Cellular Mechanisms for the Regulation of VE-cadherin Endocytosis Öffentlichkeit

Abstract

Cadherins maintain adult tissue architecture and direct morphogenic events through homotypic cellular interactions and via signaling pathways that direct cell growth and migration. In the vascular endothelium, the dynamic regulation of cadherin-based cell adhesion is crucial for vascular development. Vascular endothelial (VE)-cadherin is the central adhesion molecule in endothelial adherens junctions and its regulation is crucial for vascular function. p120-catenin (p120) binds to cadherins and regulates cadherin levels by preventing cadherin endocytosis and degradation. In vivo studies have demonstrated that p120 is required for vascular development, and mice lacking p120 in endothelial cells exhibit severe defects in vascular patterning, barrier function, and proliferation. These phenotypes are at least partially dependent on the loss of VE-cadherin observed in vessels lacking p120.

The primary goal of the research described in this dissertation is to understand the mechanism by which p120 regulates VE-cadherin endocytosis. Studies shown here have demonstrated that p120 prevents VE-cadherin from entering a clathrin-mediated endocytic pathway through a mechanism that requires binding of p120 to the cadherin cytoplasmic tail. We demonstrate that p120 prevents VE-cadherin internalization independently of its role in regulating RhoGTPase activity. Rather, we present evidence that p120 stabilizes VE-cadherin at the plasma membrane by competing with the clathrin adaptor AP-2 for interactions with the VE-cadherin juxtamembrane domain.

We have identified a key role for the clathrin adaptor complex AP-2 in regulating VE-cadherin endocytosis. AP-2 interacts with the VE-cadherin cytoplasmic tail, and is required for VE-cadherin endocytosis. Mutation of a tyrosine motif in the VE-cadherin juxtamembrane domain compromises VE-cadherin endocytosis and reduces the efficiency of AP-2 binding to the VE-cadherin tail. These findings support a model in which p120 binding to the VE-cadherin tail inhibits the entry of VE-cadherin into a clathrin-mediated endocytic pathway by preventing interactions with the clathrin adaptor AP-2.

These studies further elucidate the mechanism by which p120 functions as a setpoint for VE-cadherin expression and establish a line of inquiry to investigate the role of VE-cadherin membrane trafficking during vascular development, when it is likely to play an important role in regulating the blood vessel growth and reorganization that occurs during angiogenesis.

Table of Contents

Table of Contents:

Chapter 1: Cadherin-mediated adhesion in the vascular endothelium...1

1.1 Cadherin-mediated cell adhesion...2

1.2 The vascular endothelium...3

1.3 Structure and function of endothelial adherens junctions...6

1.4 VE-cadherin: a unique member of the cadherin family...8

1.5 The catenins: cytoskeletal linkers and signaling scaffolds...11

1.5.1 p120-catenin...11

1.5.2 β-catenin and plakoglobin...17

Chapter 2: Regulation of cadherin trafficking...25

2.1 Endocytosis of adherens junction proteins...26

2.2 Routes of cadherin endocytic trafficking...30

2.3 Regulation of cadherin trafficking by the endocytic machinery...31

2.4 RhoGTPase regulation of cadherin endocytosis...36

2.5 Regulation of cadherin endocytosis by receptor tyrosine kinases...38

2.6 Working model and central hypothesis...41

2.7 Scope and conceptual contributions of this dissertation...43

Chapter 3: p120-Catenin inhibits VE-cadherin endocytosis through a Rho-independent mechanism...54

3.1 Introduction...55

3.2 Materials and Methods...58

3.3 Results...62

3.3.1 VE-cadherin is internalized through a clathrin, AP-2, and dynamin dependent pathway...62

3.3.2 p120 prevents VE-cadherin from entering a clathrin and AP-2 enriched membrane domains...66

3.3.3 p120 inhibits VE-cadherin endocytosis independently of RhoA...67

3.4 Discussion...69

Chapter 4: Identification of a tyrosine sorting motif that regulates VE-cadherin endocytosis and AP-2 binding...87

4.1 Introduction...88

4.2 Materials and Methods...91

4.3 Results...95

4.3.1 The JMD of VE-cadherin mediates internalization and turnover...95

4.3.2 A tyrosine motif in the VE-cadherin JMD mediates the association between AP-2 and VE-cadherin...97

4.3.1 Mutagenesis of tyrosine 685prevents efficient internalization of VE-cadherin...98

4.4 Discussion...99

Chapter 5: Summary and future directions...114

5.1 Overview of this dissertation...115

5.2 Future Directions...116

5.2.1 What mechanisms regulate the dissociation of p120 from the VE-cadherin tail?...117

5.2.2 How does VE-cadherin endocytosis contribute to vascular development and disease?...119

Chapter 6: References...123
Table of Figures:

Figure 1.1 Organization of the adherens junction and the cadherin-catenin complex...22

Figure 1.2 Structure and domain organization of VE-cadherin...23

Figure 1.3 Domain organization of catenin family proteins...24

Figure 2.1 Cadherin dynamics are regulated by membrane trafficking pathways...47

Figure 2.2 Cadherin endocytosis occurs through many endocytic pathways and is regulated by different adaptors, binding partners, and signaling molecules...49

Figure 2.3 Mechanisms of clathrin-mediated endocytosis...50

Figure 2.4 Structure and organization of the heterotetrameric adaptor complex AP-2...51

Figure 2.5 Model for the regulation of VE-cadherin endocytosis by p120 preventing adaptor interactions with the cadherin tail...52

Figure 2.6 Model for the regulation of VE-cadherin endocytosis by p120-mediated regulation of RhoGTPase activity...53

Figure 3.1 VE-cadherin endocytosis is mediated through a clathrin, AP-2, and dynamin dependent pathway...73

Figure 3.2 The VE-cadherin cytoplasmic tail specifically associates with clathrin and AP-2...76

Figure 3.3 The VE-cadherin cytoplasmic tail specifically colocalizes with clathrin and AP-2...77

Figure 3.4 Exogenous expression of p120 prevents IL-2R-VE-cadcyto from colocalizing with clathrin and AP-2 in a manner dependent on the interaction of p120 with the VE-cadherin JMD...79

Figure 3.5 Inhibition of RhoA activity does not inhibit VE-cadherin endocytosis...81

Figure 3.6 p120 Rho uncoupled mutant is effective at preventing VE-cadherin endocytosis...83

Figure 3.7 A p120 Rho uncoupled mutant prevents entry of VE-cadherin into membrane compartments containing clathrin and AP-2...85

Figure 4.1 VE-cadherin sequences...105

Figure 4.2 The VE-cadherin JMD mediates turnover of VE-cadherin...107

Figure 4.3 The VE-cadherin JMD is sufficient for VE-cadherin internalization...109

Figure 4.4 Mutation of Y685 disrupts the binding between VE-cadherin and AP2...111

Figure 4.5 Tyrosine 685 is required for efficient VE-cadherin endocytosis...112

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Stichwort	clathrin AP-2 endocytosis VE-cadherin cell adhesion p120
Committee Chair / Thesis Advisor	Kowalczyk, Andrew, Emory University
Committee Members	Moberg, Kenneth H, Emory University Sale, Winfield S, Emory University Li, Lian, Pharmacology Faundez, Victor, Emory University

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