin-coated beads for1 h at 4C rotating end over end to bind biotinylated receptors. Beads were col-lected by centrifugation at 500g for 1min at 4C and washed once with NetgelNaCl (50mM Tris/HCl, pH7.5, 5mM EDTA, 0.25% gelatine, 0.02% NaN3, 500mMNaCl), once with Netgel SDS (50mM Tris/HCl, pH7.5, 5mM EDTA, 0.25% gela-tine, 0.02% NaN3, 150mM NaCl, 0.1% SDS), and once with Hunt buffer. Sampleswere subjected to reducing SDS-PAGE and levels of receptor biotinylated at 4Cor 37C were compared by immunoblotting using HA antibody.

3.5.6 Receptor degradation assay

NIH 3T3 cells expressing ApoER2 containing or lacking the cytoplasmic proline-rich insert (3T3 A+/-) were grown in 6 cm cell culture dishes (0.8106 cells/dish)using standard fibroblast growth medium. Alternatively, primary rat neurons wereplated on poly-L-ornithine coated 10 cm cell culture dishes 3 days before use.Cells were starved for 1 h in DMEM containing 20 g/ml cycloheximide to inhibitprotein synthesis. Subsequently, fibroblasts or primary neurons were incubatedfor 6 h or 24 h, respectively, in OptiMEM containing 20 g/ml cycloheximide anddifferent ligands and/or additional reagents (see figure legends) at 37C in a hu-midified environment of 7.5% CO2 for the indicated time periods (see figure leg-ends). Cell extracts were prepared in Hunt buffer and subjected to reducing SDS-PAGE and Western blotting using 20 to detect ApoER2.

3.5.7 Receptor fragmentation assay

NIH 3T3 cells expressing ApoER2 containing or lacking the proline-rich insert(3T3 ApoER2+/-) or VLDLR lacking the O-linked sugar domain (3T3 V-) wereseeded into 6 cm cell culture dishes (0.8106 cells/dish) using standard fibrob-last growth medium. After 24 h, cells were incubated for 5-6 h in OptiMEM con-taining different ligands and/or additional reagents at 37C in a humidified envi-ronment of 7.5% CO2. Alternatively, NIH 3T3 cells were grown in 6 cm cell culturedishes using standard fibroblast growth medium and transiently transfected usingpcDNAflux3-ApoER2 or pcDNAflux3-VLDLR. Transiently transfected cells weretreated with ligands and/or reagents 48 h after transfection. Cell extracts wereprepared in Hunt buffer and subjected to reducing SDS-PAGE and Western blot-


ting using 20 or 19 to detect ApoER2(+) or ApoER2(-) fragments, respectively,or HA to detect ApoER2-HA or VLDLR-HA fragments.

3.5.8 Membrane binding assay

NIH 3T3 cells expressing Dab1 and ApoER2 containing the proline-rich insert(3T3 A+/D) were seeded in 6 cm cell culture dishes (0.8106 cells/dish) andgrown for 24 h using standard fibroblast growth medium. Cells were incubatedwith OptiMEM containing different ligands for 6 h at 37C in a humidified environ-ment of 7.5% CO2 and lysed using two different methods. Hunt extracts wereprepared as before (3.4.1). Remaining dishes were washed 3 times with PBSand scraped into CAV-A buffer (250mM sucrose, 20mM Tris pH7.8, 1mM EDTA)and subjected to mechanical disruption by passing 10 times through a 23-gaugeneedle. CAV-A extracts were centrifuged for 10min at 20,000g and 4C to re-move cell debris and subsequently subjected to ultracentrifugation in a TLA-100rotor at 55,000 rpm and 4C for 90min. The supernatant was saved and used forfurther analysis, the pellet was discarded. All samples were subjected to reduc-ing SDS-PAGE and immunoblotted using 20 to detect full length and cleavedApoER2.

3.6 Immunofluorescence Microscopy

3.6.1 Subcellular receptor distribution

Sterile glass coverslips were coated by incubation with 40 g/ml poly-L-lysine inPBS for 1 h at room temperature. Subsequently, coverslips were washed 4 timeswith PBS and dried. NIH 3T3 cells were plated on the coverslips and grown for24 h at 37C in a humidified environment of 7.5% CO2 using standard fibroblastgrowth medium. At 80-90% confluency, cells were transiently transfected withpcDNAflux3-ApoER2 or pcDNAflux3-VLDLR [Hoe and Rebeck , 2005]. 48 h aftertransfection, cells were washed 3 times with PBS and fixed by incubation with 4%paraformaldehyde (PFA) fixative for 15min at room temperature. Fixed cells werewashed 3 times with PBS containing 100mM glycine and subsequently incubatedwith 0.1% Triton X-100 in PBS for 2min to permeabilize the cell membrane. Cells


were washed again with PBS and blocked for 30min with blocking solution (1%BSA in PBS) at room temperature. Afterwards, cells were incubated with primaryantibody diluted in blocking solution for 1 h before extensive washing with PBSand subsequent incubation with a specific fluorescence labeled secondary anti-body for 1 h. Cells were rinsed with PBS and once with H2O before mounting onglass slides using DAKO Fluorescent Mounting Medium (Dako Corporation). Theslides were analyzed using a 63 oil objective on a confocal fluorescence micro-scope (Laser Scanning Microscope 510, Zeiss) and the corresponding software(Zeiss LSM Image Browser).

Alternatively, primary neuronal cell were plated on glass coverslips coated with150 g/ml poly-L-ornithine and grown in DMEM F-12 containing supplements de-scribed in section 3.3.1 for 72 h at 37C in a humidified environment of 5% CO2.Primary neuronal cells were fixed, permeabilized, and stained as described forfibroblasts.

3.6.2 Reelin internalization

NIH 3T3 cells were grown and transfected on poly-L-lysine coated glass cov-erslips (see 3.6.1). Cells were transfected with either pcDNAflux3-ApoER2 orpcDNAflux3-VLDLR, coding for HA-tagged ApoER2 or VLDLR, or co-transfectedwith one of the receptors and pMSCVpuro-Dab1. 48 h after transfection, cellswere shifted to 4C for 15min. Afterwards, cells were washed with ice-cold PBSand overlaid with Reelin conditioned medium. After 1 h of incubation at 4C,Reelin containing medium was removed and cells were washed with ice-cold PBSand incubated with OptiMEM at either 4C or 37C for 10min. Subsequently, cellswere fixed, permeabilized, and stained as described in 3.6.1.

3.6.3 Immunostaining

For detection of ApoER2-HA and VLDLR-HA primary antibodies either againstthe receptors (186 and 187, respectively) or the HA-tag (HA) were used.Reelin was detected using G10. Secondary antibodies were Alexa Flour 488goat anti-mouse used for detection of G10 and HA, and Alexa Flour 594 goatanti-rabbit for detection of 186 and 187. Used antibodies and corresponding


dilutions are summarized in table 3.3. Additionally, cell nuclei were stained usingHoechst reagent diluted 1:500.

staining antibody source dilution

Reelin G10 mouse 1: 1,000ApoER2 186 rabbit 1: 20,000VLDLR 187 rabbit 1: 7,000ApoER2-HA HA mouse 1: 1,000VLDLR-HA HA mouse 1: 1,000

primary mouse antibodies ms-Alexa 488 goat 1: 1,000primary rabbit antibodies rb-Alexa 594 goat 1: 1,000

Table 3.3 Antibodies used for immunofluorescence. Primary and secondary antibodies werediluted in blocking buffer (1% BSA in PBS).

4 Results

Most of the experiments were carried out using NIH 3T3 mouse fibroblasts sta-bly expressing either ApoER2 with ligand-binding repeats 1-3, 7 and 8, contain-ing or lacking the proline-rich cytoplasmic insert (3T3 A+, 3T3 A-) or VLDLRlacking the O-linked sugar domain (3T3 V-) or co-expressing one of the recep-tors and Dab1 (3T3 A+/D, 3T3 A-/D, 3T3 V-/D). This cell model was shown tomimic the crucial events of Reelin signaling as they were observed in neurons,namely ligand-induced phosphorylation of Dab1, subsequent phosphorylation ofPKB/Akt, and degradation of phosphorylated Dab1 [Mayer et al., 2006]. For cer-tain experiments, transiently transfected NIH 3T3 cells expressing fusion proteinsof ApoER2 containing the cytoplasmic insert (ApoER2-HA) or VLDLR (VLDLR-HA) with C-terminal hemagglutinin (HA) were used. The C-terminal tag allowsdetection of the intracellular receptor fragments produced by secretase-mediatedcleavage using an antibody against HA. Since no suitable antibody against the in-tracellular domain of VLDLR was available, use of the tagged receptors was nec-essary for the study of receptor fragmentation by secretases. Furthermore, useof the HA-receptors and the corresponding anti-HA antibody constitutes a con-trol for consistent transfection efficiencies since amounts of full-length receptorsexpressed by transfected cells can be directly compared. Selected experimentswere also done with primary rat neurons to verify the results in a physiologicalsituation.

4.1 Localization of ApoER2 and VLDLR within the plasmamembrane

As described previously [Mayer et al., 2006], ApoER2 is localized to rafts whereasVLDLR is strictly excluded from these microdomains. To test if the localizationof the receptors within subdomains of the plasma membrane has an influenceon Reelin signaling, the raft disturbing agents Methyl--cyclodextrin (CDX) and

4 Results 38

nystatin (Nys) were used. Both agents disrupt caveolae/lipid rafts by depletion ofcholesterol from the membrane [di Guglielmo et al., 2003; Kilsdonk et al., 1995;Simons and Toomre, 2000]. To assure that the amounts of these agents usedare sufficient to shift the receptors out of rafts, NIH 3T3 cells expressing ApoER2were treated with the indicated amounts of either CDX, Nystatin or none of them.The cells were washed with TBS, scraped, and pelleted by centrifugation. Cellswere then solubilized in TBS containing 2% Brij 78P and protease inhibitors bypassaging 10 times through a 23-gauge needle. The lysate was mixed with equalvolume of 90% (wt/vol) sucrose in MBS and overlaid with a discontinous gradientof 35% (wt/vol) and 5% (wt/vol) sucrose in MBS. The samples were centrifugedfor 20 h at 160,000g and 9 fractions were collected from the top of the tube.Fractions were analyzed by reducing SDS-PAGE and Western blotting using anApoER2-specific antibody. Fractions 2 and 3 at the interface between the 5% andthe 35% sucrose boundaries were designated the caveolin-rich light membrane(CLM) fractions which are clearly separated from the non-caveolae membrane(NCM) in the lower fractions.

Figure 4.1 Membrane distribution of ApoER2. 3T3 A+ cells were incubated with OptiMEM(panel 1) or OptiMEM containing 5mM CDX (panel 2) or 15 g nystatin (panel 3). Cells werewashed with TBS, scraped, pelleted by centrifugation, and solubilized in TBS containing 2%Brij 78P and protease inhibitors by passaging 10 times through a 23-gauge needle. Lysates weremixed with an equal volume of 90% (wt/vol) sucrose in MBS and overlaid with a discontinousgradient of 35% (wt/vol) and 5% (wt/vol) sucrose in MBS. The samples were centrifuged for 20hat 160,000g and 9 fractions were collected from the top of the tube. Fractions were analyzed byimmunoblotting using an ApoER2 specific antibody (20). Arrowheads point at mature ApoER2( ) and ApoER2 precursor ( )

As seen in figure 4.1, ApoER2 is sorted to the raft fraction in untreated cells whileits non-glycosylated precursor which resides in the membrane of the endoplasmicreticulum can be detected in the non-caveolae fraction (panel 1). Upon additionof CDX, mature ApoER2 can only be detected in NCM fractions which indicatesefficient disruption of rafts in the plasma membrane (panel 2). The effect of nys-tatin was less pronounced; it caused only a slight reduction of CLM-associated

4 Results 39

ApoER2 (panel 3). Since higher concentrations of nystatin caused detachmentof the cells and cell death, the effect of this agent could not be increased. Pre-vious experiments demonstrated that in contrast to ApoER2, VLDLR is strictlyexcluded from the CLM fraction [Mayer et al., 2006]. Furthermore, it was shownthat membrane localization of both ApoER2 and VLDLR does not change uponReelin stimulation in NIH 3T3 A-/D and NIH 3T3 V-/D cells [Mayer et al., 2006].Since ApoER2 and VLDLR are equally po

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