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 Grades Dipl. Ing. (FH)
Eingereicht am 25. Oktober 2006
Abstract
The Reelin signaling pathway plays a crucial role in the development of the mammalian central nervous system where it is responsible for the correct positioning of neurons in laminated brain structures. The pathway is thought to inuence migration of neurons, modulate cell-cell interactions, and induce cytoskeletal rearrangements. Disruption of the pathway in mice results in ataxia, tremors, imbalance and a reeling gait. The large glycoprotein Reelin is secreted during brain development by specialized cells, the Cajal-Retzius cells, in the neocortex and external granule cells in the developing cerebellum. The key events triggering the signaling 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 rst steps in Reelin signaling are well understood, the knowledge on the modulation and regulation of the initial signal is still scarce. Here, it could be demonstrated that both receptors and Reelin are subjected to regulation by different mechanisms independently from the primary signaling event. Reelin is internalized and degraded at different rates by cells expressing either ApoER2 or VLDLR, leading to prolonged membrane association or rapid clearance from the cell surface, respectively. While VLDLR recycles to the plasma membrane after degradation of the ligand, ApoER2 is degraded via the lysosomal pathway. Furthermore, extra- and intracellular fragments of ApoER2 are produced from secretase-mediated cleavage. These fragments could also exhibit important effects in the modulation of the Reelin signaling pathway.
Zusammenfassung
Der Reelin Signalweg spielt eine entscheidende Rolle in der Entwicklung des Zentralnervensystems von Sugetieren. Er gewhrleistet die korrekte Positionierung von Neuronen in mehrschichtigen Strukturen im Gehirn, indem er Migration, Zell-Zell-Interaktionen und Umstrukturierungen des Zytoskeletts von Neuronen kontrolliert. Eine Strung dieses Signalwegs fhrt im Maus-Modell zu Koordinationsstrungen, Gleichgewichtsstrungen und einem taumelnden Gang. Das Glycoprotein Reelin wird whrend der embryonalen Hirnentwicklung von spezialisierten Zellen, den Cajal-Retzius Zellen, im Neocortex und im entstehenden Cerebellum sezerniert. Die Schlsselereignisse, die den Reelin Signalweg einleiten, sind die Bindung von Reelin an Apolipoprotein E Rezeptor 2 (ApoER2) und very low density Lipoprotein Rezeptor (VLDLR) und die anschlieende Phosphorilierung 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 offen. In der vorliegenden Arbeit konnte gezeigt weren, dass beide Rezeptoren und auch Reelin einer Regulation durch verschiedene Mechanismen unterworfen sind, die unabhngig von der Signalweitergabe agieren. Reelin wird von ApoER2und VLDLR-exprimierenden Zellen in unterschiedlicher Geschwindigkeit internalisiert und abgebaut, was entweder zu einer lnger anhaltenden MembranAssoziation oder zu einer schnellen Beseitigung von Reelin von der Zelloberche fhrt. Whrend VLDLR nach dem Abbau des Liganden an die Zellmembran zurckgefhrt wird, wird ApoER2 ber das Lysosom abgebaut. Darber hinaus wird ApoER2 einer spezischen Secretase-vermittelten Spaltung unterzogen, wodurch extra- und intrazellulre Rezeptor-Fragmente entstehen. Auch diese Fragmente knnten sich als wichtige Faktoren fr die Modulation des Reelin Signalwegs herausstelen.
Contents
List of Figures List of Tables 1 Introduction 1.1 The low density lipoprotein receptor family . . . . . . . . . . 1.1.1 Structural organization of the LDLR family members 1.1.2 The low density lipoprotein receptor . . . . . . . . . 1.1.3 The very low density lipoprotein receptor . . . . . . 1.1.4 The apolipoprotein E receptor 2 . . . . . . . . . . . 1.1.5 The LDL receptor related protein . . . . . . . . . . . 1.1.6 LRP1b . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.7 Megalin . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.8 MEGF7 . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 The Reelin signaling pathway . . . . . . . . . . . . . . . . . 1.2.1 Reelin . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 The Reelin signaling cascade . . . . . . . . . . . . . 1.2.3 Reelin signaling in brain development . . . . . . . . 1.2.4 Reelin signaling in the adult brain . . . . . . . . . . . 1.3 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Clathrin-mediated endocytosis . . . . . . . . . . . . 1.3.2 Lipid rafts and caveolae . . . . . . . . . . . . . . . . 2 Aims of this study 3 Material and Methods 3.1 Antibodies . . . . . . . . . 3.2 Plasmids . . . . . . . . . 3.3 Cell culture . . . . . . . . 3.3.1 Cells and cell lines 3.3.2 Freezing of cells . v vii 1 1 1 4 5 6 7 8 8 8 9 9 10 12 14 15 16 17 19 22 22 23 23 23 25
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Contents 3.3.3 Thawing of cells . . . . . . . . . . . . . 3.3.4 Transient transfection . . . . . . . . . . 3.3.5 Conditioned media . . . . . . . . . . . . 3.4 Protein extraction and analysis . . . . . . . . . 3.4.1 Preparation of cell extracts (Hunt buffer) 3.4.2 Electrophoresis . . . . . . . . . . . . . . 3.4.3 Western blotting . . . . . . . . . . . . . 3.5 Western blot based assays . . . . . . . . . . . 3.5.1 Membrane localization assay . . . . . . 3.5.2 Dab1 phosphorylation assay . . . . . . 3.5.3 Reelin uptake and degradation assay . 3.5.4 Reelin depletion assay . . . . . . . . . . 3.5.5 Surface biotinylation assay . . . . . . . 3.5.6 Receptor degradation assay . . . . . . 3.5.7 Receptor fragmentation assay . . . . . 3.5.8 Membrane binding assay . . . . . . . . 3.6 Immunouorescence Microscopy . . . . . . . . 3.6.1 Subcellular receptor distribution . . . . . 3.6.2 Reelin internalization . . . . . . . . . . . 3.6.3 Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv 25 25 26 26 26 26 27 28 28 29 29 30 30 31 31 32 32 32 33 33 35 35 38 42 47 50 55 56 57 61 62 67
4 Results 4.1 Localization of ApoER2 and VLDLR within the plasma membrane 4.2 Reelin internalization by ApoER2 and VLDLR . . . . . . . . . . . . 4.3 Surface expression of ApoER2 and VLDLR . . . . . . . . . . . . . 4.4 Reelin-induced degradation of ApoER2 . . . . . . . . . . . . . . . 4.5 Reelin-induced fragmentation of ApoER2 . . . . . . . . . . . . . . 5 Discussion 5.1 Membrane localization of the Reelin receptors . . . . . . . . . . . 5.2 Reelin endocytosis and degradation . . . . . . . . . . . . . . . . . 5.3 Surface expression of the Reelin receptors . . . . . . . . . . . . . 5.4 Reelin-induced degradation and specic cleavage of the receptors References
List of Figures
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Structural organization of the LDLR family members . . . . . Structure of the Reelin protein . . . . . . . . . . . . . . . . . . The Reelin signaling pathway . . . . . . . . . . . . . . . . . . Neocortical layer formation by radial migration . . . . . . . . . Defective neocortical layer formation in reeler mouse mutants Multiple mechanisms of endocytosis. . . . . . . . . . . . . . . Clathrin-mediated endocytosis . . . . . . . . . . . . . . . . . Membrane organization of lipid rafts and caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 10 11 12 14 16 17 18 36 37 38 39 40 41 42 42 43 44 45 46 47 48 49 49 50 51
4.1 Membrane distribution of ApoER2. . . . . . . . . . . . . . . . . . . 4.2 Reelin-induced phosphorylation of Dab1 is independent of receptor localization to rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Different Reelin internalization rates . . . . . . . . . . . . . . . . . 4.4 Receptor-mediated Reelin depletion from the medium . . . . . . . 4.5 Reelin fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Chimeric receptors of ApoER2 and VLDLR . . . . . . . . . . . . . 4.7 Reelin internalization by chimeric receptors . . . . . . . . . . . . . 4.8 Reelin-induced phosphorylation of Dab1 is independent of endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9 Subcellular distribution of ApoER2 and VLDLR . . . . . . . . . . . 4.10 Surface expression of ApoER2 and VLDLR . . . . . . . . . . . . . 4.11 Trafcking of ApoER2 upon Reelin stimulation . . . . . . . . . . . . 4.12 Trafcking of ApoER2 in the presence of Dab1 . . . . . . . . . . . 4.13 Multivalent ligands induce degradation of ApoER2 . . . . . . . . . 4.14 Multivalent ligands induce degradation of ApoER2 in neurons . . . 4.15 Reelin stimulation induces lysosomal degradation of ApoER2 . . . 4.16 Raft-disrupting agents do not interfere with ApoER2 degradation . 4.17 Multivalent ligands induce fragmentation of ApoER2 . . . . . . . . 4.18 The -secretase inhibitor DAPT causes accumulation of the membrane-bound intracellular fragments of ApoER2 and VLDLR . . . .
List of Figures 4.19 The intracellular fragment of ApoER2 produced by -secretase is bound to the plasma membrane . . . . . . . . . . . . . . . . . . . 4.20 Chloroquine causes accumulation of the membrane-bound intracellular fragment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.21 Methyl--cyclodextrin inhibits secretase-mediated fragmentation of ApoER2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.22 Presence of Dab1 enhances secretase-mediated ApoER2 fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.23 Fragmentation of ApoER2 does not occur at 4C . . . . . . . . . . 5.1 Reelin fragments produced by metalloproteinase cleavage . . . . .
vi
52 53 53 54 54 60
List of Tables
3.1 Composition of SDS polyacrylamide gels . . . . . . . . . . . . . . 3.2 Antibodies used in Western blotting . . . . . . . . . . . . . . . . . . 3.3 Antibodies used for immunouorescence . . . . . . . . . . . . . . 27 28 34
1 Introduction
1.1 The low density lipoprotein receptor family
The low density lipoprotein receptor (LDLR) gene family includes several structurally similar transmembrane receptors in mammals which are involved in molecular transport and signal transduction. The best known member of this gene family is the low density lipoprotein (LDL) receptor which plays an important role in cholesterol homeostasis. Other core-members of the LDLR family are the apolipoprotein E receptor 2 (apoER2), the very low density lipoprotein receptor (VLDLR), the LDL receptor related protein (LRP, also referred to as LRP1), LRP1b, megalin (also known as LRP2 or gp330) and the less well characterized MEGF7 (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 endocytosis of macromolecules and delivery of ligands to the lysosome for degradation. More recent ndings however connected these receptors to signal transduction pathways, including G-protein mediated protein kinase activation [Goretzki and Mueller , 1998], modulation of the cytoskeletal organization via cytosolic adaptor proteins [Trommsdorff et al., 1998], and neuronal migration in brain development [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 comprising four distinct domains [Gent and Braakman, 2004; Hussain et al., 1999]: the N-terminal ligand-binding domain, the epidermal growth factor (EGF) precursor homology domain, a single transmembrane domain and a cytoplasmic tail. A fth 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
region and the transmembrane domain. Combinations of these domains build up the core members of the LDLR family (gure 1.1), ranging in molecular masses from 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 receptors of this family contain various numbers of LA-repeats at the amino-terminus, followed by the EGF precursor homology domain, the O-linked sugar domain and the transmembrane domain. The cytoplasmic tails of the core members contain at least one NPxY motif. ApoER2 and VLDLR are depicted including differentially spliced regions (marked with *): several LA-repeats and the O-linked sugar domain in both receptors as well as the furin cleavage site and the proline rich insert in ApoER2. In contrast to LDLR, ApoER2, and VLDLR, MEGF7 has four EGF precursor homology domains. LRP, LRP1b and megalin are much larger than the other core members of the LDLR family and contain multiple clusters of LA-repeats and EGF precursor homology domains. Modied from Nykjaer and Willnow [2002].
The ligand-binding domains are made up of head-to-tail arranged LDL receptor type A repeats, known as LA-repeats. Each of these repeats consists of about
1 Introduction
40 amino acids including six conserved cysteine residues forming three disulde bonds. In the endoplasmic reticulum (ER), the receptor associated protein (RAP) binds to the LA-repeats to prevent premature interaction of other ligands with the receptors [Bu , 2001]. Furthermore, RAP acts as a chaperone to assist in receptor folding [Bu , 2001]. Since RAP binds to all receptors of the LDLR family, it can be used as a unique tool to study ligand-receptor interactions. The EGF precursor homology domains consist of cysteine-rich growth factor repeats of about 40 amino acids separated by YWTD repeats. The growth factor repeats (EGF-like repeats) contain six conserved cysteine residues, differing from those of the ligand-binding repeats in their pattern of disulde bond formation. Located in between are regions termed YWTD repeats, containing the consensus tetrapeptide Tyr-Trp-Thr-Asp (YWTD), which form a -propeller structure [Jeon et al., 2001; Springer , 1998]. The EGF precursor homology domains including the -propeller structures are necessary for the pH-dependent dissociation 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 receptor, and ApoER2, is the so called O-linked sugar domain, a short region rich in serine and threonine residues that undergo O-linked glycosylation. For ApoER2 and VLDLR, distinct splice variants containing or lacking this glycosylation region are expressed in different tissues [Korschineck et al., 2001; Nakamura et al., 1998]. Although it is not exactly clear which biological functions are regulated by its presence or absence, it is known that the O-linked sugar domain contributes to receptor stability and has a critical inuence on the rate of proteolytic processing of the receptors [Magran et al., 1999; May et al., 2003]. A single transmembrane domain anchoring the receptors into the membrane via a stretch of hydrophobic residues connects the extracellular regions to the cytoplasmic tail. The relatively short cytoplasmic region includes at least one NPxY (AspPro-Xxx-Tyr, where Xxx denotes any amino acid) motif which can interact with adaptor proteins and is important for the localization of the receptors to clathrincoated pits and their internalization [Chen et al., 1990]. In the more distant relatives such as LRP5, LRP6, and SorLA, not all of the mentioned 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
ligand binding and the EGF precursor domain. Furthermore, their cytoplasmic tails do not contain an NPxY motif. The structure of SorLA differs even more from the basic structure of the LDLR family members because it contains a domain with homology to a yeast receptor for vacuolar protein sorting (VPS10) and bronectin type III repeats which can also be found in neuronal adhesion molecules [Jacobsen et al., 1996].
1.1.2 The low density lipoprotein receptor The LDL receptor is the name giving and best studied member of the LDLR family. The N-terminal ligand binding domain of the mature receptor consists of seven LA-repeats, arranged in a head-to-tail fashion. The EGF precursor homology domain contains three EGF-like repeats and one YWTD region, two of the growth factor repeats being located N-terminal and one C-terminal of the YWTD sequence. These modules are followed by an O-linked sugar domain, a transmembrane segment and a short intracellular tail containing an NPxY motif [Jeon and Blacklow , 2005]. The physiological role of the LDL receptor in mammals is to maintain cholesterol homeostasis by cellular uptake and catabolism of plasma cholesterol. In the circulatory system, cholesterol and other lipids are transported as a part of lipoproteins. These particles consist of a lipid core composed of esteried cholesterol and triglycerides, surrounded by a monolayer of phospholipids and unesteried cholesterol with associated apolipoproteins. Depending on their triglyceride proportion and their buoyant density, the lipoprotein particles are divided into different subcategories: chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Dietary fat is transported from the intestinal mucosa to the liver in form of chylomicrons. In the circulation, triglycerides are extracted from the chylomicrons and degraded by lipoprotein lipases to free fatty acids which are taken up from cells as energy source or stored by adipose tissue. The chylomicron remnants are taken up by the liver via receptor mediated endocytosis. There, VLDL is synthesized from triglycerides and cholesterol delivered by the chylomicron remnants and released to the blood stream. In the circulation, the VLDL particles are converted to IDL and later LDL by further extraction of triglycerides by lipoprotein lipases. LDL nally carries cholesterol to other tissues where it is recognized
1 Introduction
and internalized by LDL receptor. HDL collects cholesterol from the tissues and delivers it back to the liver. The binding of lipoprotein particles to lipoprotein receptors is mediated by apolipoproteins. Natural ligands for the LDL receptor are apolipoprotein E (apoE) and apolipoprotein 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 clathrinmediated endocytosis (originally referred to as receptor-mediated endocytosis) [Goldstein et al., 1985]. This process is dependent on the localization of the receptors to clathrin-coated pits via the NPxY motifs in their cytoplasmic tail [Chen et al., 1990]. The coated pit pinches off the cell membrane to form a coated vesicle containing the receptor-lipoprotein-complex. After shedding the clathrin coat, the vesicle fuses with a late endosome where the receptor is exposed to a slightly acidic pH environment. This causes conformational changes in the receptor leading to the release of the LDL particle. Subsequently, the receptor and the lipoprotein are separated for further processing. The LDL receptor can either be degraded or delivered back to the cell surface by receptor recycling. The lipoprotein particle is degraded in the lysosome, liberating cholesterol which can be stored in the cell in the form of cholesterol ester or used as a component of plasma membranes or for the synthesis of bile acids or steroid hormones. Several feedback mechanisms ensure the maintenance of a constant level of cholesterol in the cell and protect it from cholesterol overaccumulation [Brown and Goldstein, 1986]. On the one hand, LDL receptor expression is downregulated, preventing further uptake of LDL and cholesterol. On the other hand, the level of cellular cholesterol biosynthesis is reduced in the presence of intracellular cholesterol by suppression of the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) synthase and HMG-CoA reductase. Furthermore, free cholesterol in the cell activates acyl-CoA-cholesterol:acyltransferase (ACAT) which facilitates the storage of excess cholesterol as cholesterol esters. Apart from delivering cholesterol to the cells in an appropriate amount, receptormediated uptake of LDL also guarantees the clearance of lipoproteins from the circulation. A genetic disorder affecting the functionality of the LDL receptor leads to the disease familial hypercholesterolemia (FH) which is characterized by lipoprotein accumulation in the blood stream due to the defective cellular uptake mechanism. Symptoms caused by elevated plasma LDL levels are premature atherosclerosis and heart attacks.
1 Introduction 1.1.3 The very low density lipoprotein receptor
The structure of the very low density lipoprotein receptor is almost identical to that of the LDL receptor except for an additional LA-repeat in the ligand binding domain. The receptor recognizes triglyceride-rich lipoproteins containing apoE which includes VLDL, IDL and chylomicrons but not LDL. VLDLR is expressed at high levels on the capillary endothelium of skeletal muscle, heart and adipose tissue [Wyne et al., 1996] where it occurs in splice variants containing or lacking the O-linked sugar domain. The variant including this domain seems to be more stable at the cell surface [Magran et al., 1999]. In the brain, a third splice variant lacking one LA-repeat can be found [Takahashi et al., 2004]. Since VLDLR is only expressed on the endothelium of tissues involved in the peripheral metabolism of triglyceride-rich lipoproteins, it was suggested that its physiological 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 triglyceriderich lipoproteins to the surface of endothelial cells, facilitating the hydrolysis of triglycerides by lipoprotein lipase (LPL) resulting in the delivery of free fatty acids to the adjacent tissue. This view is supported by the ndings that the presence of LPL enhances the binding of lipoproteins to VLDLR [Takahashi et al., 1995] and that VLDLR enhances the LPL mediated triglyceride hydrolysis [Goudriaan et al., 2004]. Moreover, it was shown that the VLDL receptor is involved in the transcytosis of LPL across endothelial cells [Obunike et al., 2001]. However, lipoprotein proles of VLDLR decient mice do not differ from those of animals expressing the receptor [Frykman et al., 1995]. Nevertheless, the knockout mice are partially resistant to diet-induced obesity [Goudriaan et al., 2001] and tend to be leaner due to a reduction in adipose tissue mass [Frykman et al., 1995] indicating reduced fatty acid transport into adipocytes. Since mice are high density lipoprotein animals, LDLR/VLDLR double knockout mice which have increased LDL and decreased HDL levels resemble the human lipoprotein prole better. Keeping these animals under a high fat diet, a signicant increase in serum triglyceride levels could be observed [Tacken et al., 2000]. Together, these results clearly show a role 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 activator inhibitor 1 complex [Argraves et al., 1995], thrombospondin [Mikhailenko et al.,
1 Introduction
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. Alternative splicing generates distinct variants of ApoER2 which differ in the number of LA-repeats and the presence or absence of the O-linked sugar domain, a furin cleavage 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 sugar domain is more susceptible to proteolytic processing [May et al., 2003]. Depending on the species and organs expressing the receptor, apoER2 can contain either three, four, ve, 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 exon at the carboxy-terminus of the ligand binding domain constitutes a furin consensus cleavage site. Cleavage by the calcium-dependent serine endoprotease furin [Thomas, 2002] at this site produces a soluble receptor fragment containing the entire ligand binding domain consisting of four LA-repeats. The secreted fragment is able to inhibit Reelin signaling in primary neurons [Koch et al., 2002]. In the 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 containing a proline-rich insertion of 59 amino acids [Brandes et al., 1997]. This insert and features in or near the transmembrane domain exclude ApoER2 from carrying out clathrin-mediated endocytosis [Sun and Soutar , 2003]. Furthermore, this region 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 protein being involved in a MAP kinase pathway. The main protein of this pathway is c-jun N-terminal kinase (JNK). Once activated, it translocates to the nucleus where it modulates target gene expression. Another adaptor protein binding to the proline-rich insert is the postsynaptic density protein PSD-95 which interacts with both ApoER2 and the N-methyl-D-aspartate (NMDA) type gutamate receptor simultaneously in the presence of Reelin. This interaction plays a role in synaptic plasticity and memory in the adult brain by regulating synaptic calcium inux via the NMDA receptor [DArcangelo, 2005].
1 Introduction 1.1.5 The LDL receptor related protein
The LDLR related protein (LRP, LRP1) is a multifunctional receptor which is expressed in a broad range of cell types, including hepatocytes, neurons, vascular smooth muscle cells, macrophages and embryonic tissues and interacts with a wide variety of ligands. This multifunctionality is likely the reason for the early embryonic lethality of mouse embryos in which the LRP gene has been disrupted in 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 balance between synthesis and clearance of the -amyloid peptide A. A is a major factor in the pathogenesis of Alzheimers disease and is generated by proteolytic processing of the -amyloid precursor protein (APP). Furthermore, LRP has an inuence on synaptic plasticity due to PSD-95 mediated interaction with NMDA receptors [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), and urokinase 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 an alternatively spliced unique exon within its cytoplasmic tail coding for 33 amino acids. In mice, the primary site of LRP1b expression is the central nervous system but it was recently shown that expression in humans is more widespread [Li et al., 2005]. LRP1b was originally discovered as a putative tumor suppressor [Liu et al., 2000]. Further ndings linking homozygous deletions, DNA methylation or abnormal transcripts of the LRP1b gene to different types of cancer have strengthened this theory [Hirai et al., 2004; Langbein et al., 2002; Pineau et al., 2003; Sonoda et al., 2004].
1 Introduction 1.1.7 Megalin
Megalin is highly expressed in the renal proximal tubules, where it plays an important role in renal resorption. Megalin was rst discribed as pathogenic antigen for Heymann nephritis, an autoimmune disease in rats [Kerjaschki and Farquhar , 1982]. Under physiological conditions, megalin mediates transepithelial transport 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 [Orlando et 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 proteins containing multiple EGF-like domains in human brain [Nakayama et al., 1998]. Recently, it was revealed that MEGF7 decient mice exhibited a severe malfunction of digit differentiation. A role of MEGF7 as a modulator of cellular signaling pathways including wingless/int proteins (Wnt), bone morphogenic proteins (Bmp), broblast growth factors (Fgf) and sonic hedgehog (Shh) was suggested, since the interaction of these pathways is required for proper limb patterning [Johnson et al., 2005].
1.2 The Reelin signaling pathway

The Reelin signaling pathway plays a crucial role in the development of the central nervous system (CNS) where it is responsible for the correct positioning of neurons in laminated brain structures. Disruption of the pathway results in ataxia, tremors, imbalance and a reeling gait that becomes apparent two weeks after birth [DArcangelo and Curran, 1998]. Although the specic mechanisms affected by Reelin signaling are not fully understood, Reelin is thought to inuence migration of neurons, modulate cell-cell interaction and induce cytoskeletal rearrangements. Analysis of mutant mice has led to the identication and characterization of genes involved in this pathway. Naturally occurring mutants showing this phenotype
1 Introduction
10
include reeler, scrambler and yotari mice. While the causative mutation of reeler mice is in Reelin, scrambler and yotari mice arise from mutations in Disabled 1, a cytoplasmic adaptor protein [Sheldon et al., 1997]. The reeler phenotype can also be observed in ApoER2 and VLDLR double knock out mice [Trommsdorff et al., 1999].
1.2.1 Reelin Reelin is a large secreted glycoprotein of 3461 amino acids with a molecular mass of approximately 430 kDa. Mouse and human Reelin are 94.2% identical [DeSilva et al., 1997]. Reelin contains a cleavable signal peptide at the N-terminus followed by eight consecutive repeats, each containing two subdomains separated by EGF-like motifs [DArcangelo et al., 1995]. It harbors two protease cleavage sites located between repeats two and three and repeats six and seven (gure 1.2). In vivo, Reelin is processed by a metalloproteinase giving rise to two fragments of approximately 300 kDa and 180 kDa which can be detected in addition to the 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 at the N-terminus (segment S) followed by an F-spondin homology domain (segment SP) and a unique region (segment H) with no sequence homology. The main body of the protein consists of eight repeats (segments 1-8), each containing an EGF-like motif at the center, anked by two subdomains (A and B). The C-terminal region of the protein is rich in basic residues and is required for secretion [DArcangelo et al., 1997]. The two arrows point to the cleavage sites responsible for Reelin processing in vivo. Modied from Tissir and Gofnet [2003].
In murine embryos, Reelin is rst detected at embryonic day 11.5 (E11.5), increases up to birth and remains high until postnatal day 11 (P11). It plays an important role in neuronal migration during development of laminar structures of the mammalian brain including the cerebral cortex and cerebellum. The highest expression of Reelin can be detected in the marginal zone of the developing cerebral cortex, where it is secreted by Cajal-Retzius neurons as well as in external granule cells of the developing cerebellum [DArcangelo et al., 1995]. Besides the brain, Reelin is also present at low levels in peripheral organs such as adult
1 Introduction
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blood, liver, and kidney [Smalheiser et al., 2000]. The function of Reelin signaling in non-neuronal tissues however is still not clear. Besides its signaling properties, Reelin acts as a serine protease on adhesion molecules of the extracellular matrix [Quattrocchi et al., 2002], thereby possibly facilitating cellular migration.
1.2.2 The Reelin signaling cascade Reelin molecules form disulde-linked homodimers [Kubo et al., 2002] which are capable 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/or VLDLR leads to dimerization or oligomerization of the corresponding receptors (gure 1.3). When clustered, the receptors are able to induce tyrosine phosphorylation 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 between the unphosphorylated NPxY motif in the cytoplasmic tails of ApoER2 and VLDLR and the phosphotyrosine binding (PTB) domain of Dab1 [Howell et al., 1999; Trommsdorff et al., 1999]. The kinase responsible for Dab1 phosphorylation is a member of the Src family kinases (SFK), with Fyn being the main Dab1 kinase 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 signicant role in Dab1 phosphorylation [Arnaud et al., 2003b]. To be a substrate for tyrosine phosphorylation, Dab1 has to be serine/threonine phosphorylated. Cyclin-dependent kinase 5 (Cdk5) has been shown to be responsible for this phosphorylation which occurs independently of the presence of Reelin [Keshvara et al., 2002]. Tyrosine phosphorylated Dab1 interacts with phosphoinositide-3-kinase (PI3-K) which leads to the activation of Akt/protein kinase B (PKB) and inhibition of glycogen synthase kinase 3 (GSK3) [Beffert et al., 2002; Bock et al., 2003]. Thereby, Reelin stimulation leads to a reduction of the phosphorylation state of the microtubule-associated protein tau, a known GSK3 substrate [Beffert et al., 2002; Ohkubo et al., 2003]. Furthermore, Reelin eventually has effects on other downstream targets of the PI3-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 a Reelin-dependent and phosphorylation state-dependent fashion. These include lissencephaly 1 (Lis1) [Assadi et al., 2003], Nck (noncatalytic region of tyrosine kinase) adapter protein (Nck) [Pramatarova et al., 2003], Crk family members
1 Introduction
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[Ballif et al., 2004; Chen et al., 2004; Huang et al., 2004], and neuronal WiskottAldrich syndrome protein (N-WASP) [Suetsugu et al., 2004], all of them being involved in cell migration.
Figure 1.3 The Reelin signaling pathway. Reelin dimers bind to both ApoER2 and VLDLR which results in dimerization or oligomerization of the receptors. Disabled 1 (Dab1) is serine/threonine phosphorylated by cyclin dependent kinase 5 (Cdk5) which makes it a substrate for tyrosine phosphorylation by Src family kinases (SFK). While Cdk5 acts independently from the Reelin signal, tyrosine phosphorylation is induced by Reelin dependent clustering of ApoER2 and VLDLR. The Src family kinases themselves can be further activated by phosphorylated Dab1 [Bock and Herz , 2003]. Furthermore, tyrosine phosphorylated Dab1 interacts with phosphoinositide-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 positioning in brain development.
After Reelin stimulated tyrosine phosphorylation, Dab1 is ubiquitinated and degraded by proteasomes [Arnaud et al., 2003a] which might be a mechanism to ensure a transient response to the Reelin signal. Non tyrosine phosphorylated Dab1 is stable with a half-life time of 12 h, while tyrosine phosphorylation decreases 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 migration of neuronal progenitors from dened proliferative zones to their nal location in
1 Introduction the cerebral cortex and the cerebellum.
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The six neuronal layers of the cerebral cortex are formed during embryonic development by migration of neurons from the subventricular zone (SVZ) outwards along radial glial bers to the pial surface of the brain (gure 1.4). This radial migration occurs in the mouse between embryonic days 11 and 18.
Figure 1.4 Neocortical layer formation by radial migration. At embryonic day 11 (E11), the preplate (PP) is established by a wave of postmitotic neurons that has migrated from the ventricular zone (VZ) to the pial surface (PS). By E13, a second postmitotic neuronal wave has migrated through 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 in an inside-out fashion, as each wave of neurons passes its predecessors to settle underneath the MZ. Neuronal positioning occurs mainly by migration along radial glial bers (shown as vertical bars). In adulthood, the SP degenerates, leaving behind a six-layered neocortex. Modied from Gupta et al. [2002].
The rst wave of postmitotic neurons leaving the SVZ establishes a neuronal layer known as the preplate which is split by the following neuronal wave, resulting in a subplate and a supercial marginal zone. The subplate is located proximal to the ventricular zone, while the marginal zone which harbors Reelin secreting CajalRetzius cells [Frotscher , 1998] is positioned just underneath the pial surface of the brain. The new layer between the subplate and the marginal zone is referred to as the cortical plate. Subsequent waves of migrating neurons traverse the subplate and the preexisting neuronal layers of the cortical plate until they encounter Reelin. There, they cease migration, detach from the radial glial bers and form new dened layers underneath the marginal zone. According to this mechanism,
1 Introduction
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cortical layers form in an inside-out manner, with the latest-born neurons occupying the outermost layers of the cortical plate. After the cortical plate has been fully established, the subplate degenerates, leaving a cortex consisting of six neuronal layers that persists throughout adulthood. In mouse mutants exhibiting the reeler phenotype, the Reelin pathway is disrupted. As demonstrated in gure 1.5, migrating neurons are unable to bypass pre-existing layers which results in an unsplit preplate (called the superplate) and inverted, disorganized cortical layers [Gupta et al., 2002]. Several hypotheses have 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 a stop signal when the neuron has reached its destination in the cortical plate; or it may interrupt the association between migrating neurons and radial glia. Another theory [Luque et al., 2003] is based upon the nding that neocortical neurons are derived from asymmetrical cell division of radial glia and the observation that they inherit the glia process, already attached to the pial basement membrane [Miyata et al., 2001; Tamamaki et al., 2001]. So, many newborn neurons might use their own basement membrane-attached radial processes to reach their target position by somatic translocation [Nadarajah et al., 2001]. It was suggested that Reelin signaling is required for the maintenance of the radial glia cytoskeletal phenotype and that it acts on the inherited radial processes of neocortical neurons to induce cytoskeletal rearrangements required for somatic translocation [Luque et al., 2003]. In the absence of Reelin, like in the reeler mouse, somatic translocation is impaired, which may explain the incapacity of reeler neurons to pass through pre-existing layers. During early postnatal development of the cerebellum, postmitotic Purkinje cells migrate outwards from the ventricular zone to the pial surface. When the cells encounter Reelin which is secreted by granule cells, they align to form a welldened cortical layer [Miyata et al., 1997]. Progenitors of granule cells in the external granule cell layer migrate inwards, passing the Purkinje cell layer, and form the internal granule cell layer, giving rise to the multilayered structure of the cerebellum. In mutant mice with disrupted Reelin signaling, neither the Purkinje cell layer nor the external granule cell layer is formed [Porcionatto, 2006].
1 Introduction
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Figure 1.5 Defective neocortical layer formation in reeler mouse mutants. In mice with mutations in Reelin, Dab1, or both VLDLR and ApoER2, the preplate (PP) does not split and forms a structure called the superplate (SPP). The cortical plate (CP) forms under the SPP and is inverted, indicating that late-migrating neurons are unable to migrate past their predecessors. Early-migrating and late-migrating neurons are positioned in the supercial and deep layers of the CP, respectively, and layering is disorganized. Modied 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 hippocampus can be modulated by Reelin signaling through ApoER2. In the postsynaptic densities of excitatory synapses, ApoER2 forms a functional complex with N-methyl-D-aspartate (NMDA) receptors. ApoER2 receptors within this complexes contain the proline-rich insert in the cytoplasmic tail which is required for the interaction of the two receptors mediated by the postsynaptic density protein PSD-95. In the presence of Reelin, the glutamate-stimulated NMDA receptormediated calcium inux is increased through tyrosine phosphorylation of NMDA subunits [Beffert et al., 2005]. Thereby, Reelin signaling contributes to the modulation of postsynaptic responsiveness, which is a key event in learning and memory. Thus, disruption of Reelin-dependent synaptic functions may lead to cognitive impairment. Numerous reports implicate involvement of Reelin signaling malfunctions in human neurodevelopmental disorders such as schizophrenia [Grayson et al., 2006], autism [Fatemi et al., 2005; Skaar et al., 2005] and autosomal recessive lissencephaly [Hong et al., 2000]. Another effect of disrupted Reelin signaling was proposed for the development of the neurodegenerative dis-
1 Introduction
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order Alzheimers disease (AD). AD is characterized by the presence of neurobrillary tangles and amyloid plaques in the brain. The tangles are intracellular aggregates of paired helical laments, mainly composed of the microtubuleassociated protein tau, in a hyperphosphorylated state. Senile plaques are formed by extracellular deposits of -amyloid peptide (A) which is generated by cleavage of the transmembrane glycoprotein -amyloid precursor protein (APP) by the -secretase Presenilin-1 (PS1). The proposal of an involvement of Reelin signaling in the development of AD is based upon several ndings. First, Reelin binds to ApoE receptors, and some ApoE gene polymorphisms are considered risk factors for AD. Moreover, the lack of Reelin is associated with hyperphosphorylation of tau [Hiesberger et al., 1999], which leads to neurobrillary tangles. Additionally, APP contains an NPxY motif similar to that found in LDLR family members, and is known to bind Dab1 [Howell et al., 1999]. Recent data also indicate 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 environment and regulates the entry and exit of molecules. Essential small molecules, such as amino acids, sugars, and ions, can traverse the plasma membrane through integral membrane protein pumps or channels. Macromolecules must be carried into the cell by endocytosis. Various endocytic pathways are employed for 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 uid and solutes, occurs in all cells. Pinocytosis includes four basic mechanisms: macropinocytosis, clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis, and clathrin- and caveolaeindependent endocytosis (gure 1.6).
1 Introduction
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Figure 1.6 Multiple mechanisms of endocytosis The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation. Modied from Conner and Schmid [2003].
1.3.1 Clathrin-mediated endocytosis Clathrin-mediated endocytosis (CME, gure 1.7) represents a constitutive mechanism in all mammalian cells involved in the continuous uptake of essential nutrients, such as LDL particles and transferrin (Tfn) when bound to their corresponding receptors [Brodsky et al., 2001; Schmid , 1997]. CME also modulates signal transduction, both by controlling the levels of surface signaling receptors, and by mediating the rapid clearance and downregulation of activated signaling receptors. A prerequisite for CME is the concentration of transmembrane receptors and their bound ligands into coated pits on the plasma membrane. These membrane regions are formed mainly by the cytosolic coat protein clathrin. Clathrin occurs as a complex of light and heavy chains, building up a threelegged structure, the so-called triskelion [Brodsky et al., 2001; Kirchhausen, 2000]. Triskelions are capable of forming polygonal cages with the help of assembly proteins. For the formation of clathrin cages at the plasma membrane, the adaptor protein complex AP2 is required. AP2 consists of four subunits [Collins et al., 2002]. The -adaptin subunit targets AP2 complexes to the plasma membrane; the -subunit interacts with clathrin to trigger its assembly; the 2-subunit binds tyrosine-based internalization motifs on the cytoplasmic domains of endocytic receptors to concentrate them into coated pits; and the 2-subunit has a structural role in stabilizing the core domain. Besides being recognized by the 2-subunit, the NPxY motif present in the cytoplasmic domain of the LDL receptor family also interacts with Disabled 2 (Dab2), which can bind to AP2, giving rise to an alternative assembly mechanism [Mishra et al., 2002; Morris and Cooper , 2001]. Moreover, the
1 Introduction
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invagination of coated pits and the formation of clathrin coated vesicles (CCVs) depends on the large GTPase dynamin. Dynamin is thought to self-assemble into a collar at the neck of the deeply invaginated coated pit and act as a mechanochemical enzyme to drive membrane ssion [Hinshaw and Schmid , 1995; Stowell et al., 1999; Sweitzer and Hinshaw , 1998]. An alternative model suggests that dynamin recruits or activates downstream effectors [Sever et al., 1999].
Figure 1.7 Clathrin-mediated endocytosis. Clathrin triskelions assemble into a polygonal lattice, which helps to deform the overlying plasma membrane into a coated pit. Adaptor protein 2 complexes, targeted to the plasma membrane, mediate this assembly. To form a clathrin coated vesicle (CCV), the GTPase dynamin is recruited to the necks of coated pits where it assembles to a collar to facilitate membrane ssion. The CCV is released into the cytoplasm. Modied from Conner and Schmid [2003].
1.3.2 Lipid rafts and caveolae Lipid rafts are small (40-60 nm diameter), highly ordered lipid microdomains of the plasma membrane. They are rich in free cholesterol and sphingolipids and move freely within the cell surface [Brown and London, 2000]. Lipid rafts can be internalized by different endocytic mechanisms, which are either caveolaedependent or -independent [Kirkham and Parton, 2005]. Caveolae are uncoated ask-shaped invaginations of the plasma membrane. They are believed to constitute a subdomain or a special type of lipid rafts [Anderson, 1998; Kurzchalia and Parton, 1999]. Like rafts, they have a composition high in free cholesterol and sphingolipids but are additionally dened by the presence of the structural protein caveolin. The formation of homo- and hetero-oligomers of caveolins [Sargiacomo 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 determine the shape of caveolae [Sargiacomo et al., 1995]. Caveolae are abundant
1 Introduction
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in some cell types (adipocytes, endothelia, muscle) but undetectable in others (lymphocytes, many neuronal cells). Various considerations lead to the conclusion that the role of caveolae-mediated endocytosis lies in the regulation of signaling processes rather than in bulk uid-phase uptake [Anderson, 1998; Razani et al., 2002]. On the one hand, caveolae are only slowly internalized (with a halftime of more than 20 minutes) and carry little volume, and on the other hand, many signaling molecules and membrane transporters are concentrated in these microdomains. Unlike LDLR and VLDLR, ApoER2 is found mostly in caveolae [Mayer et al., 2006; Riddell et al., 2001]. Evidence for caveolae-independent mechanisms of lipid raft internalization was supplied by reports on lipid raft internalization in caveolin-decient cells [Fra et al., 1994; Simons and Toomre, 2000]. Moreover, caveolae-independent lipid raft endocytosis also occurs in cells with abundant caveolae [Kirkham et al., 2005].
Figure 1.8 Membrane organization of lipid rafts and caveolae. Lipid rafts are highly ordered microdomains of the plasma membrane. In contrast to the bulk lipid bilayer which is composed mainly of phospholipids, rafts are enriched in sphingolipids and cholesterol. Caveolae are generally considered to be a type of lipid rafts. Due to an enrichment of caveolins, their assembly to oligomers (shown as dimers for simplicity), and their interaction with cholesterol, raft domains form the ask-shaped invaginations of caveolae. Modied from Galbiati et al. [2001].
Although the internalization of caveolae and rafts was considered to occur via different mechanisms, another theory has been proposed, suggesting a common pathway for both and a regulatory role for caveolin [Nabi and Le, 2003]. This theory takes into account that reduction of caveolin levels has in some cases either no effect on, or even accelerate raft-mediated uptake of molecules. This suggests that caveolin is not an essential part of the uptake mechanism; it may even act as a negative regulator of raft-mediated endocytosis. The presence of caveolin in endocytic raft domains may stabilize these structures at the plasma membrane and retard the budding of raft-derived vesicles. This would also be consistent
1 Introduction
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with the nding that caveolae are largely static. The inhibitory effect could only be overcome by specic signaling events. This proposal is supported by the nding that phosphatase inhibitors are able to induce the budding of surface-connected caveolae.
2 Aims of this study

As outlined in the introduction, two members of the LDLR family, namely ApoER2 and VLDLR are known to be involved in transmitting the Reelin signal across the plasma membrane of neuroblasts [Trommsdorff et al., 1999]. During brain development, the Reelin signaling pathway is activated upon binding of Reelin to ApoER2 and VLDLR, leading to tyrosine phosphorylation of Dab1. These events seem to constitute a linear pathway, since disruption of either the Reelin or Dab1 gene or inactivation of both ApoER2 and VLDLR genes cause the same phenotype in mice. Obviously, tyrosine phosphorylation of Dab1 is triggered by Reelin-induced receptor clustering [Strasser et al., 2004] and is mediated by Src family kinases [Arnaud et al., 2003b]. Still, the knowledge on the modulation of the initial signal and on downstream events guiding migration and positioning of neurons is scarce. Therefore, the aim of this study was to further investigate different aspects of the receptor mediated Reelin signal transduction, including endocytosis and clearance of Reelin from the cell surface and regulation of the Reelin receptors by membrane localization, intracellular trafcking, degradation, and specic cleavage. For this purpose, two different cell systems should be used: rst, a NIH 3T3 cell model mimicking the Reelin signaling pathway [Mayer et al., 2006] should be used for most of the experiments since this system offers easier availability and greater amounts of cell-material which is necessary for most biochemical analyses. Second, selected experiments should be carried out using primary rat neurons to verify the results in a more physiological situation. It was recently shown that ApoER2 and VLDLR reside in distinct subdomains of the plasma membrane [Mayer et al., 2006; Riddell et al., 2001]: ApoER2 is sorted into caveolae/lipid rafts, whereas VLDLR is strictly excluded from these microdomains. Two ndings suggested that the differential sorting of the receptors does not inuence Reelin signaling: rst, knockout of either ApoER2 or VLDLR resulted in only subtle abnormalities in mouse brain architecture, suggesting that both receptors can at least partially compensate for each other [Trommsdorff et al., 1999], and second, it was shown that both receptors are equally potent
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to phosphorylate Dab1 [Strasser et al., 2004]. Thus, the different localization of the receptors within the plasma membrane is likely not important for the signaling activity of the receptors. To verify this hypothesis, the effectiveness of Dab1 phosphorylation in the presence and absence of functional lipid rafts should be studied. This task should be addressed by the use of methyl--cyclodextrin and nystatin. Both agents disrupt rafts/caveolae by depletion of cholesterol from the membrane but have only little effect on other membrane structures such as clathrin-coated pits. Assuming that differential sorting of the receptors within the plasma membrane has no inuence on Dab1 phosphorylation, the question on possible other effects of the distinct membrane localization of both receptors arises. Differential sorting might have an inuence on endocytosis and degradation of Reelin and/or on trafcking of the Reelin receptors. Differences in endocytosis rates of LDLR family members have already been reported [Li et al., 2001]. In agreement with this hypothesis, recent results from our laboratory showed that ApoER2 and VLDLR exhibit different rates of Reelin endocytosis and degradation [Mayer , 2005]. These results were based on an assay comparing the amounts of cell bound Reelin after different incubation times with Reelin conditioned medium. Here, these results should be veried by an alternative approach based on analysis of Reelin amounts in Reelin conditioned medium during incubation of cells expressing either ApoER2 or VLDLR. Since it is not clear to date whether and how endocytosis of the receptors and Reelin modulates the signaling events and the cellular response, the potency of endocytosis-inhibited cells to induce Dab1 phosphorylation upon Reelin stimulation should be examined, . Moreover, the receptor domain responsible for the differential sorting of the receptors should be determined. For these experiments, chimeric receptors expressing various combinations of the different domains of ApoER2 and VLDLR should be used. To examine whether the different endocytosis rates of ApoER2 and VLDLR are linked to differential subcellular distribution, the surface expression of the receptors should be analysed by immunouorescence and biotinylation of surface receptors. Furthermore, a possible inuence of Reelin stimulation on the subcellular distribution of the receptors should be studied. Regarding receptor degradation, it was observed that levels of ApoER2 but not VLDLR were dramatically decreased upon Reelin stimulation [Mayer et al., 2006]. For further investigation of this event, it should be found out if ApoER2 degrada-
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tion is initiated by receptor clustering which is also responsible for Reelin-induced Dab1 phosphorylation. This question will be addressed by analysis of cell response to stimulation with multivalent or monovalent ligands. Furthermore, treatment with lysosome or proteasome inhibitors should determine the pathway of ApoER2 degradation under these circumstances. Besides degradation, the Reelin receptors are substrates for specic, ligand induced cleavage, which might add another level of modulating the Reelin signal. It was reported recently, that by the action of -secretase, an extracellular soluble receptor fragment is produced [Hoe and Rebeck , 2005]. Subsequent cleavage by -secretase leads to liberation of an intracellular fragment [May et al., 2003]. Here, this fragmentation should be further examined in terms of membrane- and raft-association of the intracellular fragment and the inuence of Dab1 on the fragmentation process. The results concerning endocytosis and degradation of Reelin and distribution, degradation, and fragmentation of the receptors are expected to lead to a better understanding of molecular events during Reelin signaling and their modulatory effects on the signal produced.
3 Material and Methods

3.1 Antibodies
For the detection of ApoER2, three polyclonal antibodies were used: rabbit 186 directed against the entire ligand binding domain (LA-repeats 1-3, 7, 8) of murine ApoER2 [Strasser et al., 2004], rabbit 19 directed against the cytoplasmic tail of ApoER2 lacking the proline-rich insert, and rabbit 20 directed against the cytoplasmic tail of ApoER2 containing the proline-rich insert [Stockinger et al., 1998]. For detection of VLDLR, rabbit 187 directed against the entire ligand binding domain of murine VLDLR was used [Strasser et al., 2004]. Furthermore, HA-tagged receptors were detected using mouse HA (Covance). Monoclonal antibody D4 was used for detection of murine Dab1 in Western blotting. Phosphorylated Dab1 was detected by the phosphotyrosine-recognizing mouse antibody PY99 (Santa Cruz). For immunoprecipitation of Dab1, polyclonal antibodies were used, i.e. rabbit 54, directed against the rst 180 amino acids of the short splice variant of murine Dab1 and rabbit 48, directed against the short splice variant of murine Dab1. Mouse GFP was purchased from Santa Cruz. Reelin was detected using the monoclonal antibody G10. D4 and G10 are a kind gift of Andre Gofnet (University of Louvain, Brussels, Belgium). For detection of endogenous proteins as loading controls, monoclonal Lis1 antibody and rabbit cav1 (transduction laboratories), 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 antimouse and goat anti-rabbit antibodies (Jackson Immuno Research). Alexa Flour 488 goat anti-mouse and Alexa Flour 594 goat anti-rabbit antibodies (Molecular Probes) were used for immunouorescence microscopy.
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3.2 Plasmids
Eukaryotic expression plasmids used for the production of stable NIH 3T3 cell lines [Mayer et al., 2006] and for transient transfection were based on the vectors pMSCVpuro and pfMSCV-IRES-GFP. The constructed plasmids comprise pMSCVpuro-ApoER2(+/-) and pMSCVpuro-VLDLR, coding for murine ApoER2 harboring LA repeats 1 to 3, 7, and 8, containing (+) or lacking (-) the proline-rich cytoplasmic insert or murine VLDLR lacking the O-linked sugar domain, respectively [Mayer et al., 2006]. Furthermore, plasmids coding for chimeric receptors were used. Chimeric receptors were constructed by swapping intracellular, transmembrane and extracellular domains between ApoER2 and VLDLR. For constructs comprising the intracellular domain of ApoER2, the variant containing the proline-rich insert (+) was used. The resulting receptors comprise Ae Atm Vi (derived from ApoER2 by replacing its cytoplasmic tail with that of VLDLR), Ae Vtm Vi (derived from VLDLR by replacing its extracellular domain with the corresponding ApoER2 domain), and vice versa, Ve Vtm Ai(+) and Ve Atm Ai(+) [Mayer , 2005]. Plasmids used coding for murine Dab1 were pfMSCV-IRES-GFP-Dab1 and pMSCVpuro-Dab1 [Mayer et al., 2006]. Furthermore, the plasmids pcDNAux3ApoER2 and pcDNAux3-VLDLR [Hoe and Rebeck , 2005] were used for expression 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 humidied environment of 7.5% CO2 in Dulbeccos modied Eagle medium (DMEM; Gibco), supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 U/ml streptomycin sulphate. Cells were split at conuency every 2-3 days.
3 Material and Methods NIH 3T3 Mouse embryonic broblasts, ATCC Number CRL-1658
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Cells were grown at 37C in a humidied environment of 7.5% CO2 in DMEM, supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin and 100 U/ml streptomycin sulphate. Cells were split at conuency 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 obtained from 16.5 day old (E16.5) rat embryonic brains. Dissected brains were washed twice with Hanks Balanced Salt Solution (HBSS, Gibco) by centrifugation for 4 min at 1,300 rpm and suspended in Dulbeccos modied Eagle medium F-12 (Gibco) containing B27 supplement (Gibco), 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin sulphate. The suspension was triturated 30 times with a re polished glass pasteur pipette and neurons were plated on 10 cm cell culture dishes coated with 150 g/ml poly-L-ornithine. Cells were grown at 37C in a humidied environment of 5% CO2 in DMEM F-12 containing all supplements for 72 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 humidied environment of 7.5% CO2 in DMEM, supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin sulphate, and 400 g/ml G418.
NIH 3T3 cell model for Reelin signaling As a model cell system for studying Reelin signaling, NIH 3T3 cells stably expressing components of the pathway were used [Mayer et al., 2006]. Cell lines stably expressing murine ApoER2 with ligand-binding repeats 1-3, 7 and 8, containing or lacking the proline-rich cytoplasmic insert (3T3 A+ or 3T3 A-, respec-
27
tively) or murine VLDLR lacking the O-linked sugar domain (3T3 V-) were used to study receptor mediated endocytosis, receptor degradation, and receptor fragmentation. 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 inuence on molecular events during Reelin signaling. Cell lines expressing chimeric receptors of ApoER2 and VLDLR comprise 3T3 Ae Atm Vi , Ae Vtm Vi , Ve Vtm Ai(+) , and Ve Atm Ai(+) where e marks the extracellular domain of ApoER2 (A) or VLDLR (V), tm stands for the transmembrane, and i for the intracellular domain. For constructs comprising the intracellular domain of ApoER2, the splice variant containing the proline-rich insert i(+) was used. Cells were grown at 37C in a humidied environment of 7.5% CO2 in DMEM, supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin sulphate, and 500 g/ml puromycine. Cells were split at conuency every 2-3 days.
3.3.2 Freezing of cells Cells were washed once with prewarmed PBS, trypsinized, resuspended in growth medium and pelleted by centrifugation at 500g for 5 min. The supernatant was removed and the cell pellet was resuspended in FCS containing 10% DMSO and aliquoted to cryotubes (Nunc). Cells were stored at -80C for several days and subsequently transfered to liquid nitrogen.
3.3.3 Thawing of cells Cells were rapidly thawed in a 37C waterbath. The cell suspension was gradually diluted with growth medium and centrifuged for 5 min at 500g. The cell pellet was 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-
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tamine 2000 transfection reagent (Invitrogen). The cells were transfected according 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 stably expressing Reelin (see 3.3.1) were used. Cells were cultivated in 15 cm cell culture dishes using standard growth medium containing 400 g/ml G418 sulphate to near conuency. The growth medium was replaced by 20 ml OptiMEM (Gibco) and cells were incubated at 37C and 7.5% CO2 for 24-48 hours. The supernatant 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. An aliquot was used for checking expression of Reelin by Western blotting using the Reelin antibody G10. Mock conditioned medium (MCM) was prepared from untransfected HEK 293 cells following the procedures described for Reelin conditioned medium.
3.4 Protein extraction and analysis

3.4.1 Preparation of cell extracts (Hunt buffer) Cells were washed 3 times with PBS (137 mM NaCl, 0.27 mM KCl, 10 mM Na2 HPO4 , 1.7 mM KH2 PO4 , pH 7.5) and subsequently scraped into ice-cold Hunt buffer (20 mM Tris/HCl pH 8.0, 100 mM NaCl, 0.5% NP40, 1 mM EDTA, Complete proteinase inhibitor cocktail (Roche)). Crude extracts were kept on ice for 30 min and centrifuged at 4C for 15 min at 20,000g. Pelleted cell debris was discarded 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 (50 mM Tris/HCl pH 6.8, 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-
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marized in table 3.1, cast, and polymerized. Gels were run in SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 25-30 mA/gel. A prestained broad range SDS-PAGE standard (Bio-Rad or Fermentas) was used as a molecular weight marker. Full length receptors (120-130 kDa) and Dab1 (80 kDa) were anaylzed using 8% gels, intracellular receptor fragments (20 kDa) using 15%, and Reelin (400 kDa) using 4% gels.
separating gel (5 ml) 4% 8% 15% dH2 O (ml) 30% acrylamid/bisacrylamid 29:1 (ml) 1.5 M Tris/HCl pH8.8 (ml) 1 M Tris/HCl pH 6.8 (ml) 10% SDS (ml) 10% APS (ml) TEMED (ml) 3 0.65 1.3 0.05 0.05 0.003 2.3 1.3 1.3 0.05 0.05 0.003 1.2 2.5 1.3 0.05 0.05 0.003 stacking gel (1 ml) 5% 0.68 0.17 0.13 0.01 0.01 0.001
Table 3.1 Composition of SDS polyacrylamide gels. Volumes are calculated for one gel (5 ml running gel and 1 ml 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 extra, Amersham) by semi-dry blotting using semi-dry transfer buffer (25 mM Tris, 192 mM glycine, 0.05% SDS). Blotting was performed at 175 mV/cm2 for 1 h at room 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 h at room temperature. Incubation with dilutions of primary antibodies in blocking buffer was done o/n at 4C. Dilutions and blocking buffers used with the different antibodies are summarized in table 3.2. Membranes were washed 5-6 times with PBST (PBS containing 0.1% Tween 20) and subsequently incubated with a dilution of the appropriate HRP-conjugated secondary antibody in PBST for one hour at room temperature. After excessive washing, the membranes were incubated for 5 min with enhanced chemiluminiscence (ECL) solution (SuperSignal West, Pierce) according to the manufacturers protocol. Excess solution was removed and membranes were exposed to X-ray lm (CL-XPosure, Pierce). Exposure times varied from 1 s to 20 min.
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detection of Reelin ApoER2(+) ApoER2(-) ApoER2-HA VLDLR-HA Phosphorylated Dab1 Dab1 GFP Lis1 Caveolin 1 primary mouse antibodies primary rabbit antibodies
antibody G10 20 19 HA HA PY D4 GFP Lis1 Cav1 ms-HRP rb-HRP
source mouse rabbit rabbit mouse mouse mouse mouse mouse mouse rabbit goat goat
dilution 1: 10,000 1: 7,000 1: 1,000 1: 2,000 1: 2,000 1: 1,000 1: 3,000 1: 500 1: 10,000 1: 15,000 1: 20,000 1: 20,000
blocking buffer BSA milk milk BSA BSA BSA BSA BSA BSA milk
Table 3.2 Antibodies used in Western blotting. Source, dilution and blocking buffers for primary 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+) were seeded in 10 cm cell culture dishes (0.8106 cells/dish) using standard broblast growth medium. After 24 h, cells were incubated for 1 h with OptiMEM containing or lacking raft disrupting agents (see gure legend) at 37C in a humidied environment of 7.5% CO2 . All subsequent steps were carried out at 4C. Cells were washed twice with ice-cold TBS and scraped into 1 ml TBS. Subsequently, cells were pelleted by centrifugation for 5 min at 1400g and 4C. For lysis, the cell pellet was resuspended in 1 ml TBS containing 2% Brij 78P (Fluka) and Complete proteinase inhibitor cocktail. Cells were solubilized by passing 10 times through a 23-gauge needle and incubated on ice for 15 min. Cell debris was removed by centrifugation (10 min, 20,000g). 0.6 ml of the cell homogenate was mixed with 0.6 ml of 90% (wt/vol) sucrose in MBS (25 mM 2-(4-morpholino)ethanesulfonic acid, 150 mM NaCl, pH 6.5) and transferred to a SW60-Ti ultracentrifuge tube. A discontinuous sucrose gradient was formed above the homogenate by layering of 2.5 ml of 35% (wt/vol) sucrose in MBS, followed by 0.6 ml 5% (wt/vol) sucrose in MBS. After centrifugation at 160,000g for 20 h in a Beckman SW60Ti rotor at 4C, 9 fractions of 0.44 ml each were collected from the top of the tube. Frac-
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tions 2 and 3 at the interface between the 5% and 35% sucrose boundaries were designated the caveolae-rich light membrane (CLM) fraction whereas the noncaveolae membrane fraction (NCM) is restricted to the lower fractions (8 and 9). Fractions were resolved by reducing SDS-PAGE and analyzed by immunoblotting using the ApoER2 specic antibody 20.
3.5.2 Dab1 phosphorylation assay NIH 3T3 cells expressing Dab1 and either ApoER2 (3T3 A+/D) or VLDLR (3T3 V-/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 broblast 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 containing or lacking additional reagents for 1 h at 37C in a humidied environment of 7.5% CO2 . Afterwards, cells were washed with ice-cold PBS and scraped into 250 l (for 6 cm dishes) or 750 l (for 10 cm dishes) Hunt buffer containing Complete proteinase inhibitor cocktail and phosphatase inhibitors (50 mM NaF, 2 mM Na3 VO4 ). Cells were lysed on ice for 30 min and lysates were centrifuged at 20,000g for 15 min at 4C. Extracts were immediately subjected to immunoprecipitation by addition of 3 l 48 and 3 l 54 and incubated o/n at 4C rotating end over end. 40 l of 50% Protein A-Sepharose CL-4B (Amersham) slurry (prepared according to the manufacturers protocol) were added and samples incubated for an additional hour at 4C rotating end over end. Protein A-Sepharose beads were collected by centrifugation at 500g for 1 min at 4C, washed three times with cold Hunt buffer and subjected to reducing SDS-PAGE. Western blotting was done using D4 and phosphotyrosine PY99 for detection of Dab1 and tyrosine phosphorylated Dab1, respectively.
3.5.3 Reelin uptake and degradation assay Reelin uptake assays were performed with NIH 3T3 cells expressing either of the Reelin receptors (3T3 A+, V-) or the chimeric receptors (3T3 Ae Atm Vi , Ae Vtm Vi , Ve Vtm Ai(+) , Ve Atm Ai(+) ). The cells were grown in 6-well plates (0.35106 cells/well) using standard broblast growth medium. After 24 hours, cells were starved for 1 h in serum-free medium and incubated on ice for 30 min. All further steps were
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carried out on ice. Cells were washed twice with ice-cold PBS and subsequently incubated with Reelin or mock conditioned medium for 1 h. Cells were washed 3 times with ice-cold PBS and either Hunt extracts were prepared immediately or cells were overlaid with OptiMEM and incubated in a 37C waterbath for different time periods before shifting cells back to ice and lysis in Hunt buffer. Extracts were subjected to SDS-PAGE and immunoblotting using G10 for detection of Reelin.
3.5.4 Reelin depletion assay NIH 3T3 cells were seeded in 6-well plates (0.35106 cells/well) and transiently transfected after 24 h using pcDNAux3-ApoER2 or pcDNAux3-VLDLR. 48 h after transfection, cells were incubated for 32 h with 3 ml of Reelin conditioned medium diluted in OptiMEM (1:3) at 37C in a humidied environment of 7.5% CO2 . Medium samples of 60 l each were taken after 8, 24, and 32 h and cells were lysed in Hunt buffer after 32 h. All samples were subjected to reducing SDS-PAGE and Western blotting using the monoclonal Reelin antibody G10 for samples of the medium or HA to detect ApoER2-HA or VLDLR-HA in the cell extracts. For quantication, integrated optical density (IOD) values of Reelin and receptor 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) using standard broblast growth medium and transiently transfected using pcDNAux3ApoER2 or pcDNAux3-VLDLR. 48 h after transfection, cells were cooled for 15 min at 4C and subsequently incubated for 2 h with 0.5 mg/ml EZ-Link SulfoNHSS-S-Biotin (Pierce) in PBS++ (PBS containing 1 mM CaCl2 and 1 mM MgCl2 ) at 4C or 37C. Cells were washed once in cold NTC buffer (20 mM Tris pH 8.8, 150 mM NaCl, 0.1 mM CaCl2 , 0.2% BSA) and once in cold PBS++. For the preparation of cell extracts, cells were scraped in 600 l cold TNE buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 0.1% Triton X-100) containing Complete proteinase inhibitor cocktail. Cells were mechanically disrupted by passaging 10 times through a 23-gauge needle. Extracts were incubated on ice for 30 min and cell debris was pelleted by centrifugation for 15 min at 4C and
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20,000g. Cleared extracts were incubated with Neutravidin-coated beads for 1 h at 4C rotating end over end to bind biotinylated receptors. Beads were collected by centrifugation at 500g for 1 min at 4C and washed once with Netgel NaCl (50 mM Tris/HCl, pH 7.5, 5 mM EDTA, 0.25% gelatine, 0.02% NaN3 , 500 mM NaCl), once with Netgel SDS (50 mM Tris/HCl, pH 7.5, 5 mM EDTA, 0.25% gelatine, 0.02% NaN3 , 150 mM NaCl, 0.1% SDS), and once with Hunt buffer. Samples were subjected to reducing SDS-PAGE and levels of receptor biotinylated at 4C or 37C were compared by immunoblotting using HA antibody.
3.5.6 Receptor degradation assay NIH 3T3 cells expressing ApoER2 containing or lacking the cytoplasmic prolinerich insert (3T3 A+/-) were grown in 6 cm cell culture dishes (0.8106 cells/dish) using standard broblast growth medium. Alternatively, primary rat neurons were plated 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 inhibit protein synthesis. Subsequently, broblasts or primary neurons were incubated for 6 h or 24 h, respectively, in OptiMEM containing 20 g/ml cycloheximide and different ligands and/or additional reagents (see gure legends) at 37C in a humidied environment of 7.5% CO2 for the indicated time periods (see gure legends). Cell extracts were prepared in Hunt buffer and subjected to reducing SDSPAGE 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-) were seeded into 6 cm cell culture dishes (0.8106 cells/dish) using standard broblast growth medium. After 24 h, cells were incubated for 5-6 h in OptiMEM containing different ligands and/or additional reagents at 37C in a humidied environment of 7.5% CO2 . Alternatively, NIH 3T3 cells were grown in 6 cm cell culture dishes using standard broblast growth medium and transiently transfected using pcDNAux3-ApoER2 or pcDNAux3-VLDLR. Transiently transfected cells were treated with ligands and/or reagents 48 h after transfection. Cell extracts were prepared in Hunt buffer and subjected to reducing SDS-PAGE and Western blot-
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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 (0.8 106 cells/dish) and grown for 24 h using standard broblast growth medium. Cells were incubated with OptiMEM containing different ligands for 6 h at 37C in a humidied environment of 7.5% CO2 and lysed using two different methods. Hunt extracts were prepared as before (3.4.1). Remaining dishes were washed 3 times with PBS and scraped into CAV-A buffer (250 mM sucrose, 20 mM Tris pH 7.8, 1 mM EDTA) and subjected to mechanical disruption by passing 10 times through a 23-gauge needle. CAV-A extracts were centrifuged for 10 min at 20,000g and 4C to remove cell debris and subsequently subjected to ultracentrifugation in a TLA-100 rotor at 55,000 rpm and 4C for 90 min. The supernatant was saved and used for further analysis, the pellet was discarded. All samples were subjected to reducing SDS-PAGE and immunoblotted using 20 to detect full length and cleaved ApoER2.
3.6 Immunouorescence Microscopy

3.6.1 Subcellular receptor distribution Sterile glass coverslips were coated by incubation with 40 g/ml poly-L-lysine in PBS for 1 h at room temperature. Subsequently, coverslips were washed 4 times with PBS and dried. NIH 3T3 cells were plated on the coverslips and grown for 24 h at 37C in a humidied environment of 7.5% CO2 using standard broblast growth medium. At 80-90% conuency, cells were transiently transfected with pcDNAux3-ApoER2 or pcDNAux3-VLDLR [Hoe and Rebeck , 2005]. 48 h after transfection, cells were washed 3 times with PBS and xed by incubation with 4% paraformaldehyde (PFA) xative for 15 min at room temperature. Fixed cells were washed 3 times with PBS containing 100 mM glycine and subsequently incubated with 0.1% Triton X-100 in PBS for 2 min to permeabilize the cell membrane. Cells
35
were washed again with PBS and blocked for 30 min with blocking solution (1% BSA in PBS) at room temperature. Afterwards, cells were incubated with primary antibody diluted in blocking solution for 1 h before extensive washing with PBS and subsequent incubation with a specic uorescence labeled secondary antibody for 1 h. Cells were rinsed with PBS and once with H2 O before mounting on glass slides using DAKO Fluorescent Mounting Medium (Dako Corporation). The slides were analyzed using a 63 oil objective on a confocal uorescence microscope (Laser Scanning Microscope 510, Zeiss) and the corresponding software (Zeiss LSM Image Browser). Alternatively, primary neuronal cell were plated on glass coverslips coated with 150 g/ml poly-L-ornithine and grown in DMEM F-12 containing supplements described in section 3.3.1 for 72 h at 37C in a humidied environment of 5% CO2 . Primary neuronal cells were xed, permeabilized, and stained as described for broblasts.
3.6.2 Reelin internalization NIH 3T3 cells were grown and transfected on poly-L-lysine coated glass coverslips (see 3.6.1). Cells were transfected with either pcDNAux3-ApoER2 or pcDNAux3-VLDLR, coding for HA-tagged ApoER2 or VLDLR, or co-transfected with one of the receptors and pMSCVpuro-Dab1. 48 h after transfection, cells were shifted to 4C for 15 min. Afterwards, cells were washed with ice-cold PBS and overlaid with Reelin conditioned medium. After 1 h of incubation at 4C, Reelin containing medium was removed and cells were washed with ice-cold PBS and incubated with OptiMEM at either 4C or 37C for 10 min. Subsequently, cells were xed, permeabilized, and stained as described in 3.6.1.
3.6.3 Immunostaining For detection of ApoER2-HA and VLDLR-HA primary antibodies either against the receptors (186 and 187, respectively) or the HA-tag (HA) were used. Reelin was detected using G10. Secondary antibodies were Alexa Flour 488 goat anti-mouse used for detection of G10 and HA, and Alexa Flour 594 goat anti-rabbit for detection of 186 and 187. Used antibodies and corresponding
36
dilutions are summarized in table 3.3. Additionally, cell nuclei were stained using Hoechst reagent diluted 1:500.
staining Reelin ApoER2 VLDLR ApoER2-HA VLDLR-HA primary mouse antibodies primary rabbit antibodies antibody G10 186 187 HA HA ms-Alexa 488 rb-Alexa 594 source mouse rabbit rabbit mouse mouse goat goat dilution 1: 1,000 1: 20,000 1: 7,000 1: 1,000 1: 1,000 1: 1,000 1: 1,000
Table 3.3 Antibodies used for immunouorescence. Primary and secondary antibodies were diluted in blocking buffer (1% BSA in PBS).
4 Results
Most of the experiments were carried out using NIH 3T3 mouse broblasts stably expressing either ApoER2 with ligand-binding repeats 1-3, 7 and 8, containing or lacking the proline-rich cytoplasmic insert (3T3 A+, 3T3 A-) or VLDLR lacking the O-linked sugar domain (3T3 V-) or co-expressing one of the receptors and Dab1 (3T3 A+/D, 3T3 A-/D, 3T3 V-/D). This cell model was shown to mimic the crucial events of Reelin signaling as they were observed in neurons, namely ligand-induced phosphorylation of Dab1, subsequent phosphorylation of PKB/Akt, and degradation of phosphorylated Dab1 [Mayer et al., 2006]. For certain experiments, transiently transfected NIH 3T3 cells expressing fusion proteins of ApoER2 containing the cytoplasmic insert (ApoER2-HA) or VLDLR (VLDLRHA) with C-terminal hemagglutinin (HA) were used. The C-terminal tag allows detection of the intracellular receptor fragments produced by secretase-mediated cleavage using an antibody against HA. Since no suitable antibody against the intracellular domain of VLDLR was available, use of the tagged receptors was necessary for the study of receptor fragmentation by secretases. Furthermore, use of the HA-receptors and the corresponding anti-HA antibody constitutes a control for consistent transfection efciencies since amounts of full-length receptors expressed by transfected cells can be directly compared. Selected experiments were also done with primary rat neurons to verify the results in a physiological situation.
4.1 Localization of ApoER2 and VLDLR within the plasma membrane

As described previously [Mayer et al., 2006], ApoER2 is localized to rafts whereas VLDLR is strictly excluded from these microdomains. To test if the localization of the receptors within subdomains of the plasma membrane has an inuence on Reelin signaling, the raft disturbing agents Methyl--cyclodextrin (CDX) and
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nystatin (Nys) were used. Both agents disrupt caveolae/lipid rafts by depletion of cholesterol from the membrane [di Guglielmo et al., 2003; Kilsdonk et al., 1995; Simons and Toomre, 2000]. To assure that the amounts of these agents used are sufcient to shift the receptors out of rafts, NIH 3T3 cells expressing ApoER2 were treated with the indicated amounts of either CDX, Nystatin or none of them. The cells were washed with TBS, scraped, and pelleted by centrifugation. Cells were then solubilized in TBS containing 2% Brij 78P and protease inhibitors by passaging 10 times through a 23-gauge needle. The lysate was mixed with equal volume of 90% (wt/vol) sucrose in MBS and overlaid with a discontinous gradient of 35% (wt/vol) and 5% (wt/vol) sucrose in MBS. The samples were centrifuged for 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 an ApoER2-specic antibody. Fractions 2 and 3 at the interface between the 5% and the 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 5 mM CDX (panel 2) or 15 g nystatin (panel 3). Cells were washed 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 were mixed with an equal volume of 90% (wt/vol) sucrose in MBS and overlaid with a discontinous gradient of 35% (wt/vol) and 5% (wt/vol) sucrose in MBS. The samples were centrifuged for 20h at 160,000g and 9 fractions were collected from the top of the tube. Fractions were analyzed by immunoblotting using an ApoER2 specic antibody (20). Arrowheads point at mature ApoER2 ( ) and ApoER2 precursor ( )
As seen in gure 4.1, ApoER2 is sorted to the raft fraction in untreated cells while its non-glycosylated precursor which resides in the membrane of the endoplasmic reticulum can be detected in the non-caveolae fraction (panel 1). Upon addition of CDX, mature ApoER2 can only be detected in NCM fractions which indicates efcient disruption of rafts in the plasma membrane (panel 2). The effect of nystatin was less pronounced; it caused only a slight reduction of CLM-associated
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ApoER2 (panel 3). Since higher concentrations of nystatin caused detachment of the cells and cell death, the effect of this agent could not be increased. Previous experiments demonstrated that in contrast to ApoER2, VLDLR is strictly excluded from the CLM fraction [Mayer et al., 2006]. Furthermore, it was shown that membrane localization of both ApoER2 and VLDLR does not change upon Reelin stimulation in NIH 3T3 A-/D and NIH 3T3 V-/D cells [Mayer et al., 2006]. Since ApoER2 and VLDLR are equally potent to induce Dab1 phosphorylation, these results suggested that the localization of the receptors to different subdomains of the plasma membrane does not inuence the generation of the primary Reelin signal. To verify this hypothesis, the effect of the raft-disrupting agents CDX and nystatin on Reelin-induced Dab1 phosphorylation was examined. Briey, 3T3 A+/D and 3T3 V-/D cells were incubated for 1 h in serum free medium to starve the cells and thereby reduce the background phosphorylation to a minimum level. Subsequently, the cells were incubated with either Reelin conditioned medium (RCM), mock conditioned medium (MCM) or RCM containing CDX or nystatin for 1 h. The cells were washed with TBS and lysed in Hunt buffer containing protease and phosphatase inhibitors. The cleared cell lysates were immunoprecipitated with an antibody against Dab1 and the resulting precipitate subjected to reducing SDS-PAGE and Western Blot using antibodies against phosphorylated tyrosine and Dab1.
Figure 4.2 Reelin-induced phosphorylation of Dab1 is independent of receptor localization to rafts. NIH 3T3 cells expressing Dab1 and either ApoER2 (3T3 A+/D, panel A) or VLDLR (3T3 V-/D, panel B) were starved for 1 h in DMEM and incubated for 1 h with Reelin conditioned medium (RCM) or mock conditioned medium (MCM). To examine a possible inuence of receptor localization on Dab1 phosphorylation, RCM was supplemented with 5 g/ml or 15 g/ml nystatin (Nys; lanes 3 and 4) or 1 mM or 2 mM methyl--cyclodextrin (CDX; lanes 5 and 6). Dab1 was immunoprecipitated from cleared cell lysates. Western blots of the immunoprecipitates were probed against phosphotyrosine (PY) and Dab1 (D4).
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As shown in gure 4.2, addition of different concentrations of CDX (lanes 5 and 6) or nystatin (lanes 3 and 4) did not reduce the amount of phosphorylated Dab1 in both cell lines expressing ApoER2 (A) or VLDLR (B). Since equal amounts of total Dab1 throughout all samples were loaded on the gel, direct comparison of the phosphorylation levels was possible. This result showed that the presence or functional integrity of lipid rafts is not required for Reelin-induced Dab1 phosphorylation.
4.2 Reelin internalization by ApoER2 and VLDLR

To study the kinetics of Reelin uptake by ApoER2 and VLDLR, NIH 3T3 cells expressing either of the receptors were pre-cooled and incubated with RCM for 1 h at 4C. The cells were either washed and lysed immediately or incubated with OptiMEM at 37C for a dened time period prior to lysis. Samples were analyzed by immunoblotting using an antibody against Reelin. At 4C, Reelin is bound to the receptors expressed at the cell membrane but not internalized. When shifted to 37C, Reelin becomes internalized and subsequently degraded. Therefore, the amount of cell associated Reelin after different time periods at 37C was analyzed to determine the rate of Reelin internalization. Figure 4.3 shows that cells expressing VLDLR (V-) internalize and degrade Reelin much faster than cells expressing ApoER2 (A+).
Figure 4.3 Different Reelin internalization rates. NIH 3T3 cells expressing ApoER2(+) (A+) or VLDLR(-) (V-) were pre-cooled and incubated with RCM for 1 h on ice. The cells were either washed and lysed immediately or incubated with OptiMEM at 37C for 12.5, 25, 37.5, 50 or 62.5 min prior to lysis. Samples were analyzed by reducing SDS-PAGE and immunoblotting for Reelin (G10).
To conrm this result, an alternative approach was used. The faster Reelin is internalized and degraded by the cells, the faster the Reelin amount in the medium is decreased. Therefore, analysis of Reelin levels in the medium after different time periods of incubation can be used to study uptake kinetics. Briey, NIH 3T3 cells expressing HA-tagged ApoER2 or VLDLR were incubated with a dened volume of Reelin conditioned medium at 37C. After 8, 24, and 32 h of incubation, small aliquots of the medium were analyzed. Samples were subjected to
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reducing SDS-PAGE and Western blotting using the monoclonal Reelin antibody G10. Furthermore, cells were lysed after 32 h, cell extracts separated by reducing SDS-PAGE and analyzed by immunoblotting to measure the amounts of receptor expressed in the respective cell lines. Figure 4.4 (A) shows that Reelin present in the medium is taken up and degraded signicantly faster by cells expressing VLDLR than by cells expressing ApoER2. To correlate the loss of Reelin from the medium with the amounts of ApoER2 or VLDLR expressed by the cells, Reelin decrease during 24 h was normalized to the respective receptor levels. Figure 4.4 (B) shows normalized Reelin amounts in the medium at the last time point. During 24 h of incubation, ApoER2 expressing cells decreased the Reelin concentration in the medium to approximately 52% while in the medium of VLDLR expressing cells only about 10% of the initial Reelin amount remain.
Figure 4.4 Receptor-mediated Reelin depletion from the medium. NIH 3T3 cells expressing HA-tagged ApoER2 or VLDLR were incubated with a dened volume of Reelin conditioned medium at 37C. Medium samples were taken after 8, 24, and 32 h and cells were lysed in Hunt buffer after 32 h. All samples were subjected to reducing SDS-PAGE and Western blotting using the monoclonal anti-Reelin antibody G10 for samples of the medium or HA to detect ApoER2 or VLDLR in the cell lysates. (A) Diagram of Reelin decrease in the medium of receptor-expressing cells. Western blots of two Reelin depletion experiments were quantied using Gel-Pro Analyzer. Average values of the integrated optical density (IOD) of the bands were normalized to the Reelin amount at the rst time point. (B) Reelin depletion during 24 h. The decrease of Reelin in the medium during 24 h was calculated from the difference of Reelin IODs at the rst and last time point. Reelin decrease was calculated for ApoER2- or VLDLR-expressing cells, and normalized to the respective amounts of receptor expressed by the cells.
For the determination of the amount of Reelin in the medium, only bands corresponding to full-length Reelin were quantied. In the analyzed samples of the medium however, not only full length Reelin was detected, but also bands corresponding to a Reelin fragment running slightly below the full-length protein on
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SDS-PAGE gels [Lambert de Rouvroit et al., 1999]. Interestingly, the Reelin fragment was found to accumulate in the medium in the presence of cells expressing ApoER2 while incubation of Reelin with VLDLR expressing cells did not lead to a relative increase of the amount of the fragment (gure 4.5 (A)). Figure 4.5 (B) shows the amounts of the Reelin fragment produced in ApoER2 an VLDLR expressing cells, normalized to the respective receptor amounts. During 24 h, the amount of the Reelin fragment in the medium is increased to more than 400% while VLDLR does not signicantly alter the fragment amount.
Figure 4.5 Reelin fragmentation. NIH 3T3 cells expressing HA-tagged ApoER2 or VLDLR were treated and analyzed as described for gure 4.4. (A) Accumulation of Reelin fragment in the medium of receptor-expressing cells. Western blots of two experiments were quantied using Gel-Pro Analyzer. Average values of the integrated optical density (IOD) of the bands were normalized to the amount of the Reelin fragment at the rst time point. (B) Accumulation of Reelin fragment during 24 h. The accumulation of Reelin fragment in the medium during 24 h was calculated from the difference of Reelin fragment IODs at the rst and last time point. Reelin fragment accumulation was calculated for ApoER2- or VLDLR-expressing cells, and normalized to the respective amounts of receptor expressed by the cells.
To determine which parts of the receptors are responsible for their respective endocytosis rate, 3T3 cells expressing chimeric receptors were used. Chimeric receptors were constructed by Harald Mayer [Mayer , 2005] by swapping intracellular, transmembrane and extracellular domains between ApoER2 and VLDLR (gure 4.6). The resulting receptors comprise Ae Atm Vi (derived from ApoER2 by replacing its cytoplasmic tail with that of VLDLR), Ae Vtm Vi (derived from VLDLR by replacing its extracellular domain with the corresponding ApoER2 domain), and vice versa, Ve Vtm Ai(+) and Ve Atm Ai(+) [Mayer , 2005].
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Figure 4.6 Chimeric receptors of ApoER2 and VLDLR. VLDLR (shown in blue) and ApoER2 (shown in orange) and chimeric receptors derived from them (shown in orange and blue, according to the domain composition). Chimeric receptors were derived from ApoER2 containing the proline-rich insert (ApoER2(+)) and VLDLR lacking the O-linked sugar domain (VLDLR(-)). The chimeric receptors of ApoER2 and VLDLR with swapped cytoplasmic tails are Ae Atm Vi and Ve Vtm Ai(+) . Ae Vtm Vi and Ve Atm Ai(+) are contructed by swapping the extracellular domains of ApoER2(+) and VLDLR(-).
NIH 3T3 cells expressing ApoER2, VLDLR or either of the chimeric receptors were used for Reelin uptake experiments as described for gure 4.3. As shown in gure 4.7, uptake kinetics of Ae Atm Vi and Ae Vtm Vi resembled those of ApoER2(+) (panel A), Ve Atm Ai(+) and Ve Vtm Ai(+) showed endocytosis rates similar to VLDLR (panel B). These results led to the assumption that the extracellular domain of the receptors is regulating their endocytosis rates. To determine if endocytosis of Reelin has a direct effect on the signaling pathway, the potency of endocytosis-inhibited cells to induce Dab1 phosphorylation upon Reelin stimulation was examined. To prevent endocytosis, the cells were shifted to 4C. NIH 3T3 cells co-expressing Dab1 and either ApoER2 or VLDLR were
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Figure 4.7 Reelin internalization by chimeric receptors. NIH 3T3 cells expressing ApoER2(+) or VLDLR(-) or one of the chimeric receptors Ae Atm Vi , Ae Vtm Vi , Ve Vtm Ai(+) , and Ve Atm Ai(+) were used. Cells were treated as described for gure 4.3. Samples were separated by reducing SDS-PAGE and analyzed by immunoblotting for Reelin using G10.
starved for 1 h in serum-free medium and incubated for 1 h in RCM or MCM at either 4C or 37C. Dab1-immunoprecipitates of the cell lysates were separated by reducing SDS-PAGE and immunoblotted for detection of phosphotyrosine and Dab1. Figure 4.8 shows that preventing endocytosis by incubating the cells at 4C does not signicantly alter the level of phosphorylated Dab1 at equal levels of total Dab1 in the cells.
Figure 4.8 Reelin-induced phosphorylation of Dab1 is independent of endocytosis. NIH 3T3 cells expressing Dab1 and either ApoER2 (A) or VLDLR (B) were starved for 1 h in DMEM and incubated for 1 h with Reelin conditioned medium (RCM; lanes 1 and 3) or mock conditioned medium (MCM; lanes 2 and 4) at either 37C (lanes 3 and 4) or 4C (lanes 1 and 2). Dab1 was immunoprecipitated from cleared cell lysates and the resulting precipitate was separated by reducing SDS-PAGE. Western blots were probed for phosphotyrosine (PY) and Dab1 (D4).
4.3 Surface expression of ApoER2 and VLDLR

To determine the fraction of the total cellular pool of the receptors located at the cell membrane, a surface biotinylation assay was applied. Briey, NIH 3T3 cells expressing HA-tagged versions of either ApoER2 or VLDLR were shifted to 4C to prevent endocytosis. After pre-cooling the cells for 15 min, they were washed
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and subsequently with Sulfo-NHS-SS-Biotin. Cells were incubated with the agent for 2 h either at 4C or shifted to 37C. Since Sulfo-NHS-SS-Biotin is not able to pass the plasma membrane, only receptors present at the cell surface are biotinylated. When cells are shifted to 37C, receptors are internalized and recycle to the cell surface. Thus, all receptors within a cell are exposed to biotinylation within 2 h. After the incubation time, the cells were lysed and mechanically disrupted by passaging them 10 times through a 23-gauge needle. Cleared lysates were subjected to immunoprecipitation with neutravidin beads to extract biotinylated receptors from the total receptor pool of the cell. The samples were analyzed by immunoblotting detecting the HA-tag of the receptors. Biotinylated receptors detected in cells incubated at 4C represent the receptor pool present at the cell surface at steady state conditions. Samples derived from cells incubated at 37C for 2 h were used as a reference since all receptors present in a cell are biotinylated under these conditions. As shown in gure 4.9, the receptor fractions present at the cell membrane are equal to the respective total amounts of both ApoER2 (lanes 1 and 2) and VLDLR (lanes 3 and 4), suggesting that both receptors are preferentially present on the cell surface. In the case of ApoER2 however, two bands were detected. The lower band corresponds to unglycosylated precursor, localized at the membrane of the endoplasmic reticulum. The higher band represents the glycosylated mature form of ApoER2, localized at the cell surface. Since Sulfo-NHS-SS-Biotin is not able to pass the plasma membrane, it should not be possible to biotinylate and subsequently precipitate and detect the ApoER2 precursor. In the case of VLDLR, the band for the biotinylated precursor is much weaker.
Figure 4.9 Subcellular distribution of ApoER2 and VLDLR. NIH 3T3 cells expressing HA-tagged versions of either ApoER2 (lanes 1 and 2) or VLDLR (lanes 3 and 4) were incubated for 2 h with Sulfo-NHS-SS-Biotin at 4C (lanes 1 and 3) or 37C (lanes 2 and 4). Cells were lysed and mechanically disrupted by passaging 10 times through a 23-gauge needle. Cleared lysates were subjected to immunoprecipitation with neutravidin beads to extract biotinylated receptors. Samples were analyzed by immunoblotting using HA antibody.
The subcellular distribution of ApoER2 and VLDLR was also examined by immunouorescence microscopy using primary rat neurons as well as transiently transfected NIH 3T3 cells expressing HA-tagged ApoER2 and VLDLR. Transient
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transfection was chosen for the rst experiments because untransfected cells always present in such cultures constitute an intrinsic control for the specicity of the antibodies. The cells were cultivated and transfected on coated glass coverslips. The slides were either coated with poly-L-lysine for the cultivation of NIH 3T3 cells or with poly-L-ornithine for the use with primary neurons. The cells were washed with PBS, xed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X100. Cells were immunostained for ApoER2 or VLDLR using the antibodies 186 and 187, respectively, and a secondary Alexa Fluor 594 labeled goat anti-rabbit antibody. For detection of the receptors by their HA-tags, HA and a secondary Alexa Fluor 488 labeled goat anti-mouse antibody were used. As shown in gure 4.10, both ApoER2 and VLDLR could be mostly detected at the cell surface in the broblast cell system (b, c, e, f). In primary neurons, ApoER2 shows the same subcellular distribution as seen in 3T3 cells (a). Localization of VLDLR in primary neurons however, was not as clear (d).
Figure 4.10 Surface expression of ApoER2 and VLDLR. Primary rat neurons (a and d) and NIH 3T3 cells expressing HA-tagged ApoER2 (b and c) or VLDLR (e and f) were cultured on coated glass coverslips, washed with PBS, xed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X100. Cells were immunostained for ApoER2 or VLDLR using the antibodies 186 (a and b) and 187 (d and e), respectively, and a secondary Alexa Fluor 594 labeled goat anti-rabbit antibody or stained for the HA-tag of the receptors using HA (c and f) and a secondary Alexa Fluor 488 labeled goat anti-mouse antibody.
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Furthermore, immunouorescence microscopy was used to study the distribution of ApoER2 upon Reelin stimulation. For this purpose, NIH 3T3 broblasts expressing HA-tagged ApoER2 were pre-cooled for 15 min at 4C and incubated with a dened volume of ice-cold Reelin conditioned medium for 1 h. Subsequently, the medium was removed, the cells were overlaid with a dened volume of OptiMEM and incubated for 10 min at either 37C or 4C. The cells were xed and permeabilized as described before and stained using antibodies against ApoER2, VLDLR, and Reelin.
Figure 4.11 Trafcking of ApoER2 upon Reelin stimulation. NIH 3T3 broblasts expressing HA-tagged ApoER2 were cultured on poly-L-lysine coated glass coverslips. Prior to staining, the cells were cooled for 15 min at 4C and incubated with ice-cold Reelin-conditioned medium for 1 h. The medium was removed, the cells were overlaid with a dened volume of OptiMEM and incubated for 10 min at either 37C or 4C. The cells were xed and permeabilized as described for gure 4.10 and stained using antibodies against ApoER2 (186) and Reelin (G10). 186 was detected using a secondary Alexa Fluor 594 labeled goat anti-rabbit antibody; G10 was detected by a secondary Alexa Fluor 488 labeled goat anti-mouse antibody.
As shown in gure 4.11, cells kept at 4C were able to bind but not to internalize Reelin. In this case, Reelin can be detected at the plasma membrane (b). ApoER2 is also present at the cell surface (a) and colocalizes with Reelin (c). When cells were incubated at 37C for 10 min, endocytosis of Reelin occurs. Reelin is removed completely from the cell surface and accumulates within
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the cell (e). Interestingly, ApoER2 no longer colocalizes with Reelin (f) but is still detected on the cell surface to a large amount (d). In respect to VLDLR, no consistent data about subcellular distribution upon Reelin stimulation could be obtained yet. To study the inuence of Dab1 on endocytosis of Reelin and subcellular distribution of ApoER2, the assay was repeated using NIH 3T3 cells expressing Dab1 and HA-tagged ApoER2. The cells were treated and analyzed as described above. As demonstrated in gure 4.12, co-expression of Dab1 does not alter the subcellular distribution of ApoER2 and endocytosis of Reelin. Again, Reelin binds to the cell at 4C (b), thereby colocalizing with ApoER2 (c). Shifting the cells to 37C resulted in endocytosis and accumulation of Reelin within the cell (e) and unaffected ApoER2 localization to the plasma membrane (d).
Figure 4.12 Trafcking of ApoER2 in the presence of Dab1. NIH 3T3 broblasts expressing Dab1 and HA-tagged ApoER2 were cultured and treated as described for gure 4.11. Cells were stained using antibodies against ApoER2 (186) and Reelin (G10). 186 was detected using a secondary Alexa Fluor 594 labeled goat anti-rabbit antibody; G10 was detected by a secondary Alexa Fluor 488 labeled goat anti-mouse antibody.
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4.4 Reelin-induced degradation of ApoER2

It was recently observed that ApoER2 but not VLDLR is degraded upon Reelin stimulation in NIH 3T3 broblasts [Mayer et al., 2006]. To examine if this degradation is induced by receptor clustering, the effect of an antibody against the ligand binding domain of ApoER2 (186) on the receptor was studied. This antibody was previously demonstrated to cluster ApoER2 and to induce Dab1 phosphorylation in primary neurons [Strasser et al., 2004] and broblasts [Mayer et al., 2006]. Briey, NIH 3T3 cells expressing ApoER2 were starved for 1 h in DMEM. In addition, the medium was supplemented with cycloheximide to prevent protein synthesis. Subsequently, the cells were incubated for 6 h in OptiMEM containing cycloheximide and varying concentrations of 186. As negative control, the unspecic antibody 20 which is directed against the intracellular tail of ApoER2 was used. Furthermore, a fusion protein of ApoER2 and maltose binding protein (MBP-ApoER2) was used to compete with cellular ApoER2 for 186 binding which should result in an inhibition of ligand induced degradation. Moreover, cells were stimulated with the monovalent ligand RAP which is not able to induce receptor clustering. Cell were lysed in Hunt buffer and lysates were separated by reducing SDS-PAGE. Western blots were probed with 20 to detect ApoER2 and with Lis1 to detect endogenous Lis1 as a loading control.
Figure 4.13 Multivalent ligands induce degradation of ApoER2. NIH 3T3 cells expressing ApoER2(-) were incubated for 1 h in DMEM containing cycloheximide. Subsequently, the cells were incubated for 6 h in OptiMEM containing cycloheximide and different ligands. Western blots of the cell lysates were probed with 20 to detect ApoER2 and with Lis1 to detect endogenous Lis1 as a loading control. (A) Cells were treated with varying concentrations of 186 (lanes 25), 20 (lane 1) or 186 and 200 g/ml MBP-ApoER2 (lane 6). (B) Cells were treated with 20 (lane 1), 186 (lane 2), or 100 g/ml RAP (lane 3)
As shown in gure 4.13 (A), the antibody 186 is able to mimic the effect of Reelin and induces degradation of ApoER2. This effect is enhanced by increased antibody concentrations (lanes 2-5) and is inhibited by addition of MBP-ApoER2
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as competitor for antibody-binding (lane 6). Neither the unspecic antibody 20 (lane 1) nor the monovalent ligand RAP (B, lane 3) induce receptor degradation . The experiment was also carried out with primary neurons to conrm that the effect of 186 induced ApoER2 degradation is not a unique feature of the broblast cell system. To maximize the effect of 186, primary rat neurons were incubated for 24 h with the antibody. As controls, neurons were incubated with Reelin conditioned medium (RCM) or mock conditioned medium (MCM) for the same time period. All media were supplemented with cycloheximide and were changed after 8 h with all supplements to assure a constant level of all supplements. After 24 h, cells were lysed in Hunt buffer and lysates were subjected to reducing SDSPAGE and immunoblotting using 20 and Lis1. Figure 4.14 shows that both Reelin and 186 induced degradation of ApoER2 to similar extents (lanes 1 and 3) when compared with the receptor level present when neurons were incubated with MCM (lane 2).
Figure 4.14 Multivalent ligands induce degradation of ApoER2 in neurons. Primary rat neurons were incubated for 24 h with Reelin conditioned medium (RCM; lane 1), mock conditioned medium (MCM; lane 2) or MCM containing 186 (lane 3). All media were supplemented with cycloheximide and were changed to fresh medium containing the same supplements after 8 h. Samples were analyzed as described for gure 4.13.
To determine which degradation pathway is responsible for ligand induced ApoER2 loss, NIH 3T3 cells expressing ApoER2 were starved for 1 h in DMEM containing cycloheximide and incubated for 6 h in RCM or MCM, containing cycloheximide or RCM or MCM containing cycloheximide and either chloroquine (CQ), ammonium chloride, or MG132. Cells were lysed in Hunt buffer and cell extracts were subjected to reducing SDS-PAGE and immunoblotting using 20 and Lis1. Chloroquine and ammonium chloride inhibit the function of the lysosome [de Duve et al., 1974; Lie and Schoeld , 1973], whereas MG132 interferes with proteasomal degradation [Lee and Goldberg , 1998]. As demonstrated in gure 4.15 (A), ApoER2 was degraded upon Reelin stimulation in the presence of MG132 (lane 6), while addition of chloroquine (lane 4) or ammonium chloride (lane 2) led to inhibition of ApoER2 degradation. Panel (B) shows degradation of ApoER2 in Reelin-stimulated cells without lysosome- or proteasome inhibitor treatment. This result indicates that the receptor is degraded via the lysosomal pathway upon Reelin stimulation.
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Figure 4.15 Reelin stimulation induces lysosomal degradation of ApoER2. NIH 3T3 cells expressing ApoER2(-) were starved for 1 h in DMEM containing cycloheximide and incubated for 6 h in conditioned medium containing cycloheximide. Cells were lysed in Hunt buffer and lysates were subjected to reducing SDS-PAGE and immunoblotting using 20 and Lis1. (A) Cells were incubated with Reelin conditioned medium containing cycloheximide and either 25 M chloroquine (CQ; lane 4), 10mM ammonium chloride (NH4 Cl; lane 2), or 25 M MG132 (lane 6) or with mock conditioned medium containing the same supplements (lanes 1, 3, and 5). (B) Cells were incubated with mock conditioned medium (lane 1) or Reelin conditioned medium (lane 2), both containing cycloheximide.
To determine if lipid rafts have a critical inuence on the degradation of ApoER2, NIH 3T3 cells expressing ApoER2 were treated with methyl--cyclodextrin or Nystatin. Cells were starved in DMEM containing cycloheximide for 1 h and subsequently incubated with Reelin conditioned medium or mock conditioned medium both supplemented with cycloheximide and containing or lacking methyl-cyclodextrin or nystatin, respectively. Cell extracts were prepared in Hunt buffer and separated by reducing SDS-PAGE. Western blots were probed with 20 to detect ApoER2 and Lis1 to detect endogenous Lis1. As shown in gure 4.16, neither methyl--cyclodextrin (A) nor nystatin (B) inhibited degradation of ApoER2.
Figure 4.16 Raft-disrupting agents do not interfere with ApoER2 degradation. (A) NIH 3T3 cells expressing ApoER2(+) were starved in DMEM containing cycloheximide for 1 h and subsequently incubated with Reelin conditioned medium or mock conditioned medium supplemented with cycloheximide and containing or lacking methyl--cyclodextrin for 6 h. Cell extracts were prepared in Hunt buffer and separated by reducing SDS-PAGE. Western blots were probed with 20 to detect ApoER2 and Lis1 to detect endogenous Lis1. (B) NIH 3T3 cells expressing ApoER2(-) were starved in DMEM containing cycloheximide for 1 h and subsequently incubated with RCM or MCM supplemented with cycloheximide and containing or lacking nystatin. Cell extracts were analyzed as described for (A).
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4.5 Reelin-induced fragmentation of ApoER2

As reported recently, the Reelin receptors are substrates for a specic, ligand induced cleavage by secretases. By the action of -secretase, an extracellular soluble receptor fragment is produced [Hoe and Rebeck , 2005]. The remaining membrane bound intracellular fragment with a molecular weight of about 25 kDa is subsequently processed by -secretase [May et al., 2003]. To further investigate the initiation of the cleavage, NIH 3T3 cells expressing ApoER2 containing or lacking the cytoplasmic proline rich insert were incubated with mono- or multivalent ligands for 5 h. Cells were lysed in Hunt buffer and subjected to reducing SDS-PAGE and immunoblotting using 20 for ApoER2(+) and 19 for ApoER2(-) fragments. As loading control, caveolin1 or Lis1 were used to detect endogenous proteins. As shown in gure 4.17, only the multivalent ligands Reelin and 186 but not the monovalent ligand RAP were able to induce ApoER2(+) cleavage (A and B). Negative controls using MCM, the unspecic antibody 20, or competition by soluble MBP-ApoER2 inhibited the production of the membranebound intracellular fragment. Panel (C) shows the specic cleavage of ApoER2(-), demonstrating that the proline-rich insert in the cytoplasmic domain does not inhibit this process. Since different antibodies had to be used for the detection of ApoER2(+) and ApoER2(-) fragments, the amounts of the produced fragments can only be compared qualitatively. From these experiments it is obvious that both variants of the receptor are subjected to cleavage.
Figure 4.17 Multivalent ligands induce fragmentation of ApoER2. NIH 3T3 cells expressing ApoER2 containing (A and B) or lacking (C) the cytoplasmic proline rich insert were incubated for 5 h with RCM, MCM, MCM containing 20 g/ml RAP, 20, 186, or 186 and 200 g/ml MBPApoER2. Cells were lysed in Hunt buffer and subjected to reducing SDS-PAGE and immunoblotting using 20 for ApoER2(+) and 19 for ApoER2(-). As loading control, caveolin1 (A and B) or Lis1 (C) were used to detect endogenous proteins.
Since no suitable antibody against the cytoplasmic tail of VLDLR was available, VLDLR could not be detected by this assay. Thus, HA-tagged versions of both receptors were used which allowed detection of both ApoER2 and VLDLR and com-
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parison of the amounts of fragment produced. The -secretase inhibitor DAPT was used to robustly detect the fragments produced by -secretase. Extracellular cleavage of the receptors by -secretase produces membrane-bound intracellular fragments of the receptors. Inhibiting subsequent cleavage by -secretase and degradation of the produced unstable intracellular fragment leads to accumulation of the membrane bound fragment. NIH 3T3 cells expressing either of the tagged receptors were incubated for 6 h with Reelin conditioned medium or RCM containing DAPT. Subsequently the cells were lysed in Hunt buffer and the samples immunoblotted using HA antibody directed against the C-terminal tag of the receptors. As demonstrated in gure 4.18, VLDLR (lanes 3 and 4) produces very little membrane-bound intracellular fragment in comparison to ApoER2 (lanes 1 and 2). This is consistent with earlier investigations reporting that fragments of VLDLR are consistently harder to detect than those of ApoER2 [Hoe and Rebeck , 2005]. In both cases, the amounts of the respective fragments are increased by addition of DAPT (lanes 2 and 4).
Figure 4.18 The -secretase inhibitor DAPT causes accumulation of the membrane-bound intracellular fragments of ApoER2 and VLDLR. NIH 3T3 cells expressing HA-tagged ApoER2 (lanes 1 and 2) or VLDLR (lanes 3 and 4) were incubated for 6 h with Reelin conditioned medium (lanes 1 and 3) or RCM containing 10 M DAPT (lanes 2 and 4). Cells were lysed in Hunt buffer, the samples were separated by reducing SDSPAGE and immunoblotted using HA antibody.
To conrm the assumption that the intracellular fragment accumulating in the presence of DAPT is indeed membrane bound, four dishes of NIH 3T3 A+/D cells were incubated with 186 to induce fragmentation or with MCM as a negative control. After 6 h of incubation, one dish each containing stimulated and unstimulated cells were processed further and the cells were lysed in Hunt buffer. The cells of the other two dishes were lysed in CAV-A buffer (no detergent) and subjected to mechanical disruption and ultracentrifugation for 90 min. Proteins of all samples were separated by reducing SDS-PAGE and immunoblotted to detect full length ApoER2 and its cleavage products. Since cells expressing GFP-tagged Dab1 were used, GFP could be used as a loading control. Cell extracts derived from lysis in Hunt buffer should contain the total protein pool of the cell, including membrane-bound and cytosolic fragments of ApoER2. CAV-A extracts contain only the cytoplasmic protein fraction. As shown in gure 4.19, full length ApoER2
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is detected in both stimulated and unstimulated cells but can only be detected in the corresponding Hunt extracts (lanes 2 and 4). Also the intracellular fragment of ApoER2 was detected in Hunt extracts (lanes 2 and 4) but not in CAV-A extracts (lanes 1 and 3) which demonstrated that the intracellular fragment accumulating in the presence of DAPT is membrane bound. As expected, more of the fragment is produced in stimulated than in unstimulated cells.
Figure 4.19 The intracellular fragment of ApoER2 produced by -secretase is bound to the plasma membrane. Four dishes of NIH 3T3 A+/D cells were incubated with mock conditioned medium (lanes 1 and 2) or MCM containing 186 (lanes 3 and 4) for 6 h. One dish each of 186- and MCM-treated cells was lysed in Hunt buffer (lanes 2 and 4). The other dishes were lysed in CAV-A buffer and subjected to mechanical disruption and ultracentrifugation for 90 min (lanes 1 and 3). All samples were separated by reducing SDS-PAGE and immunoblotted using 20 to detect full length and cleaved ApoER2 and GFP to detect the GFP tag of Dab1 as a loading control.
As demonstrated above, DAPT leads to accumulation of the membrane bound intracellular ApoER2 fragment. In the next step, chloroquine was added to inhibit a possible lysosomal degradation of the soluble intracellular fragment produced by -secretase cleavage. Briey, NIH 3T3 A+/D cells were incubated for 5 h with OptiMEM containing either 186 or 74, and containing or lacking chloroquine. 186 is able to induce fragmentation of ApoER2 whereas 74 serves as a negative control. Cells were lysed in Hunt buffer and the samples were immunoblotted using 20 antibody to detect the intracellular fragment of ApoER2. Endogenous Lis1 was used as a loading control. Addition of chloroquine did not cause an accumulation of the soluble fragment (not shown). The experiment was not yet repeated with MG132 to inhibit proteasomal degradation. Interestingly, in the presence of chloroquine an increase of the membrane-bound intracellular fragment produced by the action of -secretase could be detected as shown in gure 4.20. This can be explained by higher receptor levels due to a lack of unspecic degradation as shown above in gure 4.15 which provides more substrate for secretase cleavage, resulting in more fragment.
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Figure 4.20 Chloroquine causes accumulation of the membrane-bound intracellular fragment. NIH 3T3 cells expressing ApoER2(+) and Dab1 were incubated for 5 h with OptiMEM containing either 186 or 74, and containing or lacking chloroquine. Cells were lysed in Hunt buffer, the samples were separated by reducing SDS-PAGE and immunoblotted using 20 antibody. Lis1 was detected using Lis as a loading control.
To study the importance of functional rafts for secretase-mediated receptor fragmentation, NIH 3T3 cells expressing ApoER2 were incubated with RCM or MCM either with or without addition of methyl--cyclodextrin. After 5 h of incubation, the cells were lysed in Hunt buffer and the cleared lysates were analyzed by SDSPAGE and subsequent Western blotting using the antibody 20. Figure 4.21 depicts the inuence of CDX on the fragmentation of ApoER2. Comparing accumulation of the membrane-bound intracellular fragment of ApoER2 upon Reelin stimulation with and without raft disruption by CDX (lanes 2 and 4), demonstrated that the amount of the fragment is signicantly decreased in the absence of functional rafts.
Figure 4.21 Methyl--cyclodextrin inhibits secretase-mediated fragmentation of ApoER2. NIH 3T3 A(+) cells were incubated with RCM (lanes 2 and 4) or MCM (lanes 1 and 3) either with or without addition of methyl--cyclodextrin. After 5 h of incubation, the cells were lysed in Hunt buffer and the cleared lysates were analysed by SDS-PAGE and subsequent Western blotting using the antibody 20. Endogenous caveolin 1 was used as loading control.
Since Dab1 recruits the Reelin receptors to the cell surface [Morimura et al., 2005], the inuence of Dab1 on ApoER2 cleavage was studied. Briey, HEK 293 cells were transfected with ApoER2(+) or ApoER2(-) constructs and either a Dab1 construct or an empty vector as mock transfection. 48 h after transfection, the cells were incubated for 5 h with 186 or 20. Cell lysates were prepared in Hunt buffer and subjected to reducing SDS-PAGE and immunoblotting. The blots were probed with 20 to detect the membrane-bound intracellular fragment of ApoER2(+), 19 for detection of the corresponding fragments derived from ApoER2(-), and Lis1 as endogenous loading control. Figure 4.22 illustrates that co-expression of Dab1 increases fragmentation of both ApoER2 containing (A) or lacking the cytoplasmic insert (B).
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Figure 4.22 Presence of Dab1 enhances secretase-mediated ApoER2 fragmentation. HEK 293 cells were transfected with pMSCVpuro-ApoER2(+) or pMSCVpuro-ApoER2(-) and either pfMSCV-IRES-GFP-Dab1 or pfMSCV-IRES-GFP. 48 h after transfection, the cells were incubated for 5 h with 186 (lanes 1 and 3) or 20 (lanes 2 and 4). Cell lysates were prepared in Hunt buffer and subjected to reducing SDS-PAGE and immunoblotting. Blots were probed with 20 to detect intracellular fragment of ApoER2(+), 19 for detection of ApoER2(-) derived fragment, and Lis1 as endogenous loading control.
To examine a possible inuence of endocytosis on secretase-mediated ApoER2 cleavage, NIH 3T3 A+/D cells were incubated for 5 h with OptiMEM containing either 186 or 74 at 4C or 37C. Subsequently, the cells were lysed in Hunt buffer, samples were separated by reducing SDS-PAGE and analyzed by immunoblotting using 20 antibody to detect the intracellular fragment of ApoER2. Lis1 was used as a loading control. As shown in gure 4.23, fragmentation of ApoER2 is strictly inhibited in cells at 4C.
Figure 4.23 Fragmentation of ApoER2 does not occur at 4C. NIH 3T3 cells expressing ApoER2 and Dab1 were incubated for 5 h with OptiMEM containing either 186 or 74 at 4C or 37C. Cells were lysed in Hunt buffer and the samples were separated by reducing SDS-PAGE and immunoblotted using 20 antibody. Lis1 was detected as a loading control.
5 Discussion
Members of the LDLR family have originally been described as receptors involved in endocytosis of macromolecules [Hussain et al., 1999]. The LDLR, the best studied member so far, binds and internalizes LDL particles, which subsequently dissociate from the receptor within the endosomes and is transported to the lysosome [Davis et al., 1987]. The receptor can be recycled back to the plasma membrane and internalized cholesterol present as LDL can be utilized by the cell. Recently, many other functions of LDLR family members distinct from endocytosis have been discovered [Herz et al., 2000]. Two receptors of this family, namely ApoER2 and VLDLR, have been shown to be involved in the Reelin signaling pathway. This pathway plays a crucial role in the development of the mammalian central nervous system where it is responsible for the correct positioning of neurons in laminated brain structures. The molecule activating the signaling cascade is Reelin, a large glycoprotein of about 430 kDa forming disulde-linked homodimers under physiological conditions [Kubo et al., 2002; Utsunomiya-Tate et al., 2000]. It is secreted during brain development by Cajal-Retzius cells in the neocortex and external granule cells in the developing cerebellum. During development of the cerebral cortex, Reelin guides the layering of postmitotic neurons migrating from the subventricular zone along radial glial bers to the pial surface of the brain, a process leading to a six-layered structure of the cortex which persists throughout adulthood. A key event triggering the signaling pathway is binding of Reelin to ApoER2 and VLDLR expressed by migrating target neurons [DArcangelo et al., 1999; Hiesberger et al., 1999]. Binding of Reelin dimers leads to clustering of the receptors [Strasser et al., 2004], an event which is required for the subsequent phosphorylation of the intracellular adaptor protein Dab1 [Hiesberger et al., 1999] by Src family kinases [Arnaud et al., 2003b]. While functional Reelin signaling results in the formation of dened neuronal layers, disruption of the pathway leads to disorganization of the neuronal layers, as seen in reeler, scrambler, yotari and ApoER2/VLDLR double knock-out mice.
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5.1 Membrane localization of the Reelin receptors

It was shown that ApoER2, in contrast to LDLR, localizes to caveolae/rafts [Riddell et al., 2001]. Since also Src family kinases (SFK) which play a critical role in the phosphorylation of Dab1 [Arnaud et al., 2003b] can be detected in these microdomains [Anderson, 1998], two of the main components of the intracellular signaling complex responding to Reelin are localized to caveolae/rafts. Furthermore, Dab1 which is located to the plasma membrane by binding phosphoinositides simultaneously to binding transmembrane glycoproteins via its PTB domain [Howell et al., 1999], was also suggested to show a preference for caveolae/rafts. This suggestion is based on the high afnity of Dab1 to phosphatidylinositol4,5-diphosphate (PtdIns4,5P2) which is mainly found in caveolin-rich light membranes [Pike and Casey , 1996]. In agreement with this hypothesis, it was shown in a NIH 3T3 cell model that a signicant fraction of Dab1 is targeted to caveolae/rafts [Mayer , 2005]. Interestingly, studies on VLDLR showed that this receptor is strictly excluded from these microdomains and that this membrane localization is neither altered by binding of Reelin nor is the receptor recruited to lipid rafts by Dab1 [Mayer et al., 2006]. Since ApoER2 and VLDLR are equally potent to induce Dab1 phosphorylation [Strasser et al., 2004], these results suggested that the localization of the receptors to different subdomains of the plasma membrane does not inuence the Reelin signal. The main aim of this thesis was to verify this hypothesis by experiments testing the potency of ApoER2 to phosphorylate Dab1 in the absence of functional lipid rafts. Only low amounts of lipid rafts can be obtained from primary neurons of embryonic mouse or rat brains. Since this has turned out not to be sufcient for analysis of the membrane distribution of the receptors, cell lines based on murine NIH 3T3 broblasts expressing Dab1, VLDLR, ApoER2, Dab1 and VLDLR, and Dab1 and ApoER2 were used [Mayer et al., 2006]. This cell system was shown to mimic the key events of Reelin signaling, i.e. Reelin induced Dab1 phosphorylation, which can also be obtained by stimulation with a receptor clustering antibody and inhibited by treatment with the selective SFK inhibitor PP2 [Arnaud et al., 2003b], and subsequent phosphorylation of PKB/Akt. Previous experiments demonstrated that ApoER2 fractions residing in distinct membrane microdomains of overexpressing NIH 3T3 cells can be separated by density gradient centrifugation based on the light buoyant density of lipid rafts. In a steady state,
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the ApoER2 precursor which resides in the membrane of the endoplasmic reticulum, was detected in non-caveolae membrane (NCM) fractions whereas mature ApoER2 was always found in the caveolin-rich light membrane (CLM) fraction. Addition of the raft disrupting agent methyl--cyclodextrin (CDX) led to a shift of mature ApoER2 into non-caveolae membrane fractions. Indeed, CDX treatment did not alter Dab1 phosphorylation levels in NIH 3T3 cells expressing Dab1 and ApoER2 which conrmed that lipid rafts or caveolae are not necessary structures in the primary signaling event of the Reelin pathway. This is in contrast to previous results reporting CDX to inhibit Reelin induced Dab1 phosphorylation in neurons [Bock et al., 2003]. In those experiments however, overall phosphorylation was also signicantly reduced. Here, a CDX concentration with minimal effect on overall phosphorylation but leading to a complete loss of ApoER2 from the CLM fraction, was used. Since differential sorting of the Reelin receptors obviously has no inuence on the primary signaling event, i.e. phosphorylation of Dab1, it could rather have regulatory effects on the propagation of the signal and the respective cellular response. These mechanism could involve the rate of Reelin endocytosis by ApoER2 and VLDLR, which was shown to depend on the membrane localization of the receptors [Mayer , 2005].
5.2 Reelin endocytosis and degradation

For a long time, members of the LDLR family have been regarded as cell surface endocytosis receptors that function in delivering their ligands to lysosomes for degradation [Hussain et al., 1999]. Closer analysis of the endocytic potential of the receptors of this family using RAP as a ligand revealed striking differences in the uptake kinetics between different members of the family [Li et al., 2001]. These results were consistent with reports suggesting that some of the receptors play a role in signal transduction rather than in endocytosis [Herz et al., 2000]. It was demonstrated that, in comparison to LRP and LRP minireceptors, ApoER2 and VLDLR exhibited a very slow RAP internalization rate [Li et al., 2001]. Direct comparison of the uptake kinetics of the two Reelin receptors in our laboratory showed pronounced differences between endocytosis rates of ApoER2 and VLDLR [Mayer , 2005]. These preliminary results were based on an assay analyzing amounts of cell-bound Reelin. Cells were incubated with equal amounts of Reelin conditioned medium at 4C resulting in binding of Reelin to the surface
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receptors, and subsequently shifted to 37C, allowing internalization and degradation of the ligand. Cell-associated Reelin was degraded signicantly faster in cells expressing VLDLR than in cells expressing ApoER2, indicating a faster internalization rate for VLDLR. Here, these results were conrmed, rst by use of the assay described above, and second, by use of an alternative approach measuring Reelin amounts in conditioned medium during incubation of ApoER2- or VLDLR-expressing cells. These experiments demonstrated that, ApoER2 was indeed not able to internalize Reelin as fast as VLDLR. To examine if the differential endocytosis rates might inuence the primary signaling event, the potency of endocytosis-inhibited cell to phosphorylate Dab1 was studied by phosphorylation assays at 4C. Under these conditions, neither ApoER2 nor VLDLR expressing cells showed changes in Dab1 phosphorylation. Thus, differential endocytosis rates of the Reelin receptors are not a regulator for the efciency of the primary signaling event. The role for these differences might otherwise lie in the clearance of Reelin from the cell surface which would have an regulatory effect on the propagation of the Reelin-induced signal. Fast clearance of Reelin from the cell surface, as seen for VLDLR, could remove the initiating stimulus of the signaling pathway, resulting in a down-regulation of the signal. This hypothesis might explain the differences of brain defects seen in VLDLR versus ApoER2 knockout mice. In histological sections of the neocortex of wild type mice, six neuronal layers can easily be distinguished where layer 1 is normally cell free [Trommsdorff et al., 1999]. In VLDLR knockout mice, layer 1 is also cell free. The remaining layers however, are no longer clearly separated and the neurons are arranged in a radial pattern. The lamination defect is even more pronounced in ApoER2 knockout mice, where it is difcult to distinguish the individual layers 2-6. In contrast to the VLDLR k.o. however, tight horizontal neuronal layers are formed. As ApoER2 k.o. neurons are still expressing VLDLR, Reelin can bind to VLDLR and induce Dab1 phosphorylation. However, as VLDLR shows a very high endocytosis rate for bound Reelin, Reelin might become internalized and degraded very fast. This fast Reelin clearance could lead to insufcient local Reelin concentrations which could partly explain the observation that in ApoER2 knockout mice, the cortical layering defect is more pronounced than in VLDLR knockout mice. In contrast, VLDLR-/- neurons are expressing ApoER2 that can bind Reelin. Endocytosis of Reelin by ApoER2 is slow compared to its uptake by VLDLR, possibly resulting in a prolonged activation of the Reelin signaling
5 Discussion pathway.
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The reason for the differential endocytosis rates of the Reelin receptors may lie in their structural features, their membrane localization, or the endocytic pathway used for their internalization. As described recently, Reelin is internalized via clathrin-mediated endocytosis by both receptors and does not require functional lipid rafts [Cuitino et al., 2005; Mayer , 2005]. Contrary, sorting of ApoER2 to rafts slows Reelin internalization by this receptor so that endocytosis rates for ApoER2 and VLDLR are equal in the absence of lipid rafts [Mayer , 2005]. This suggests that the endocytosis rates of the receptors are regulated by their sorting within the plasma membrane and not by intrinsic features of the receptors. This further suggests that the receptor domain responsible for the sorting of the receptors will also determine their endocytic behaviour. It was shown that inuenza haemagglutinin has the ability to accumulate in caveolin-rich light membranes [Scheiffele et al., 1998] which can be prevented by alterations of the transmembrane domain [Scheiffele et al., 1997]. Furthermore, it was demonstrated that the transmembrane domain of megalin promotes its association with lipid rafts [Marzolo et al., 2003]. Chimeric proteins containing the megalin transmembrane domain were shown to be present in lipid rafts, whereas chimeric proteins comprising the LRP transmembrane domain and the megalin cytoplasmic tail were excluded from rafts. These observations emphasize the importance of the transmembrane domain for raft-association. In contrast, the targeting of the epidermal growth factor receptor was found to be controlled by its extracellular domain [Yamabhai and Anderson, 2002]. Previous experiments with Reelin receptors in our laboratory showed that neither the membrane localization nor the endocytosis rates of ApoER2 and VLDLR are determined by their cytoplasmic tail, since swapping the intracellular domains of the two receptors did not alter their sorting and internalization [Mayer , 2005]. To address the question if the transmembrane or extracellular receptor domain is responsible for the differential endocytosis rates of ApoER2 and VLDLR, chimeric receptors comprising different combinations of extracellular, transmembrane, and intracellular domains of the two receptors were used. Reelin binding and degradation experiments with these receptors clearly demonstrated that the extracellular receptor domain controls the respective endocytosis rates since chimeric receptors always exhibited uptake kinetics resembling that of the wild type receptor providing the extracellular domain. Since raft localization of ApoER2 was demonstrated to be the factor limiting the endocytosis
5 Discussion
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rate of the receptor, it is likely that the effect of the extracellular domain on the differential endocytosis rates is due to its inuence on the differential sorting of the Reelin receptors. Nevertheless, the role of the extracellular domain in controlling receptor localization has to be further investigated to verify this hypothesis. Performing the Reelin depletion experiments which were used to study the receptor-mediated decrease of Reelin from the medium, not only different endocytosis rates but also another interesting difference between ApoER2- and VLDLR-mediated Reelin uptake was observed. Incubation of ApoER2- but not VLDLR-expressing cells, led to the accumulation of a Reelin fragment in the medium. As described previously, Reelin fragments are produced from the fulllength protein by metalloproteinases [Lambert de Rouvroit et al., 1999]. The increased Reelin cleavage in the presence of ApoER2 might be due to the slower endocytosis and prolonged surface presence of Reelin when bound to ApoER2 in comparison to VLDLR. The different Reelin fragments and their functional abilities were studied previously [Jossin et al., 2003]. By the action of metalloproteinase, Reelin is cleaved at two sites, generating ve fragments detectable by antibodies against the N-or C-terminus of Reelin (gure 5.1).
Figure 5.1 Reelin fragments produced by metalloproteinase cleavage. Cleavage by the action of metalloproteinase produces ve fragments detectable by antibodies against the N-or C-terminus of Reelin. Metalloproteinase cleavage sites (indicated by arrowheads) are located between repeats 2 and 3, and 6 and 7.
The fragment accumulating in Reelin conditioned medium in the presence of ApoER2 corresponds to the N-terminal and central part of the protein with a molecular weight of about 300 kDa. It was shown that the central fragment is essential for the function of Reelin and that the fragment comprising the N-terminal and central part of the protein is sufcient for binding of Reelin receptors and inducing Dab1 phosphorylation [Jossin et al., 2003]. Thus, cleavage of Reelin in
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the presence of ApoER2 does not lead to a loss of Reelin function and signaling capacity. However, it is not clear yet what the functions of the fragment are.
5.3 Surface expression of the Reelin receptors

It could be that differential intracellular trafcking of both receptors is the reason for the observed differences in localization and endocytosis rates of the Reelin receptors. To address this question, surface expression of both receptors was analyzed. These experiments were based on surface biotinylation using SulfoNHS-SS-biotin and subsequent precipitation of biotinylated proteins. Since this agent is unable to pass the plasma membrane, only surface bound receptors are biotinylated when endocytosis is inhibited by incubation at 4C. Cells incubated at 37C were used as a reference, since receptor recycling allows biotinylation of the total cellular receptor pool. For both ApoER2 and VLDLR, the entire amount of mature receptor in the cells seemed to be located at the plasma membrane. Surprisingly, a second band, normally corresponding to the unglycosylated precursor of ApoER2, was detected on Western blots. The ApoER2 precursor resides in the membrane of the endoplasmic reticulum (ER), a localization which should exclude it from interaction with Sulfo-NHS-SS-biotin. There are three possible explanations for the presence of the second ApoER2 band: rst, the biotinylation agent can pass the plasma membrane and in consequence labels the receptor precursor resident in the ER. In this case, the results of the surface biotinylation experiments are not valid, since the whole receptor pool in the cell was biotinylated even at 4C. Second, the additional band could be derived from unspecic precipitation. To rule this out, the experiment has to be repeated together with a negative control which is not subjected to biotinylation. Third, there could be another variant of ApoER2 which was not recognized to date, corresponding to the size of the precursor of the receptor. This possibility has to be further investigated. Another attempt to determine the subcellular distribution of the Reelin receptors was based on immunouorescence microscopy. In NIH 3T3 broblast overexpressing either ApoER2 or VLDLR, both receptors were preferentially detected at the cell surface in the absence of Reelin. In primary neurons, localization of endogenous ApoER2 resembled the situation in broblasts. Localization of VLDLR
5 Discussion
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in primary neurons however, was not as clear, since the receptor was partly detected in the cytoplasm of these cells. This result however, could possibly be due to the antibody used which did not always provide consistent staining in the broblast system either. In broblast however, surface expression could be veried by the transient expression of an HA-tagged version of the receptor and the use of an anti-HA antibody. Taken together, immunouorescence experiments point to a preferential surface expression of both ApoER2 and VLDLR. Of course, results concerning VLDLR localization have to be conrmed in neurons to fully approve this hypothesis. In subsequent experiments, it was shown that binding of Reelin to ApoER2 did not alter the localization of the receptor since ApoER2 colocalized with Reelin at the cell surface in cells incubated at 4C. Interestingly, ApoER2 was still detected at the cell membrane after incubation for 10 min at 37C, whereas Reelin accumulated within the cell. Similar experiments with broblasts co-expressing Dab1 and ApoER2 led to the same result. According to these experiments, Reelin dissociates from ApoER2 quickly after internalization and the receptor recycles back to the cell surface. Since it was shown that ApoER2 is degraded upon Reelin stimulation [Mayer et al., 2006], the recycling of the receptor is unlikely. To resolve this discrepancy further experiments by the use of other methods are necessary.
5.4 Reelin-induced degradation and specic cleavage of the receptors

It was observed recently, that levels of ApoER2 but not VLDLR were dramatically decreased upon Reelin stimulation [Mayer et al., 2006]. Here, ligand-induced degradation of ApoER2 was conrmed and could be mimicked by the use of an antibody binding to the ligand binding domain of ApoER2. This antibody has already been shown to induce Dab1 phosphorylation by receptor clustering [Strasser et al., 2004]. Binding of the monovalent ligand RAP and use of an unspecic antibody did not result in receptor degradation demonstrating that the results are specic. The same experiments were also done in primary neurons, leading to similar results. These consistent data clearly demonstrate that ApoER2 is indeed degraded as a consequence of receptor clustering induced by binding of a multivalent ligand. Furthermore, the dependence of receptor loss on the function of lysosomes could be demonstrated, pointing to a lysosomal degra-
5 Discussion
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dation of ApoER2. Since the ligand-induced receptor loss is an exclusive feature of ApoER2, it might be part of the regulatory machinery modulating the initial Reelin signal. As described before (5.2), the differential endocytosis rates of the receptors may provide a partial explanation for the differences in the phenotype of ApoER2 and VLDLR knockout mice. Wile binding of Reelin to ApoER2 leads to slow endocytosis and a prolonged signaling, VLDLR mediates a fast clearance of Reelin from the cell surface and the surrounding environment, resulting in a down-regulation of the pathway. The nding that ApoER2 but not VLDLR becomes degraded upon Reelin binding contributes to this scheme. To assure fast and efcient uptake of Reelin, VLDLR recycles back to the cell surface after degradation of the ligand to bind more Reelin. ApoER2 however, permits a more pronounced activation of the signaling pathway and is subsequently degraded which would prevent unrestricted propagation of the Reelin signal. Besides lysosomal degradation, the Reelin receptors are substrates for specic, ligand-induced cleavage, which might add another level of modulating the Reelin signal. As reported recently, an extracellular soluble receptor fragment is produced by the action of -secretase [Hoe and Rebeck , 2005]. The remaining membrane-bound intracellular fragment is subsequently processed by -secretase [May et al., 2003] to release a soluble intracellular fragment. Here, especially the production and function of the membrane-bound intracellular fragment was studied. Membrane-bound intracellular fragments generated by secretasemediated processing of ApoER2(+), ApoER2(-) and VLDLR could be detected in receptor-overexpressing broblast cell lines. Comparison to fragmentation of HAtagged receptors showed that the amount of fragments derived from ApoER2 is signicantly higher than that derived from VLDLR. This is consistent with earlier publications, reporting that VLDLR fragments were consistently harder to detect [Hoe and Rebeck , 2005]. The amount of fragments produced from ApoER2 variants containing or lacking the proline-rich cytoplasmic insert could not be quantitatively determined since the corresponding fragments had to be detected by the use of different antibodies. Nevertheless, it was shown that both variants are substrates for secretase-mediated cleavage. Furthermore, secretase-mediated cleavage could be induced by multivalent but not monovalent ligands, pointing to a process requiring receptor clustering. The identity of the detected fragment was proven by experiments using the -secretase inhibitor DAPT. As expected, addition of DAPT led to an accumulation of the produced fragment, since further
5 Discussion
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processing was inhibited. Moreover, it was shown that the respective fragment is associated with the plasma membrane. Together, these results clearly demonstrate that the observed fragment is indeed the membrane-bound intracellular fragment resulting from -secretase cleavage and that it is further processed by -secretase. The respective fragment could not be observed in primary neurons to date. The reason for this could be that the amount of the fragment produced in these cells is too small for detection by Western blotting. To further study the membrane-bound intracellular fragment and its production, the inuence of functional lipid rafts and the intracellular adaptor protein Dab1 were examined. When lipid rafts were disrupted using methyl--cyclodextrin, Reelin stimulation led to a signicantly smaller amount of the membrane-bound intracellular fragment than under normal conditions which indicates that the presence of functional lipid rafts greatly facilitates secretase-mediated cleavage. This can possibly explain the inefcient fragmentation of VLDLR, since VLDLR is not located to lipid rafts. Since Dab1 promotes surface expression of ApoER2 and VLDLR [Morimura et al., 2005] and could therefore also contribute to enhanced processing of the receptors, secretase-mediated cleavage in the presence of Dab1 was studied. As expected, Dab1 increased fragmentation of both ApoER2 containing and lacking the cytoplasmic proline-rich insert signicantly. Finally, production of the membrane-bound intracellular fragment in cells incubated at 4C was studied. If this fragment would be present under these conditions, an inuence of endocytosis on the fragmentation process could be ruled out. The observed results however, showed an inhibition of secretase-mediated ApoER2 cleavage at 4C. This result does not allow a clear interpretation, since it is possible that other mechanisms, as the secretase action itself, are inhibited at 4C. Therefore, this experiment has to be repeated at 14C, a temperature that still inhibits endocytosis but has less pronounced effects on other cellular functions. Nevertheless, this result allowed another assumption. Since secretasemediated fragmentation is inhibited at 4C and the efciency of Dab1 phosphorylation is not altered by incubation at this temperature, it can be assumed that the cleavage of the receptors is not a prerequisite for the primary signaling event. Attempts to detect the intracellular fragment produced by the action of -secretase were not successful. Since this could be explained by a fast degradation of the respective fragment after -secretase cleavage, experiments were repeated in the presence of lysosomal inhibitors. Inhibition of the lysosome however, was
5 Discussion
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not sufcient to increase the amount of the intracellular fragment. The inuence of proteasome inhibitors has not been studied yet. Further experiments will be necessary to prove the hypothesis that this fragment is indeed rapidly degraded. In the course of experiments using the lysosome inhibitor chloroquine, it was observed that the amount of membrane-bound intracellular fragment of ApoER2 was increased under these conditions. This can be explained by the stabilization of the receptor in the presence of chloroquine. As a result, the increased amount of ApoER2 in the cells provides more substrate for secretase-mediated cleavage and leads to higher amounts of the fragment. This result led to another interesting assumption. Upon chloroquine treatment, the amount of full-length ApoER2 is equal in Reelin-stimulated and unstimulated cells. Nevertheless, the amount of membrane-bound intracellular receptor fragment produced by -secretase cleavage is signicantly increased upon Reelin stimulation in these cells. Thus, Reelin induces secretase-mediated cleavage in the presence of chloroquine but does not lead to a decrease of full-length receptor amounts under these conditions. These results suggest that secretase-mediated receptor fragmentation does not result in a signicant decrease of full-length receptor, indicating that specic cleavage does not contribute to the regulation of total receptor levels. Thus, the specic secretase-mediated cleavage of ApoER2 has to play another role. One possibility might be the importance of the receptor fragments in the formation of an ApoER2-specic signalosome. Although to date, co-precipitation of the receptor fragment with Dab1 was not successful, the intracellular membrane-bound fragment could form a complex with Dab1 or phosphorylated Dab1, respectively. This complex might be liberated by the action of -secretase to generate a soluble signalosome which would be able to interact with proteins which are not associated with the plasma membrane. Another possibility is that the liberated intracellular fragment of ApoER2 is recruited into the nucleus where it might affect gene expression. A similar process has been described for Notch, which is cleaved by TACE (tumor necrosis factor alpha converting enzyme) and -secretase upon ligand-binding to liberate an intracellular fragment, which subsequently moves to the nucleus [Okochi et al., 2002]. Also the secretase-mediated fragmentation of APP (amyloid precursor protein) and the role of the produced fragment in signaling to the nucleus resembles this mechanism [Gao and Pimplikar , 2001; Leissring et al., 2002]. Another possible function for a soluble cytosolic receptor fragment would be a role as dominant negative modulator of the Reelin pathway by competing with the membrane-bound receptors for Dab1 and thereby preventing
5 Discussion
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Dab1 from phosphorylation. Since the amount of soluble intracellular fragment in the cytoplasm is too small to detect, this mechanism does not seem plausible. In contrast, the soluble extracellular receptor fragment produced by -secretase cleavage could be important for a similar mechanism. This fragment, comprising the ligand binding domain of the receptor, could compete with full-length receptor for Reelin binding, as it was described for the fragment produced from an ApoER2 variant carrying a furin-cleavage site [Koch et al., 2002].
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Curriculum vitae
Personal data Name Date of birth Place of birth Nationality Sarah Duit August 25th , 1982 Klosterneuburg, Austria Germany
Education 1988-1992 1992-2000 June 2000 2000-2001 2002-2006 Primary school Secondary school, Realgymnasium Klosterneububrg Matura Study of Chemistry, University of Vienna Study of Biotechnology, University of Applied Science (fh campus wien), Vienna Biocenter
Diploma thesis 2005-2006 Diploma thesis at the Institute of Medical Biochemistry, Department of Molecular Genetics, Medical University of Vienna, Vienna Biocenter, Laboratory of Ao. Prof. Dr. J. Nimpf
Publications Mayer H, Duit S, Hauser C, Schneider WJ, Nimpf J. (2006). Reconstitution of the Reelin signaling pathway in broblasts demonstrates that Dab1 phosphorylation is independent of receptor localization in lipid rafts. Mol Cell Biol, 26: 19-27.
Danksagung
Ich mchte mich an dieser Stelle bei Johannes und meinen Laborkollegen Harald, Harry, Pavel, Beate, Nuno, Sophia und Viktoria fr die Untersttzung und das lockere und (mit sehr seltenen Ausnahmen) angenehme Arbeitsklima bedanken. Ganz besonders danke ich Harald fr die nette und kompetente Betreuung und seine Geduld beim Entwirren diverser geistiger Knoten. Einen herzlichen Dank richte ich auch an Harry fr seine bestndige Hilfsbereitschaft im Labor. Bei Viktoria und Maria mchte ich mich fr die entspannenden und interessanten Rauchpausen und Gesprche bedanken, die meinen Laboralltag sehr bereichert haben. Ausserdem danke ich meinem Freund Ren, der mich manchmal besser versteht als ich selbst, und meiner Familie, die immer fr mich da ist.

Molecular Events During Reelin Signaling

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Molecular Events During Reelin Signaling

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FH-Studiengang Biotechnologie

Vienna Biocenter 1030 Wien

Molecular events during Reelin signaling

Diplomarbeit zur Erlangung des akademischen Grades Dipl. Ing. (FH)

Eingereicht am 25. Oktober 2006

1 Introduction 1.1.3 The very low density lipoprotein receptor

1 Introduction 1.1.5 The LDL receptor related protein

1 Introduction 1.1.7 Megalin

1.2 The Reelin signaling pathway

1 Introduction the cerebral cortex and the cerebellum.

2 Aims of this study

2 Aims of this study

2 Aims of this study

3 Material and Methods

3 Material and Methods

3.3 Cell culture

3 Material and Methods

3 Material and Methods

3.4 Protein extraction and analysis

3 Material and Methods

3 Material and Methods

antibody G10 20 19 HA HA PY D4 GFP Lis1 Cav1 ms-HRP rb-HRP

3.5 Western blot based assays

3 Material and Methods

3 Material and Methods

3 Material and Methods

3 Material and Methods

3.6 Immunouorescence Microscopy

3 Material and Methods

3 Material and Methods

4.1 Localization of ApoER2 and VLDLR within the plasma membrane

4.2 Reelin internalization by ApoER2 and VLDLR

4.3 Surface expression of ApoER2 and VLDLR

4.4 Reelin-induced degradation of ApoER2

4.5 Reelin-induced fragmentation of ApoER2

5.1 Membrane localization of the Reelin receptors

5.2 Reelin endocytosis and degradation

5.3 Surface expression of the Reelin receptors

5.4 Reelin-induced degradation and specic cleavage of the receptors

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