Mechanisms of outside-in JAM-A mediated signaling Open Access

Severson, Eric Allan (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/xk81jk79h?locale=en
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Abstract

Abstract

Mechanisms of outside-in JAM-A mediated signaling

By Eric A. Severson

 

JAM-A is a transmembrane component of tight junctions that regulates multiple processes, yet studies on the mechanism of JAM-A function are lacking. We hypothesized that the ability of JAM-A to dimerize was necessary for the mechanism of JAM-A function. Overexpression of dimerization-defective JAM-A mutants or treatment with a dimerization inhibiting antibody in 293T cells reduced cell migration across permeable filters. Analyses of cells expressing the JAM-A dimerization-defective mutant proteins revealed diminished β1 integrin protein. A functional link between JAM-A and β1 integrin was confirmed by restoration of cell migration to control levels after overexpression of β1 integrin in JAM-A dimerization-defective cells. To extend upon these results, we reported that JAM-A is physically and functionally associated with the PDZ domain containing signaling molecules Afadin and PDZ-GEF2, but not ZO-1, in the intestinal epithelial cell line SK-CO15. Both Afadin and PDZ-GEF2 were observed to co-localize and co-immunoprecipitate with JAM-A. Loss of JAM-A, Afadin or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, β1 integrin protein and the rate of epithelial cell migration. The effects observed were secondary to decreased levels of Rap1A since knockdown of Rap1A resulted in decreased β1 integrin protein and cell migration. These findings suggest that JAM-A dimerization leads to the close apposition of Afadin and PDZ-GEF2. The proximity of PDZ-GEF2 and Afadin results in activation of Rap1A. Active Rap1A stabilizes β1 integrin protein levels, which controls the rate of cell migration. These results illustrate a novel mechanism for JAM-A signaling based on its structural motifs.

Table of Contents

Introduction...........................................................................................1
Chapter 1: Structure and function of JAM proteins....................................... .2

1.1 Introduction...................................................................................3
1.2 Nomenclature................................................................................4
1.3 Relationship to other IgSF family members.............................................5
1.4 Structure of JAM-A, JAM-B, JAM-C....................................................6
1.5 Tissue and Cellular Expression/Localization.........................................................7
1.6 Homophilic Extracellular Interactions..................................................................10
1.7 Intracellular Protein-Protein Interactions..................................................11
1.8 Cellular Function Mediated by JAM-A, JAM-B and JAM-C.........................12
1.81 Determination of cell polarity.................................................12
1.82 Barrier function..................................................................14
1.83 Cell adhesion, integrin regulation and cell migration......................15
1.84 Angiogenesis.....................................................................18
1.85 Role of JAM-A in leukocyte transmigration .................................18
1.86 Role of JAM-B/JAM-C in leukocyte transmigration.......................20
1.87 Role of JAM-A in recovery from colitis and cellular proliferation......22
1.9 The relationship of JAM function and human disease................................23
1.10 Conclusions...............................................................................24
Chapter 2: Cis-dimerization mediates function of Junction Adhesion molecule A..25
2.1 Abstract.....................................................................................26
2.2 Introduction.................................................................................27
2.3 Experimental Procedures.................................................................30
2.4 Results.......................................................................................35
2.5 Discussion..................................................................................55
Chapter 3: Association of Junctional Adhesion Molecule A (JAM-A) with Afadin
and PDZ-GEF2 is required for activation of Rap1A, maintenance of cellular levels
of 1 integrin protein and regulation of cell migration....................................64

3.1 Abstract.....................................................................................65
3.2 Introduction.................................................................................66
3.3 Results.......................................................................................68
3.4 Discussion..................................................................................85
3.5 Methods.....................................................................................92
Chapter 4: Conclusions..........................................................................98
4.1 Summary of Results.......................................................................99
4.1.1 Introduction........................................................................99
4.1.2 JAM-A and outside-in signaling: evidence for dimerization-mediated
regulation of cell function........................................................99
4.1.3 The role of JAM-A extracellular dimerization and scaffolding complex
formation in intracellular signaling............................................101
4.2 Different functions of JAM-A may be mediated through activation of distinct
scaffolding and signaling molecules...................................................103
4.3 JAM-A, PDZ-GEF1, Rap1B and cell morphology..................................104
4.4 Other potential interacting domains on JAM-A......................................106
4.5 Therapeutic potential for targeting of JAM-A homodimerization.................106

4.6 Conclusions...............................................................................107

Chapter 5: References..........................................................................109

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