IDENTIFICATION AND CHARACTERIZATION OF PROTEINS THAT INTERACT WITH ZONULA OCCLUDENS PROTEINS P JAYA KAUSALYA INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 IDENTIFICATION AND CHARACTERIZATION OF PROTEINS THAT INTERACT WITH ZONULA OCCLUDENS PROTEINS P JAYA KAUSALYA (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEGMENTS This work would not be possible without the support of family and colleagues. I wish to thank my parents and brother for their love, understanding and encouragement. I wish to thank A/P Walter Hunziker for being my mentor and I am grateful for his guidance and patience. I also, wish to thank my supervisory committee members, Prof Hong Wan Jin and A/P Cai Ming Jie for their guidance. I thank Dr. Manuela Reichert for her help during the initial stages of my project and collaborators, Dr. Dominik Muller and co-workers and Dr. Michael Fromme and co-workers. I am grateful to Dr. K. Willecke and Dr. Gonzalez-Mariscal for plasmids. I thank past and present members of WH lab and other IMCB members, in particular Drs. Wong Siew Cheng, Joy Tan, Lu Lei and Yu Xianwen for useful discussions and support. Last but not least, I thank God for all the opportunities given to me. TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS 10 MEDICAL DEFINITIONS 13 SUMMARY 14 CHAPTER 1: INTRODUCTION . 15 1.1: TIGHT JUNCTIONS 18 1.1.1: Tight junction structure and morphology . 18 1.2: Functions of Tight Junctions . 20 1.3: Models of Tight Junctions 23 1.4: Protein components of TJ 25 1.4.1: TJ transmembrane proteins . 26 1.4.1.1: Occludin . 27 1.4.1.1.1: Occludin as a structural and functional component of TJ 27 1.4.1.1.2: Protein-protein interactions of occludin . 30 1.4.1.2: Claudins . 31 1.4.1.2.1: Claudins as structural and functional components of TJs 34 1.4.1.2.2: Model for claudins in ion- and solute permeability 38 1.4.1.3: Junction Adhesion Molecule (JAM) 39 1.4.2: Peripherally-associated scaffolding proteins 42 1.4.2.1: PDZ domains . 43 1.4.2.2: Mechanism of binding and specificity of PDZ domains . 44 1.4.2.3: Structure and function of PDZ domain 45 1.4.2.4: Roles of PDZ domains . 49 1.4.2.5: SH3 and GUK domains . 50 1.4.2.6: The ZO protein family . 51 1.4.3: Regulatory proteins . 56 1.4.4: Transcriptional and post-transcriptional regulators 57 1.5: Assembly of Tight Junctions . 58 1.6: Diseases linked to the TJ function . 61 1.6.1: Diseases associated with TJ peripheral proteins . 61 1.6.2: Diseases associated with TJ integral membrane proteins . 62 1.7: Model systems to study junction assembly and function 65 1.7.1: General techniques 65 1.7.1.1: Epithelial cells as a model cell system . 65 1.7.1.2: Permeable supports . 66 1.7.1.3: Immunofluorescence analysis for TJ protein localization . 68 1.7.2: Analysis of TJ function . 69 1.7.2.1: Analysis of fence function . 69 1.7.2.2: Analysis of gate function . 70 CHAPTER 2: Identification and characterization of proteins that interact with ZO-1 PDZ domains using yeast-two-hybrid 71 Section 2.1: Connexin45 directly binds to the PDZ domains of ZO-1 and localizes to the tight junction region in epithelial MDCK cells . 73 2.1: Gap Junction and Connexins . 73 Section 2.2: Results . 76 Section 2.2.1: The C-terminus of Cx45 interacts with the PDZ domains of ZO-1 and ZO-3 in a yeast two-hybrid assay . 76 2.2.2: Characterization of epithelial MDCK cells transfected with Cx45 cDNA. 78 2.2.3: Cx45 directly associates with ZO-1 in vivo 80 2.2.4: Cx45 co-localizes with ZO-1 in the tight junction region in polarized MDCK cells 81 Section 2.3: Discussion . 84 Section 2.4: Association of ARVCF with ZO-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF . 89 2.4.1: Cadherins and Catenins 89 2.4.2: The p120 family 93 2.4.2.1: ARVCF 94 2.5: Results 96 2.5.1: ARVCF interacts with ZO-1 and ZO-2 but not ZO-3 96 2.5.2: ARVCF and ZO-1 interact in vivo and partially co-localize in MDCK cells . 102 2.5.4: ARVCF can associate with E-cadherin via its armadillo domains or through binding to PDZ domains proteins . 111 2.5.5: Plasma membrane and nuclear localization of ARVCF require the interaction with PDZ domain proteins and are regulated by cell-cell adhesion . 116 2.5.6: ARVCF, ZO-1 and E-cadherin are recruited to sites of initial cell-cell contact . 118 2.5.7: The PDZ domains of ZO-2 mediate the efficient nuclear localization of ARVCF . 122 Section 2.6: Discussion . 126 CHAPTER 3: Characterization of Claudin-16/Paracellin-1 and mutants associated with familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) 132 Section 3.1: Claudin-16 132 3.1.1: Magnesium Homeostasis and Reabsorption . 132 3.1.2: Claudin-16/Paracellin-1 and renal magnesium wasting disorder . 133 3.1.3: Magnesium Resorption Mechanism . 135 3.1.4: Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis linked to mutations in CLDN16 gene 137 3.1.5: Objective of study . 140 Section 3.2: Results on CLDN16 and mutations . 141 3.2.1: Characterization of an antibody to the first extracellular loop of CLDN16 . 141 3.2.2: Cldn16 internalizes via clathrin-dependent pathway 142 3.2.3: Subcellular and surface expression of Cldn16 mutations . 146 3.2.4: Cldn16 mutants that fail reach cell surface localize to the ER and Golgi 148 3.2.5: Characterization of trafficking defects of Cldn16 mutants displaying a predominant intracellular steady-state localization 151 3.2.5: Cldn16 mutants retained in the ER are subject to proteosomal degradation . 156 3.2.6: T233R mutation disruption PDZ binding motif and is mistargeted to the lysosomes 161 3.2.7: Cldn16 mutants delivered to lysosomes use different routes . 166 3.3.8: Pharmacological chaperones rescue cell surface expression of several Cldn16 mutants . 168 3.3.9: Cldn16 mutants present in TJ are defective in paracellular Mg2+ transport . 171 3.3.10: Clinical phenotypes . 175 Section 3.4: Discussion on Cldn16 and mutations 176 Chapter 4: Concluding Remarks . 185 Chapter 5: Materials and Methods 188 5.1 Antibodies and Reagents . 188 5.2: Plasmids constructions . 189 5.2.1: Cloning of ZO and mutants 189 5.2.2: Cloning of Connexin 45 and mutant constructs 189 5.2.3: Cloning of ARVCF and mutant constructs . 189 5.2.4: Cloning of Cld16 and mutants . 190 5.3: Yeast Two-Hybrid Screen . 190 5.4: Cell Cuture and Transfection of cells 191 5.5: Co-immunoprecipitation assays . 192 5.5.1:Cx45 coimmunoprecipitations . 192 5.5.2: ARVCF coimmunoprecipitation . 192 5.5.3: Cldn16 coimmunoprecipitation 193 5.6: GST Fusion Purification . 193 5.7: Pull-down assay . 194 5.7.1: ARVCF GST pull down assays 194 5.7.2: Cldn16 pull down assays 194 5.8: Immunofluorescence Labeling 195 5.9: Calcium-switch and cell-cell contact detection protocol . 196 5.10: Blot overlay 196 5.11: Cytochalasin D treatment for ARVCF or mutant transfected cells . 196 5.12: Endocytosis/ Internalization of CLDN16 and FHHNC mutants . 197 5.13: 20˚C block and cyclohexamide experiments . 197 5.14: Nocodazole treatment 197 5.15: Pharmacological inhibition of endocytosis 198 5.16: Proteosomal Degradation Western Analysis . 198 References . 199 LIST OF FIGURES Fig. 1: Junctional complexes. Fig. 2: Structure of Tight junctions. Fig. 3: Transcellular and Paracellular pathways. Fig. 4: Barrier and Fence function of tight junctions. Fig. 5: Proposed protein and lipid models of TJs. Fig. 6: The composition of tight junctions. Fig. 7: Integral membrane proteins of tight junctions. Fig. 8: Schematic representation of the structure and the membrane topology of claudins. Fig. 9: Creating a paracellular seal. Fig.10: Proposed model of ion and size discriminations and flux of charged and noncharged molecules. Fig. 11: Interaction of two of the major protein complexes in the TJs. Fig. 12: The ZO protein family in TJ that are part of MAGUK family. Fig. 13: Structure of PDZ3 domain of PSD-95 with a peptide ligand. Fig. 14: Possible modes of interaction of PDZ containing proteins. Fig. 15: ZO-3 interacts with PATJ, which is part of the Crumbs-Pals-PATJ complex. Fig.16: Stages of cell-cell contact and polarization. Fig. 17: Permeable supports use in the study of polarity. Fig. 18: TJ Barrier analysis. Fig. 19: Gap junctions between neighboring cells where ions and cAMP molecules are transferred. Fig. 20: Gap Junction Structure. Fig. 21: Characterization of MDCK cells expressing wild type or mutant Cx45. Fig. 22: Cx45 directly interacts with ZO-1 via the C-terminal SWVI. Fig. 23: Cx45 co-localizes with ZO-1 to the tight junction region in MDCK cells. Fig. 24: Main steps involved in connexin synthesis, assembly and turnover. Fig. 25: Schematic diagram of cadherin molecule showing its functional domains. Fig. 26: The armadillo family. Fig. 27: Cadherin-catenin complexes. Fig. 28: Schematic diagram of the domain structure of ARVCF. Fig. 29: Schematic diagrams of ARVCF, ZO-1 and ZO-2. Fig. 30: Binding of ARVCF and ZO-1. Fig. 31: A. ARVCF co-precipitates with ZO-1 from transfected MDCK cells. B. Endogenous ARVCF and ZO-1 coprecipitate from MDCK cells. Fig. 32: Colocalization of ARVCF and ZO-1 in transfected MDCK cells. Fig. 33: ARVCF partially colocalizes with ZO-1 to a discrete region between the lateral plasma membrane and TJ of polarized MDCK cells. Fig. 34: Colocalization and coprecipitation of ARVCF and ZO-1 or E-cadherin in transfected MDCK cells treated with cytochalasin D. Fig. 35: Binding of ARVCF and E-cadherin. Fig. 36: Plasma membrane recruitment and nuclear localization of ARVCF require the PDZ-binding motif and are regulated by cell-cell adhesion using calcium switch assay. Fig. 37: I. Recruitment of ARVCF to sites of cell-cell contact in MCF7 cells. II. Effect of ZO-1 mutants on ARVCF localization in MCF7 cells. Fig. 38: Role of ZO-2 in nuclear localization of ARVCF. Fig. 39: Putative E-cadherin/ARVCF/ZO-1 associations and C-terminal PDZ binding motifs in members of the p120ctn protein family. Fig. 40: Magnesium resorption in nephron segments. Fig. 41: Sequence, structure and expression of claudin-16. Fig. 42: A schematic model of magnesium resorption in the cortical ascending limb (cTAL) of Loop of Henle. Fig. 43: Predicted topology of CLDN16 and the location of the different mutations reported in humans. Fig. 44: Clathrin mediated endocytosis of Cldn16. Fig. 45: Cell surface expression of Cldn16 mutants linked to FHHNC. Fig. 46: Steady-state localization of Cldn16 mutants to different subcellular organelles. Fig. 47: Characterization of intracellular trafficking defects of different Cldn16 mutants. Fig. 48: Colocalization of Cldn16 mutants with ubiquitin is increased in the presence of a proteasome inhibitor. Fig. 49: ER-retained Cldn16 mutants are subject to proteasomal degradation. Fig. 50: Binding of wild type and mutant CLDN16 to ZO-1. Fig. 51: Subcellular localization of wild type and mutant CLDN16. Fig. 52: Cldn16 mutants that localize to lysosomes follow different pathways. Fig. 53: Chemical chaperones rescue cell surface expression of several Cldn16 mutants. Fig. 54: TJ localization of Cldn16 mutants expressed on the cell surface. Fig. 55: Measurements of Mg2+ permeability (A) and transepithelial resistance (B). Fig. 56: Predicted topology of Cldn16 and location of the different mutations linked to FHHNC reported. Fig. 57: Multiple roles of ZO proteins in the different adhesion junctional complexes. LIST OF TABLES Table 1: Claudin gene family Table 2: PDZ domain classes and examples of PDZ ligands. Table 3: Summary on disease associated with TJ peripheral proteins. Table 4: Summary on diseases associated with TJ integral membrane proteins. Table 5: Interaction of the PDZ domains of ZO-1, ZO-2 and ZO-3 with a construct encoding a C-terminal region of Cx45 or a Cx45 mutant in which the SVWI amino acids encoding a putative PDZ-binding motif were mutated to alanine. Table 6: Interaction of the PDZ domains of ZO-1 and ZO-2 with a construct encoding the C-terminal region of ARVCF (amino acids 670-893) or a mutant thereof (ARVCF∆P) in which the SWV amino acids encoding a putative PDZ-binding motif were changed to alanines. Table 7: Summary of the clinical features of FHHNC. Table 8: Summary of steady-state localization and defects of Cldn16 mutants linked to FHHNNC ABBREVIATIONS Å: Angstrom AF-6: ALL-1 fusion partner at chromosome AJ: Adherens Junction ALLN: N-acetyl–leu-leu-norleucinal Ap: Apical ARVCF: Armadillo repeat in Velo cardio facial syndrome Arm: Armadillo ASIP: atypical PKC isotype specific interacting protein BBB: Blood brain barrier Bl: Basolateral Ca2+: calcium CAR: Coxsackie and adenovirus receptors CPE: Clostridum perfringens enterotoxin Cld or Cldn: Claudin CLDN16: claudin-16 cTALH: cortical segment of the thick ascending limb of Henle Cx: Connexin Dlg: Drosophila Disc-Large DHR: Disc-large homology regions EEA1: Early Endosomal marker EMT: Epithelial-mesenchymal transition FHHNC: Familial Hypomagnesaemia with hypercalciuria and nephrocalcinosis 10 Izumi,Y., Hirose,T., Tamai,Y., Hirai,S., Nagashima,Y., Fujimoto,T., Tabuse,Y., Kemphues,K.J., and Ohno,S. (1998). 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Curr Biol 9, 880-888. 223 [...]... intermixing of apical and basolateral plasma membrane components TJs consist of integral membrane proteins such as claudin, occludin and JAM proteins that are linked via cytoplasmic peripheral proteins e.g ZO-1, -2 and -3 to the actin cytoskeleton This study identifies the roles of zonula occludens (ZO) proteins and characterizes their interacting partners Novel proteins that interact with ZO-1 were identified... strands represent integral membrane proteins that polymerize linearly within the lipid bilayer with their extracellular domain contacting that of proteins in strands on adjacent cells (Fig 5A) The ‘lipid model’, in contrast, depicts strands as cylindrical micelles with the polar head groups of the lipids directed inward and the hydrophobic tail immersed in the lipid matrix of the plasma membrane of. .. recent identification of TJ integral and peripheral proteins, the ‘protein model’ gained additional support In particular, the identification of the TJ integral proteins, occludin and claudin, and the observation that overexpression of these proteins in MDCK cells increases TER of MDCK cell monolayers (Balda et al., 1996; Van Itallie et al., 2001) strongly implicate a central role for proteins in TJ function... laterally and tightly associates in trans with another TJ-strand in the apposing membrane of an adjacent cell to form a paired strand completely obliterating the intercellular space (Fig 2C) The number of TJ strands as well as the frequency of their ramification vary from one cell type to another and are likely of significance for the functional properties of a particular TJ 18 A B C D Fig 2: Structure of. .. at the level of tight junction 22 1.3: Models of Tight Junctions Two types of models have been proposed to explain the features of TJ strands seen in freeze fractures (Fig 5) TJ strands were proposed to be composed of either proteins (Cereijido et al., 1978; Chalcroft and Bullivant, 1970; Griepp et al., 1983; PolakCharcon et al., 1978) or lipids (Kachar and Reese, 1982; Kan, 1993; Pinto and Kachar,... or regulatory proteins, e.g Rab13, Rab3B, Sec6/8, aPKC, PP2A and PTEN, and (4) Transcriptional and post-transcriptional regulators, e.g symplekin, ZONAB and HuASH Fig 6: The composition of tight junctions Tight junction proteins are composed of integral membrane proteins that are linked to the actin cytoskeleton via peripheral membrane proteins (Figure reprinted from Johnson et al., 2005 with permission... TJ transmembrane proteins Several integral membrane proteins have been identified in recent years and only three classes of integral membrane proteins, namely occludin, claudin and JAM proteins (Fig 7), will be discussed here Claudins and JAMs are protein families, with individual members showing distinct tissue distributions and functions, thus adding to the complexity and uniqueness of tight junctions... an intracellular seal that acts as a barrier and fence to prevent paracellular diffusion and intermixing of proteins and lipids between the apical and basolateral plasma membrane domains, respectively AJs and desmosomes are mechanically linked to AJ and desmosomes on neighboring cells as well as to the actin and intermediate filament cytoskeleton, respectively, providing strength and rigidity to the... model (A) involves integral membrane proteins that polymerize within the lipid bilayers and interacts with proteins on neighboring cells The lipid model (B) involves inverted lipid cylindrical micelles, which constitute TJ strands Figure reproduced with permission from Macmillan Magazines Ltd (Tsukita et al., 2001) 24 1.4: Protein components of TJ The protein components of TJ can be divided in at least... motion of the eyeball Tenany: An abnormal condition characterized by periodic painful muscular spasms and tremors, caused by faulty calcium metabolism and associated with diminished function of the parathyroid glands 13 SUMMARY Zonulae Occludens (ZO) or tight junctions (TJ) are specialized plasma membrane domains that regulate the paracellular transepithelial permeability and prevent the intermixing of . IDENTIFICATION AND CHARACTERIZATION OF PROTEINS THAT INTERACT WITH ZONULA OCCLUDENS PROTEINS P JAYA KAUSALYA INSTITUTE OF MOLECULAR AND CELL BIOLOGY. BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 2 IDENTIFICATION AND CHARACTERIZATION OF PROTEINS THAT INTERACT WITH ZONULA OCCLUDENS PROTEINS P JAYA KAUSALYA. in TJ that are part of MAGUK family. Fig. 13: Structure of PDZ3 domain of PSD-95 with a peptide ligand. Fig. 14: Possible modes of interaction of PDZ containing proteins. Fig. 15: ZO-3 interacts