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Progress in Biophysics & Molecular Biology 81 (2003) 144
Review
Tight junction proteins
L. Gonz!alez-Mariscal*, A. Betanzos, P. Nava, B.E. Jaramillo
Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV),
Ave. Polit!ecnico Nacional 2508, M!exico DF, 07000, Mexico
Abstract
A fundamental function of epithelia and endothelia is to separate different compartments within the
organism and to regulate the exchange of substances between them. The tight junction (TJ) constitutes the
barrier both to the passage of ions and molecules through the paracellular pathway and to the movement of
proteins and lipids between the apical and the basolateral domains of the plasma membrane. In recent years
more than 40 different proteins have been discovered to be located at the TJs of epithelia, endothelia and
myelinated cells. This unprecedented expansion of information has changed our view of TJs from merely a
paracellular barrier to a complex structure involved in signaling cascades that control cell growth and
differentiation. Both cortical and transmembrane proteins integrate TJs. Among the former are scaffolding
proteins containing PDZ domains, tumor suppressors, transcription factors and proteins involved in vesicle
transport. To date two components of the TJ filaments have been identified: occludin and claudin. The latter
is a protein family with more than 20 members. Both occludin and claudins are integral proteins capable of
interacting adhesively with complementary molecules on adjacent cells and of co-polymerizing laterally.
These advancements in the knowledge of the molecular structure of TJ support previous physiological models
that exhibited TJ as dynamic structures that present distinct permeability and morphological characteristics
in different tissues and in response to changing natural, pathological or experimental conditions.
r 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Tight junctions; Claudin; Occludin; JAM, MAGUK, ZO; Cingulin
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Transmembrane proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1. TJ tetraspan proteins found in epithelial and endothelial cells . . . . . . . . . . . . . . . 5
2.1.1. Occludin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.2. Claudins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
*Corresponding author. Tel.: +52-55-57-47-70-00 x 5110 or 5155; fax: +52-55-57-47-70-00 x 5702.
E-mail address: [email protected] (L. Gonz!alez-Mariscal).
0079-6107/03/$ - see front matterr 2003 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 7 9 - 6 1 0 7 ( 0 2 ) 0 0 0 3 7 - 8
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1. Introduction
In multicellular organisms fluids with different molecular compositions (e.g. urine, milk, gastricjuice, blood, etc.) are contained in compartments delineated by epithelia (e.g. renal tubules) and
endothelia (blood vessels). These cellular sheets constitute the frontier between the organism
internal milieu and the compartments contents.
Epithelia and endothelia have tight junctions (TJ) that regulate the passage of ions, water and
molecules through the paracellular pathway. This characteristic is generally referred to as the gate
property of the TJ. The establishment of TJ at the uppermost portion of the lateral plasma
membranes is the result of a polarized insertion of proteins, yet TJ act as a fence that maintains
cell polarity, by blocking the free diffusion of proteins and lipids between the apical and
basolateral domains of the plasma membrane.
2.2. TJ tetraspan proteins found within myelin sheaths. . . . . . . . . . . . . . . . . . . . 10
2.2.1. OSP/claudin 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.2. PMP22/gas-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.3. OAP-1/TSPAN-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.3. TJ proteins that belong to the immunoglobulin superfamily . . . . . . . . . . . . . . . 11
2.3.1. JAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.3.2. CAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.3. P0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3. Plaque proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1. PDZ-containing proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1.1. The MAGUK proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1.2. MAGI, the MAGUK inverted proteins of the TJ . . . . . . . . . . . . . . . . 20
3.1.3. PAR proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.4. MUPP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1.5. AF-6/Afadin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1.6. PATJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.2. TJ proteins lacking PDZ domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.1. Cingulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.2. Symplekin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.3. 7H6 antigen/barmotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.4. Rab proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.5. Pilt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.6. JEAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.7. huASH1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.8. Heterotrimeric G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4. Molecular assembly of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.1. Assembly of TJ molecules in epithelial and endothelial monolayers . . . . . . . . . . . 28
4.2. Molecular maturation of TJ during trophoectoderm differentiation . . . . . . . . . . . 294.3. TJ assembly during earlyXenopus development . . . . . . . . . . . . . . . . . . . . . 29
5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
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On ultrathin section electron micrographs, TJs are viewed as a series of fusion points between
the outer leaflets of the membrane of adjacent cells. At these kissing points, the intercellular
space is completely obliterated. On freeze-fracture replica electron micrographs TJs appear as a
network of continuous and anastomosing filaments on the protoplasmic face (P) of the plasmamembrane, with complementary grooves on the exoplasmic face (E) (Gonzalez-Mariscal et al.,
2001) (Fig. 1).
Two models have been developed to explain the chemical nature of TJ strands. In the protein
model, the filaments are formed by integral membrane proteins that associate with a partner in the
apposing membrane of the adjacent cell. In the lipid model instead, strands are proposed to be
formed of cylindrical micelles with the polar groups of the lipids directed inwards, and the
hydrophobic tails immersed in the lipid matrix of the plasma membrane of both contacting cells
(Kachar and Reese, 1982; Pinto and Kachar, 1982). The lipid model is however inconsistent with
the observation that changing the total composition of phospholipids, sphingolipids and
cholesterol in epithelial cells does not alter the appearance of TJ strands nor the gate or fencefunction of TJ (Calderon et al., 1998). Nevertheless, a rapid reduction of cell cholesterol by
methyl-b-cyclodextrin has been reported to generate a decline in transepithelial electrical
resistance (TER), an increased mannitol flux and an augmented number of TJ particles associated
with the E face (Francis et al., 1999). These results therefore suggest that although the TJ strands
might be composed of proteins, the structure is sensitive to rapid changes on its lipidic
environment.
In recent years, a constellation of cortical and integral proteins of the TJ has been
discovered. Of the former, 16 different molecules have so far been identified. Some function
as scaffolds, that link the integral proteins of the TJ to the actin cytoskeleton, while others
act as cross linkers of transmembrane junctional proteins. Still others are involved in
vesicular trafficking to the TJ or in cell signaling through their association to kinases andRas. Exciting new information has revealed that some submembranous junctional proteins even
have a role in gene expression due to their nuclear shuttling and specific binding to transcription
factors.
At the TJ three integral proteins are found: occludin, claudins and JAM. The former
two constitute the backbone of TJ strands while JAM appears to be important for the
routine trafficking of T-lymphocytes, neutrophiles and dentritic cells from the lymphoid
and vascular compartments to the tissues during immune surveillance and inflammatory
responses.
TJs were initially described in epithelia and endothelia. However recent observations have
demonstrated that they are also present in myelinated cells. Thus, during development of thecentral (CNS) and peripheral nervous system (PNS), oligodendrocyte and Schwann cells stop
dividing and respectively start to wrap axons in a loose spiral, resulting in the typical
multilamellar structure that electrically isolates the axon and allows saltatory conduction to
proceed. TJs present between the glia and the axons and within the myelin sheaths have proteins
found in the TJs of epithelia and endothelia as well as other nervous system specific molecules that
will also be described in this review.
Since a wealth of information on TJ proteins has emerged in recent times, and keeping updated
on so many different proteins has become a hard task, we have written this review, to serve as a
guide for readers interested in the field of cellcell adhesion.
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Fig. 1. Structure of tight junctions. (A) Freeze-fracture electron microscopic image of TJs in an epithelial monolayer.
TJs appear as a network of anastomosing strands on the P face (P) with complementary grooves on the E face (E). Scale
bar, 100 nm:(B) Ultrathin section of a TJ. Ruthenium red added to the apical surface of epithelial monolayers cannotpass beyond the tight junction (arrow). Scale bar, 10 nm (courtesy of Dr. Bibiana Ch !avez de Ram!rez).
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2. Transmembrane proteins of the TJ
2.1. TJ tetraspan proteins found in epithelial and endothelial cells
In this section we will refer to integral TJ proteins whose structure predicts four transmembrane
regions with two extracellular domains, and with both amino and carboxyl terminal ends oriented
towards the cytoplasm. While certain TJ tetraspans have both extracellular loops of
approximately the same size (e.g. occludin), some have one extracellular loop larger than the
other (e.g. claudins and tetraspanins) (Fig. 2).
2.1.1. Occludin
The name of this integral protein of the TJ derives from the Latin word occludere which
means to occlude (Furuse et al., 1993). Two crucial lines of evidence have shown that occludin is a
Occludin TetraspaninClaudin
254 aaCOOH
21-42 aaCOOH149 aa
NH2
10 aa
45 aa 44 aa
NH2
51 aa14 aa
13 aa
9-24 aaNH2
C
4-40aaCOOH
4 aa
13-30aa
78-150aa
C
C
C
G
X
XP
Y G
G
G Y
YG
G
-
-
-
++
+
+
+-
Y
Y
Y
Y
Y
Y
C
Fig. 2. Schematic representation of tetraspan proteins of the TJ. Tetraspan proteins have four transmembrane regions,
two extracellular domains, and their amino and carboxyl terminal ends are oriented towards the intracellular region.
Both extracellular loops of occludin are of approximately the same size, lack charged residues and are very rich in
tyrosine (Y). More than half of the first loop residues are tyrosines and glycines (G). In claudins the first extracellular
loop is longer than the second one. Both claudin loops display a number of charged residues ;which are expectedto influence the passage of ions through the paracellular space. Tetraspanins have a longer second extracellular loop
and can be further differentiated from claudins, by the presence within the long loop of CCG and PXXCC motifs as
well as two or four cysteine residues (C), one of which is consistently located 11 residues from the predicted start of the
fourth transmembrane domain.
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constituent of TJ filaments: (A) Occludin has the capacity of forming TJ-like filaments when
transfected into cells that lack TJ (e.g. L-fibroblasts) (Furuse et al., 1998b). (B) Freeze-fracture
immunoreplica electron microscopy (EM) has revealed the presence of occludin within TJ fibrils
(Fujimoto, 1995). Occludin comprises four transmembrane domains, two extracellular loops ofsimilar size, and three cytoplasmic domains: one intracellular short turn, a small amino terminal
domain and a long carboxyl terminal region. Both extracellular loops are enriched in tyrosine
residues, and in the first one more than half of the residues are tyrosines and glycines (Fig. 2).
Several lines of evidence assign occludin an important role at TJs. Thus, the over-expression of
mutant forms of occludin in epithelial cells leads to changes in the gate and fence function of TJs
(Balda et al., 1996b; McCarthy et al., 1996; Bamforth et al., 1999) as in the transepithelial
migration of neutrophils (Huber et al., 2000). In addition, the administration of synthetic peptides
corresponding to the extracellular loops of occludin, to epithelial cells, results in the
disappearance of TJs, inhibition of cell adhesion and up regulation of b-catenin and of the
b-catenin/TCF downstream target gene c-myc (Lacaz-Vieira et al., 1999; Medina et al., 2000; VanItallie and Anderson, 1997; Vietor et al., 2001; Wong and Gumbiner, 1997).
Occludin migrates as a tight cluster of 6282 kDa bands on SDS gels as a result of
phosphorylation on serine, threonine (Sakakibara et al., 1997; Wong, 1997; Wong and Gumbiner,
1997) and tyrosine residues (Chen et al., 2002b; Tsukamoto and Nigam, 1999). In these
phosphorylations PKC (Andreeva et al., 2001), CK2 (Cordenonsi et al., 1999b), p34cdc2=cyclin Bcomplex (Andreeva et al., 2001; Cordenonsi et al., 1997; 1999b) and the non-receptor tyrosine
kinase c-Yes (Chen et al., 2002b) are involved. In epithelial cell lines highly phosphorylated
occludin molecules are selectively concentrated at TJs whereas non- or less-phosphorylated
occludin molecules localize in the cytoplasm (Andreeva et al., 2001; Sakakibara et al., 1997;
Tsukamoto and Nigam, 1999). Studies inXenopusembryos, have however revealed opposite data,
as occludin dephosphorylation correlates with de novo assembly of TJs (Cordenonsi et al., 1997).Furthermore, in endothelial cells, shear stress significantly reduces occludin content and increases
its tyrosine phosphorylation with a concomitant increase in hydraulic conductivity (DeMaio et al.,
2001). Therefore occludin phosphorylation may play opposite roles in distinct biological systems
or alternatively, phosphorylation of different residues may have dissimilar consequences.
The last 150 amino acids of the carboxyl tail of occludin interact directly with F-actin (Wittchen
et al., 1999). This constitutes a property not shared by other TJ integral proteins that require the
mediation of scaffolding proteins for actin association. Occludin is also capable of binding
directly through this carboxyl segment, to the MAGUK proteins ZO-1 (Furuse et al., 1994), ZO-2
(Itoh et al., 1999b; Wittchen et al., 1999). and ZO-3 (Haskins et al., 1998). These terminal 150
amino acids of the occludin tail, are remarkably conserved among interspecies and are predictedto form a typical a-helical coiled-coil structure (Ando-Akatsuka et al., 1996). Employment of a
novel bait peptide method, revealed that occludin could interact with itself through this domain.
Additionally this region associates with the regulatory proteins PKC-z; non-receptor tyrosinekinase c-Yes and phosphatidylinositol 3-kinase as with the gap junction component connexin 26
(Nusrat et al., 2000). However since these interactions were detected by an in vitro procedure their
physiological significance remains unclear.
The type I WW binding motif (PPXY) present in the amino terminal portion of occludin
interacts with four WW motifs present in Itch, a E3 ubiquitin protein ligase involved in occludin
degradation (Traweger et al., 2002).
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Recently an occludin related gene (ORG) has been identified on the Y chromosome of
Drosophila melanogaster (Carvalho et al., 2001), and two alternatively splicing forms of occludin
have lately been described (Muresan et al., 2000). Isoform 1B contains a unique N terminal
sequence of 56 amino acids, whose function remains unknown.Although occludin is a clear constituent of TJ filaments, and its abundance is related to the
degree of sealing of epithelia (e.g. more in distal than in the proximal segments of the nephron)
(Gonzalez-Mariscal et al., 2000), its precise role in TJ remains unclear, specially after observing
that occludin knock out mice display well developed TJ (Saitou et al., 2000).
2.1.2. Claudins
The paradoxical results obtained with occludin deficient mice described above, led Tsukita and
co-workers to search for other integral components of TJ. Using the same liver fraction employed
to identify occludin, and by means of a sucrose step gradient, a single 22 kDa band was
discovered as a putative novel TJ integral protein. Peptide sequencing revealed two proteins in thisband that were subsequently named claudin 1 and 2 (Furuse et al., 1998a). The name claudin
derives from the Latin word claudere which means to close.
By data base searching and cDNA and genomic cloning the claudin family has expanded to 24
members (Table 1) (Tsukita et al., 2001). All claudins encode 2027 kDa proteins with four
transmembrane domains, two extracellular loops where the first one is significantly longer than
the second one, and a short carboxyl intracellular tail (Fig. 2). The last amino acids of this tail are
highly conserved within the family and constitute PDZ binding motifs: claudins 19 and 17 S/
TYV, claudins 10 and 15 AYV, claudin 11 AHV, claudin 12 HTT, claudin 13 LDV, claudins 14,
18 and 20 DYV, claudin 16 TRV, and claudin 19 DRV. Through these motifs claudins are linked
to the TJ PDZ containing proteins ZO-1, ZO-2, ZO-3 (Itoh et al., 1999a), PATJ (Roh et al.,
2002a) and MUPP1 (Hamazaki et al., 2002).When individual claudins 13, 5 or 11 were introduced into mouse L fibroblasts,
intramembrane strands appeared in freeze-fracture replicas (Furuse et al., 1998b; Morita et al.,
1999a, b). Thus suggesting that claudins constitute the backbone of TJ strands. Different claudin
species are capable of generating different freeze-fracture patterns. Thus, transfection of claudins
1 or 3 generates continuous smooth intramembrane strands on the protoplasmic surface (P face)
of the replicas (Furuse et al., 1999), whereas claudins 2 or 5 form discontinuous chains of particles
associated to the exoplasmic face (E face) (Morita et al., 1999b). Transfection with claudin 11
generates instead parallel intramembrane strands on the P face that scarcely branch (Morita et al.,
1999a).
Heterogeneous claudins can interact within a single TJ strand. For example, by immunoreplicaEM the co-incorporation of distinct transfected claudins into individual intramembrane strands
has been confirmed. The particular combination of claudins within a TJ strand might give rise to
different freeze-fracture patterns. Thus strands formed with claudins 1 and 3 are continuous and
associated to the P face, while strands formed with claudins 1 and 2 or 3 and 2 have evenly
scattered particles in the E face grooves. At the paracellular space the extracellular loops of
different species of claudins belonging to neighboring cells can also interact, except in some
combinations. Thus, when L transfectant singly expressing claudin 1, 2 or 3 were co-cultured,
claudin 3 strands associated with claudin 1 and 2 strands of the apposing cell, whereas claudin 1
did not interact with claudin 2 strands (Furuse et al., 1999).
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Claudins depict a differential distribution in distinct tissues, supporting the idea that they are
responsible for the ample variety in electrical resistance and paracellular ionic selectivity displayed
by epithelia and endothelia. The nephron is a good model to exemplify this point, since it is
integrated by tubules with low and high TER (6 O cm2 in proximal segments vs. 8702000 Ocm2
in the collecting duct) that specialize in the resorption of specific ions. Northern blot analysis hasrevealed the expression of all claudins except 6, 9, 13 and 14 in a total kidney extract. However,
the study of kidney cryosections (Enck et al., 2001; Kiuchi-Saishin et al., 2002) or of micro-
dissected tubules (Reyes et al., 2002) has revealed the differential distribution of these proteins:
claudins 5 and 15 in endothelia, claudins 2, 10 and 11 at the proximal segment, claudins 1, 3 and 8
at the distal tubule and claudins 1, 3, 4 and 8 at the collecting segment.
The expression of different type of claudins appears to be finely tuned during development. For
example: (A) Claudin 6 is present in embryonic epithelia (Turksen and Troy, 2001) and its over
expression in transgenic mice generates a defective epidermal permeability barrier (Turksen and
Troy, 2002). It has been proposed that the unstable temperature control and dehydration
Table 1
Characteristic features of different claudins
Claudin Distinctive characteristics
1 Present in high-resistance epithelia (e.g. collecting segment) and absent in leaky epithelia (e.g. proximal
tubule) (Reyes et al., 2002). Crucial for the mammalian epidermal barrier (Furuse et al., 2002). Absent in
most human breast cancer cell lines (Hoevel et al., 2002).
2 Present in leaky epithelia (e.g. proximal tubule) and absent in tight epithelia (e.g. collecting segment)
(Reyes et al., 2002; Enck et al., 2001). Present in the choroids plexus epithelium (Wolburg et al., 2001).
3 Also known as RVP1 (Briehl and Miesfeld, 1991). Present in the tighter segments of the nephron (Kiuchi-
Saishin et al., 2002). Its expression is elevated in regressing ventral prostate and in prostate
adenocarcinomas (Long et al., 2001). Capable of CPE binding (Sonoda et al., 1999).
4 Its expression decreases paracellular conductance through a selective decrease in sodium permeability
(Van Itallie et al., 2001). Present in the tighter segments of the nephron (Kiuchi-Saishin et al., 2002). Over
expressed in pancreatic and gastrointestinal tumors (Michl et al., 2001). The selective CPE binding gave
rise to its alternative name CPE-R (Sonoda et al., 1999).
5 Receives the alternative name of TMVCF, as it is frequently deleted in Velo cardio facial syndrome
(Sirotkin et al., 1997). Constitutes TJ strands in endothelial cells (Morita et al., 1999b). Transiently
expressed during the development of the retinal pigment epithelium (Kojima et al., 2002).
6 Present in embryonic epithelia (Turksen and Troy, 2001). Its over expression in transgenic mice generates
a defective epidermal permeability barrier (Turksen and Troy, 2002).
7 Down regulated in head and neck squamous cell carcinomas (Al Moustafa et al., 2002).
8 Present in the tighter segments of the nephron (Kiuchi-Saishin et al., 2002).
11 Also named OSP. Present in oligodendrocytes and Sertoli cells (Morita et al., 1999a).
14 Expressed in the sensory epithelium of the organ of Corti. Mutations in the gene cause autosomal
recessive deafness (Wilcox et al., 2001).
15 Present in endothelial cells (Kiuchi-Saishin et al., 2002).
16 Also known as Paracellin-1. Critical for Mg2 and Ca2 resorption in the human thick ascending limb of
Henle (Blanchard et al., 2001; Simon et al., 1999).18 A downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Expressed in lung
and stomach (Niimi et al., 2001).
Claudins 9, 10, 12, 13, 17 and 1924 have not been yet well characterized.
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frequently observed in premature infants, might be related to the expression of this claudin in their
epidermis. (B) Claudin 5 is transiently expressed during the development of the retinal pigment
epithelium (Kojima et al., 2002). (C) Claudin 11 is expressed in Sertoli cells, immediately after the
peak of expression of the sex determining region in the Y gene (Hellani et al., 2000).Claudin 16 is mutated in human patients with hypomagnesemia hypercalciuria syndrome
(HHS) (Simon et al., 1999). These patients manifest a selective defect in paracellular Mg2 and
Ca2 reabsorption in the thick ascending limb of Henle (TAL), while maintaining an intact NaCl
resorption ability (Blanchard et al., 2001). Hence claudin 16 might function as a paracellular
channel selective for Mg2 and Ca2 (Goodenough and Wong, 1999). Other claudins have also
proved to be ionic selective. Such is the case of claudin 4, that when transfected into epithelial
cells, decreases the paracellular conductance through a selective decrease in Na permeability
without a significant effect on Cl permeability (Van Itallie et al., 2001). The proposal of ion
channels or pores within the TJ strands is more than two decades old, and arose with Claudes
observation that TER increases with the number of TJ strands present in the epithelia, not in alinear fashion as would be expected from the addition of resistors in series, but exponentially
(Claude, 1978; Gonzalez-Mariscal et al., 2001). The ionic selectivity at the TJ could therefore be
determined by the specific claudins that constitute the pore. On analyzing the extracellular loops
of claudins an enormous variability in distribution and number of charged residues is found. For
example the isoelectric points of the first loop range from 4.17 in claudin 16 to 10.49 in claudin 14,
and in the second extracellular loop from 4.05 in claudins 2, 7, 10 and 14 to 10.5 in claudin 13.
Based on the pKIs of the extracellular loops sequences, claudin 16 is predicted to act as a cation
pore, whereas claudins 4, 11 and 17 should function as anionic channels (Mitic and Van Itallie,
2001).
Variations in the tightness of the TJ appear to be determined by the combination and mixing
ratios of different claudin species. Thus when MDCK cells expressing claudin 1 and 4 wereincubated with the claudin 4 binding protein,Clostridium perfringensenterotoxin (CPE), claudin 4
was selectively removed from TJs, generating a significant decrease in TER (Sonoda et al., 1999).
When claudin-2 instead was introduced into high-resistance MDCK cells (MDCK I), their TJs
became leaky and were similar functionally and morphologically to those in low-resistance cells
(MDCK II), which normally contain high levels of claudin 2 (Furuse et al., 2001).
The role of claudins in carcinogenesis is controversial. Thus, claudin 4 is over-expressed in
pancreatic cancer and gastrointestinal tumors. Treatment with TGFb or CPE, leads to a
significant reduction of tumor growth (Michl et al., 2001), thus suggesting that proteins involved
in cellcell contacts such as claudins may facilitate processes of invasion and migration. On the
other hand certain claudins remain low or undetectable in a number of tumors and cancer celllines. For example claudin 1 expression is lost in most human breast cancers without presenting
alterations in its promoter or coding sequences (Hoevel et al., 2002; Kramer et al., 2000), and
claudin 7 is down regulated in head and neck squamous cell carcinomas (Al Moustafa et al.,
2002).
The crucial task of claudins in the gate function of TJs is highlighted by the following evidence:
(A) In the mammalian epidermis, claudin-1 co-localizes with occludin in the most apical regions
of the second layer of the stratum granulosum, while claudin 4 is present in deeper layers of the
stratum. In claudin 1 deficient mice, the epidermal barrier is severely affected leading to
dehydration, wrinkled skin and death of the animals within 1 day of birth. In these mice the
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occludin positive and claudin-1 deficient skin layers allow the passage of paracellular tracers,
suggesting that the combination of claudin-1 and occludin is needed for the establishment of an
effective paracellular barrier (Furuse et al., 2002). (B) In human breast cancer cells that have lost
the expression of claudin-1, transfection of this claudin decreases the paracellular flux of tracersdespite the absence of occludin (Hoevel et al., 2002).
2.2. TJ tetraspan proteins found within myelin sheaths
2.2.1. OSP/claudin 11
OSP/claudin 11 is a crucial component of TJs in CNS myelin and between Sertoli cells (Gow
et al., 1999; Spector et al., 1998). Claudin 11 null mice have no TJ in their oligodendrocytes and
Sertoli cells, show slow CNS conductance, hind limb weakness, and sterility in male animals (Gow
et al., 1999).
In contrast to conventional TJ that are formed by networks of anastomosing strands, thosefound in the CNS myelin and in Sertoli cells are comprised by parallel filaments (Southwood and
Gow, 2001), thus suggesting that claudin 11 polymerization restricts the formation of branching
TJ fibrils.
OSP/claudin 11 is the third most abundant central nervous system (CNS) myelin protein
(Bronstein et al., 1997). During prenatal development OSP/claudin 11 is profuse in developing
meninges and mesenchymal cells, especially around regions of chondrocyte formation (Bronstein
et al., 2000). Postnatally, it is only expressed in oligodendrocytes and testis. In adult animals,
expression of OSP/claudin 11 by the testis is inhibited by the hormone FSH and the cytokine
TNFa (Hellani et al., 2000).
Although abundant evidence supports a major role for claudin 11 at the TJ of Sertoli cells, the
participation of occludin in these junctions cannot be ruled out. In fact, the administration of asynthetic peptide corresponding to the second extracellular loop of occludin perturbs the blood
testis barrier and reversibly disrupts spermatogenesis (Chung et al., 2001). Therefore both
occludin and claudin 11 might be needed for Sertoli TJ to develop.
2.2.2. PMP22/gas-3
Theperipheralmyelinprotein PMP22/gas-3 was originally identified as a growtharrestspecific
protein of fibroblasts (Schneider et al., 1988). PMP22/gas-3 is a 22 kDa tetraspan glycoprotein of
160 amino acids. PMP22/gas-3 expression is closely synchronized with Schwann cells
differentiation and localizes in the myelin sheath (Baechner et al., 1995). It has been characterized
as a strong adhesive component for compact myelin formation. Deletions, duplications ormutations of PMP22/gas-3 account for the majority of heritable demyelinating peripheral
neuropathies in mice (Trembler) and humans including CharcotMarie-Tooth disease type IA,
Dejerine Sottas syndrome and heredity neuropathy with liability to pressure palsies (HNPP)
(Suter and Nave, 1999). PMP22/gas-3 mRNA has been detected in a variety of non-neural tissues,
the epithelial cells of the lungs and intestine being the higher expressers (Baechner et al., 1995).
In epithelial cells PMP22/gas-3 co-localizes with occludin and ZO-1 at the TJs, and its over-
expression in L cell fibroblasts mediates the formation of ZO-1 positive intercellular junctions
(Notterpek et al., 2001). Therefore it is tempting to speculate that PMP22/gas-3 plays a role in the
establishment and maintenance of TJs in epithelia and within the Schwann cell membrane. The
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amino acid sequence and predicted structure have posed the question of whether PMP22/gas-3 is
a claudin family member that functions as the peripheral nervous system (PNS) homologue of
claudin 11. In this respect it should be pointed that: (1) the sequence of PMP22/gas-3 is fairly
shorter compared to those of claudins (160 vs. 207264 amino acids); (2) PMP22/gas-3 has a weakhomology even with claudin-10 to which sequence it resembles the most (25% identity); (3)
claudin family members display at their carboxyl termini the PDZ binding motifs YV or fXf
(where X denotes any amino acid and f a hydrophobic one), while PMP22/gas-3 ends with RE,
which is not a consensus for PDZ binding; and (4) PMP22/gas-3 expressing cells do not show any
homophilic cell adhesion (Takeda et al., 2001). Therefore it has been suggested that PMP22/gas-3
does not contribute by itself to form and maintain the compact myelin sheath, and instead does so
via its heterophilic interaction with P0, a member of the immunoglobulin superfamily
(Berditchevski, 2001), known to form compact myelin sheaths by homophilic adhesion (DUrso
et al., 1990).
PMP22/gas-3 has recently been described as a member of the evolving epithelial membraneprotein family (EMP13) which appears to function in regulating cell growth and differentiation.
Since members of this family, are somehow similar to the claudin family, it has been suggested
that they derive from a common ancestor (Jetten and Suter, 2000).
2.2.3. OAP-1/TSPAN-3
OAP-1/TSPAN-3 is a tetraspanin of 28 kDa with 254 amino acids. Tetraspanins can be
differentiated from other tetraspan proteins such as claudins, for having their first extracellular
loop shorter than the second one. They are also characterized by the presence within the long
extracellular loop, of CCG and PXXCC motifs as well as of two or four cysteine residues, one of
which is consistently located 11 residues from the predicted start of the fourth TM domain(Berditchevski, 2001) (Fig. 2).
OAP-1/TSPAN-3 forms a complex with b1 integrin and OSP/claudin-11 within myelin sheaths
that regulates proliferation and migration of oligodendrocytes (Tiwari-Woodruff et al., 2001).
2.3. TJ proteins that belong to the immunoglobulin superfamily
2.3.1. JAM
The junctional adhesion molecule JAM is a glycosilated 43 kDa protein found at the TJs of
epithelial and endothelial cells. It has three distinct structural domains: an extracellular region of
215 amino acids that contains two variable type Ig domains; a single transmembrane domain, anda short intracellular tail (45 aa) that features a classical type II PDZ binding motif (Martin-Padura
et al., 1998). Through its carboxyl termini JAM interacts with the PDZ domains of AF6 (Ebnet
et al., 2000), ASIP/Par-3 (1st domain) (Itoh et al., 2001; Ebnet et al., 2001) and ZO-1 (domains 2
and 3) (Bazzoni et al., 2000). JAM co-immunoprecipitates with cingulin, and this association
requires the amino terminal globular head of cingulin (Bazzoni et al., 2000; Ebnet et al., 2000).
The X-ray structure of JAM suggests a homophilic adhesion model in which U-shaped JAM
dimmers stick out almost perpendicular to the cell surface. Contact is established between the first
variable type amino terminal loops that lie almost parallel to the cell surface (Fig. 3) (Kostrewa
et al., 2001).
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Antibodies against JAM inhibit TER recovery in a transient calcium depletion assay,
suggesting the participation of JAM in TJ sealing. The same antibodies have no effect when
applied to confluent monolayers with well-formed TJ, thus indicating the inaccessibility of JAM
within the sealed TJs (Liu et al., 2000).
In freeze-fracture immunoreplicas JAM shows an intimate spatial relationship with TJ strands.However, JAM transfection into fibroblasts does not generate the appearance of TJ fibrils (Itoh
et al., 2001). This can be explained by the observation that integral membrane proteins with a
single membrane-spanning domain like JAM, cannot be detected as intramembrane particles
(IMP) in freeze-fracture replicas. Therefore JAM molecules in epithelial cells may associate
laterally as dimmers, that in turn could aggregate with TJ strands made of linear polymers of
claudin and occludin.
JAM transfection instead generates the appearance of IMP devoid areas in the freeze-fracture
replicas. This pattern is remarkable, as it resembles the in vivo appearance of the membrane
during the beginning of TJ assembly, described by several groups more than two decades ago
COOH COOH
COOH COOH COOH
NH2 NH2
NH2NH2
NH2NH2
215a
a
45
aa COOH
*
*
*
Fig. 3. Homophilic model of interaction of JAM molecules. U-shaped JAM dimmers (indicated with a discontinuousgreen line) stick out almost perpendicular to the cell surface, while their first amino terminal loops (red) lie almost
parallel to the cell surface and contact each other in a common central plane (asterisks). Paracellular JAM interactions
occur between the first loops of JAM molecules located in apposing cell membranes (arrows). JAM network is thus
constructed by repeating the structural motif of the U-shaped dimmers over several neighboring cells.
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(Humbert et al., 1976; Montesano et al., 1975; Tice et al., 1977). The development of this pattern
could speculatively suggest a role for JAM in restricting the free diffusion of proteins within the
membrane. This is a fundamental characteristic of TJs and was described long before the
molecular components of the TJ were first identified (Dragsten et al., 1981; Mandel et al., 1993).Endothelial TJs in addition to their role in regulating solute permeability, serve to impede
leukocyte egress. However, during inflammation leukocytes traverse the microvasculature. The
role of JAM in this process is revealed by the ability of a neutralizing antibody to modulate
monocyte transmigration through the vessel wall (Lechner et al., 2000; Martin-Padura et al.,
1998). New JAMs have recently been described, suggesting the existence of a JAM protein family.
JAM2/VE-JAM present at the cellular borders of venules and vessels, functions as an adhesion
protein capable of capturing human T cells (Cunningham et al., 2000; Palmeri et al., 2000). It
displays homo and heterotypic interactions. The latter happen on T cells, when JAM3 functions
as the counter receptor of JAM2 (Arrate et al., 2001).
Most recently JAM has also been described as a receptor for Reovirus attachment protein s1(Barton et al., 2001; Tyler et al., 2001).
2.3.2. CAR
Thecoxsackievirus andadenovirusreceptor (CAR) is a 46 kDa integral membrane protein with
one transmembrane region, a long cytoplasmic tail, and an extracellular region composed of two
Ig-like domains (Tomko et al., 1997).
CAR seems to be a functional component of TJs since: (A) In epithelial cells it co-
immunoprecipitates with ZO-1 and co-localizes with it at the TJ. The carboxyl terminal domain of
CAR contains the type I PDZ binding motif SXV, that could account for the observed JAM/ZO-1
interaction. (B) In transfected fibroblasts, CAR mediates homotypic cell aggregation and recruitsZO-1 to cellcell contacts. (C) CAR over expression in epithelial cells leads to an increase in TER
accompanied by a reduced passage of macromolecules through the paracellular pathway (Cohen
et al., 2001).
CAR binds to IgG and IgM present in serum (Carson and Chapman, 2001) and is over
expressed at sites of inflammation (Ito et al., 2000). Therefore it is tempting to speculate that CAR
like JAM might participate in the transmigration of cells of the immune system. However, an
identity lower than 30% is maintained between the extracellular regions of CAR and JAM.
2.3.3. P0
Protein 0 (P0) is the major myelin protein of the PNS. Mutations in the P0 gene, cause thedemyelinating peripheral neuropathy CharcotMarie-Tooth disease, the more severe Dejerine
Sottas syndrome and congenital hypomyelination.
In transfected epithelial cells, P0 behaves as a homophilic adhesion molecule (DUrso et al.,
1990; Filbin et al., 1990). This PNS protein is capable of triggering epithelial reversion in
carcinoma cells, highlighting its importance as a cell adhesion molecule (Doyle et al., 1995).
Together with the tetraspan PMP22, P0 is involved in the formation and compaction of myelin.
These two proteins co-localize at the intercellular borders of transfected epithelial cells and when
PMP22 and P0 are expressed in separate but neighboring epithelial cells, P0 is recruited at the
apposed plasma membrane of the PMP22 expressor cell (DUrso et al., 1999). Crystallographic
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studies of P0 explain this PMP22/P0 heterotypic interaction, proposing a tetrameric arrangement
of P0 molecules arranged around a central hole that accommodates PMP22 (Shapiro et al., 1996).
The interaction between two distinct types of myelin proteins, opens the possibility that other
heterotypic contacts might be present in epithelial TJ, for example between JAM and claudins oroccludin. However it should be pointed that the crystallographic structure predicted for JAM is of
a U-shaped dimmer instead of a tetramer.
3. Plaque proteins of the TJ
3.1. PDZ-containing proteins
PDZ are 8090 amino acid modules that bind to specific motifs [e.g. S/TXV, FXF;for a review
see (Bezprozvanny and Maximov, 2001; Songyang et al., 1997)] found at the carboxyl terminalend of several proteins, although some PDZ domains are capable of recognizing internal motifs
(Shieh and Zhu, 1996). The PDZ motif also mediates interactions with PDZ motifs in other
proteins, thus this module in the neural NO synthase (nNOS) binds to the PDZ domain of PSD-
95 (Brenman et al., 1996). At the TJ, ZO-1 associates through its second PDZ to the second PDZ
present in ZO-2 and ZO-3 as will be described below in further detail. PDZ domains are critical
for the clustering and anchoring of transmembrane proteins (Kim et al., 1995). Thus proteins that
contain multiple PDZ domains (PSD95/DLG/ZO-1) function as scaffolds that bring together
cytoskeletal, signaling, and integral proteins at specific regions of the plasma membrane (Fig. 4).
3.1.1. The MAGUK proteins of the TJThe race for the discovery of the molecular components of the TJ started with the identification
by Daniel Goodenough and Mark Mooseker groups of a 225 kDa protein associated to the TJ,
and consequently named ZO-1 (zonula occludens 1) (Stevenson et al., 1986). When the cDNA of
ZO-1 was unraveled, its homology with the tumor suppressor protein disc large (Dlg) ofDrosophilaand with the postsynaptic density protein PSD95/SAP90 was recognized (Itoh et al.,
1993; Willott et al., 1993). Later, when the sequence of the other ZO molecules of the TJ, namely
ZO-2 (Jesaitis and Goodenough, 1994) and ZO-3 (Haskins et al., 1998) was acknowledged, it
became clear they too belonged to a protein family named MAGUK (membrane associated
guanylate kinase homologues). Proteins in this family are recognized for having structurally
conserved PDZ, SH3 and GK domains.SH3 are 5070 amino acid and non-catalytic protein domains that bind to GK modules or to
ligands at least seven residues in length that contain a PXXP sequence. The GK module is
homologous to the enzyme guanylate kinase that catalyzes the conversion of GMP to GDP at the
expense of ATP. However since the sequence of ZO proteins does not predict binding neither to
GMP nor to ATP, the GK module in these proteins is assumed to be enzymatically inactive.
Instead protein binding properties have been ascribed to this module (Kim et al., 1997). It has also
been hypothesized that the GK domain could activate G-protein coupled pathways. In this
respect it should be mentioned that TJ assembly is regulated by G proteins (Balda et al., 1991;
Saha et al., 2001).
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TJ proteins, particularly ZO-1 and ZO-2, also contain a long carboxyl terminal region with an
acidic module, a proline-rich domain and several alternative splicing sites. This area absent in
other MAGUK proteins might be responsible for the unique properties of MAGUK TJ
molecules. In fact transfection with ZO-1 mutants that maintain the MAGUK core but lack the
PDZ1 PDZ2 PDZ3+ SH3 GK - PR
claudins ZO-1occludin
actin, 4.1
cingulin, atypical PKC, AP-1 and C/EBP
PDZ1 PDZ2 PDZ3+ SH3 GK -PR
claudins ZO-1
occludin and cinguin
ZO-1
ZO-2
ZO-3
MAGI-1
MAGI-2
PDZ1 PDZ2 PDZ3 PDZ5PDZ4PDZ0 GK WW
GEP
PDZ1 PDZ2 PDZ3 PDZ5PDZ4PDZ0 GK WW
PTEN
MAGI-3PDZ1 PDZ2 PDZ3 PDZ5PDZ4PDZ0 GK WW
PTEN
MUPP1PDZ1
AF-6PDZmyosin VDRBD1 PRRBD2 kinesin D PRPR actin BD
ZO-1cingulin
JAM
PATJ
CAR, cingulin and AF-6
PDZ1 PDZ2 PDZ3+ SH3 GK - PR
claudins ZO-2, ZO-3 ZONAB ZAK
occludin actinJAM 4.1
claudinsJAM
PDZ2 PDZ3 PDZ4 PDZ5 PDZ6 PDZ7 PDZ8 PDZ9 PDZ10 PDZ11 PDZ12 PDZ13
PDZ1 PDZ2 PDZ3 PDZ4 PDZ5 PDZ6 PDZ7 PDZ8
actin
PAR-3
PAR-6PDZ1
atypical PKC
CR1 CR2 CRIB
PDZ1 PDZ2 PDZ3
atypical PKC
CR3CR1
JAM
ZO-3 claudins
PATJ
PDZ9 PDZ10MRE
Pals1
Pals1PDZ1 SH3 GKU1 L27N L27C 4.1 B
PATJ, MUPP1 CRB1
MRE
Pals1
Fig. 4. PDZ-containing proteins found at the TJ. PDZ domains are represented by ovals while the other domains are
all schematized by dotted boxes. Intermolecular associations with other TJ and cytoskeletal proteins are indicated with
brackets.
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carboxyl region generates a transformation from epithelia to a mesenchymal like type (Ryeom
et al., 2000).
3.1.1.1. ZO-1. ZO-1 is a 210225 kDa protein found at the submembranous domain of TJs inepithelia and endothelia. Cells that do not form TJs such as fibroblasts show ZO-1 disperse in the
cytoplasm and concentrated at cadherin-based adherens junctions (Itoh et al., 1993) through
interactions with a-catenin and the nectinafadin system (Yokoyama et al., 2001).
At the TJ ZO-1 is associated through its first PDZ domain to the carboxyl terminal end of
claudins (Itoh et al., 1999a), by the second and third PDZs to JAM (Ebnet et al., 2000) and by its
GK module to occludin (Fanning et al., 1998; Schmidt et al., 2001). ZO-1 immunoprecipitates
with CAR, a protein that contains PDZ and SH3 recognition motifs (Cohen et al., 2001). ZO-2
and ZO-3 independently associate to ZO-1 through a PDZ-2/PDZ-2 interaction (Wittchen et al.,
1999). ZO-1 binds to the actin cytoskeleton (Fanning et al., 1998; Itoh et al., 1997; Wittchen et al.,
1999) and to actin binding protein 4.1 (Mattagajasingh et al., 2000) through its carboxyl terminalend. Other cortical proteins of the TJ such as AF-6 (Yamamoto et al., 1997) and cingulin
(Cordenonsi et al., 1999a) bind to ZO-1. ZO-1 associates to the adherens junction proteins a-
catenin (Itoh et al., 1997) and to the gap junction proteins connexins 43 (Barker et al., 2002;
Toyofuku et al., 1998) and 45 (Kausalya et al., 2001).
ZO-1 is a phosphoprotein, however the effect of phosphorylation will remain controversial,
until studies on the participation of different kinases over distinct residues on the protein, clarify
the results so far obtained. ZO-1 in low resistance cells is significantly more phosphorylated than
in high-resistance monolayers (Stevenson et al., 1989), and hypoxia in brain micro-vessels induces
an enhanced phosphorylation of ZO-1 that correlates with a decreased expression and
dislocalization of ZO-1 (Fischer et al., 2002). However, a low phosphorylated ZO-1 has been
detected in cells that lack TJs or have them disassembled due to lack of calcium (Howarth et al.,1994). With regards to tyrosine phosphorylation, some recent studies have demonstrated that
vascular endothelial growth factor increases paracellular permeability and augments ZO-1
tyrosine phosphorylation (Antonetti et al., 1999). However, in A431 cells, epidermal growth
factor induces tyrosine phosphorylation of ZO-1 and concentrates this protein at TJs (Van Itallie
et al., 1995) and during TJ assembly ZO-1 becomes tyrosine phosphorylated (Chen et al., 2000).
ZO-1 associates and is a substrate of ZAK, a serine/threonine kinase (Balda et al., 1996a) and
of PKC (Avila-Flores et al., 2001). MAPK signaling pathway regulates tyrosine phosphorylation
of ZO-1, as MEK1 inhibition in Ras transformed epithelial cells restores epithelial morphology
and increases tyrosine phosphorylation of ZO-1 and occludin (Chen et al., 2000).
Three alternative splicing domains have been identified in ZO-1, all of which are located at thecarboxyl region of the molecule. The first named motifa is an 80 amino acid domain (Balda and
Anderson, 1993). In epithelia and endothelia both thea anda isoforms are expressed. Yet, the
a is quantitatively more abundant in epithelia while the opposite is true for endothelia (Balda
and Anderson, 1993; Underwood et al., 1999). These isoforms seem to perform different roles.
For example, the a isoform is present in cells that display no TER like podocytes (Balda and
Anderson, 1993; Kurihara et al., 1992), in Sertoli cells (Balda and Anderson, 1993), whose
junctions can be described as dynamic since they move along the entire lateral cell border and
break and reseal around migrating spermatocytes, and in mouse blastomeres that lack TJs.
Instead, the a isoform is expressed later upon the formation of the blastocoele and the
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development of TJs (Sheth et al., 1997). Therefore the a isoform appears to be related to the
establishment of functional TJs (Table 2), while the a is related to structurally dynamic
junctions.
The other alternative splicing domains identified in ZO-1 areb1;b2and g;with respective motifsof 7, 20 and 45 amino acids. Although they are expressed in a variety of tissues their functional
significance still remains unclear (Gonzalez-Mariscal et al., 1999).
The sequence of ZO-1 contains two putative nuclear export signals (NES) and three nuclear
export signal (NES) (Gonzalez-Mariscal et al., 1999), thus suggesting shuttling of ZO-1 between
the nucleus and the plasma membrane (Islas et al., 2002). Furthermore, cells with decreased
cellcell contact, such as those in sparse or mechanically injured monolayers, display a strong
presence of ZO-1 at the nuclei (Gottardi et al., 1996). ZO-1 specifically interacts through its
SH3 domain, with a Y box transcription factor named ZONAB, which binds to promoter
sequences of cell cycle regulators. This interaction modulates paracellular permeability and gene
expression in reporter assays (Balda and Matter, 2000), speculatively suggesting that ZOmolecules establish a cross talk between the nucleus and the TJ that balances epithelial cell
differentiation and growth.
Numerous studies employing cytokines, hormones and growth factors have been done, that
relate ZO-1 abundance with the degree of tightness of the junction. For example, IL-15 up-
regulates ZO-1 and fastens intestinal monolayers (Nishiyama et al., 2001), while IL-3 and IL-4 in
lung epithelia, decrease ZO-1 expression and the barrier function of TJs (Ahdieh et al., 2001).
Pathogens and their toxins also modify ZO-1 expression an epithelial permeability. Thus,
Entamoeba hystolytica, alters the TER and paracellular flow of enteric monolayers and induces
degradation of ZO-1 (Leroy et al., 2000), and Clostridium difficile toxin A increases paracellular
permeability of colonic epithelia and delocalizes ZO-1 from the TJ (Chen et al., 2002a). In
contrast, glycoprotein E of Varicella-Zoster increases translocation of ZO-1 to the cell membraneand augments the TER (Mo et al., 2000). Many of these correlations should however be taken
with caution as changes in ZO-1 expression do not necessarily imply that this protein is the direct
target of the treatment employed. Instead alterations in ZO-1 could arise as a consequence of the
modification of another key TJ component. One of the few cases in which the direct participation
of ZO-1 in the development of tighter junctions has been demonstrated is constituted by the study
of glucocorticoid treatment in trabecular endothelial cells of the eye. In this case, inhibition of
ZO-1 expression with an specific antisense, abolished the dexamethasone-induced increase in
resistance, supporting the idea that ZO-1 is involved in development and maintenance of TER
(Underwood et al., 1999).
The role played by ZO-1 in tumorigenesis remains widely unexplored. However, ZO-1 isstarting to be considered a tumor suppressor since deletions or mutations in its gene produce
overgrowth, and down regulation of its expression is found coupled to breast cancer progression
(Hoover et al., 1998). The participation of ZO-1 in tumor suppression is complex, as many
additional factors appear to be intertwined. For example, in breast cancer cells, insulin-like
growth factor I receptor (IGF-IR) induces E-cadherin mediated cellcell adhesion by up-
regulating ZO-1. The expression of IGF-IR and ZO-1 increase growth and survival of the
primary tumor but in contrast, may reduce cell metastasis (Mauro et al., 2001). Vitamin D3
promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and ZO-1,
inhibition of b-catenin signaling and translocation of ZO-1 from the nucleus to the plasma
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membrane (Palmer et al., 2001). In opposition to the above observations, ZO-1 is over expressed
in primary and metastasic pancreatic cells (Kleeff et al., 2001). The reason for this astonishing
difference is not known, but reveals that ZO-1 may act as a tumor suppressor only in specific
cancers.
Table 2
Appearance of TJ proteins during embryonic development
Protein Stage at which first detected Assembly into TJ Model References
Claudin-1 ND 32-cell stage Mouse Fleming et al. (2001)
Claudin-5 Barely detectable on
embryonic day 5 (early
stage)
Embryonic day 10 (near the
beginning of the
intermediate stage)
Chick
RPE
Kojima et al. (2002)
Xcla Throughout all embryonic
stages
Blastula stage Xenopus Brizuela et al. (2001)
Occludin 7275 kDa band,
throughout all embryonic
stages, decreases from late
blastocyst onwards 65
67 kDa band, throughout
all embryonic stages,
increases from early
blastocyst onwards 58 kDa
band, throughout all
embryonic stages, decreases
from compact 8-cell
embryos onwards
Early 32-cell stage, just
prior to blastocele
cavitation
Mouse Sheth et al. (2000b)
Throughout all embryonic
stages
2-cell stage after cingulin
and ZO-1 incorporation
Xenopus Fesenko et al. (2000)
JAM ND 8-cell stage Mouse Fleming et al. (2001)
ZO-1 a Throughout all embryonic
stages
Punctuate staining at
compact 8-cell embryos
Mouse Sheth et al. (1997)
Throughout all embryonic
stages
2-cell stage, after cingulin
incorporation
Xenopus Fesenko et al. (2000)
ZO-1 a Beginning of the blastocyst
stage
32-cell stage, just prior to
the early blastocyst stage
Mouse Sheth et al. (1997)
Throughout all embryonic
stages
2-cell stage, after cingulin
incorporation
Xenopus Fesenko et al. (2000)
AF-6 ND Observed at 7.5 days post-
coitum
Mouse Zhadanov et al. (1999)
Cingulin Throughout all embryonic
stages
16-cell stage Mouse Javed et al. (1993)
Throughout all embryonic
stages
First cell division, 2-cell
stage
Xenopus Cardellini et al. (1996)
Rab13 Throughout all embryonic
stages
Punctuate staining at
compact 8-cell embryos
Mouse Sheth et al. (2000a)
ND, not determined; RPE, retinal pigment epithelium; Xcla, Xenopus claudin.
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In Drosophila, a ZO-1 homologue named Tamou, is involved in the signaling pathway that
activates the expression of the repressor gene emc which participates in neural development
(Takahisa et al., 1996).
3.1.1.2. ZO-2. ZO-2, a 160 kDa molecule, was originally identified as a TJ protein due to its co-
immunoprecipitation with ZO-1 (Gumbiner et al., 1991). Further studies demonstrated that
this association proceeds through the respective second PDZ domains of each molecule (Wittchen
et al., 1999). ZO-2 interacts as well with other tight and adherens junction associated mole-
cules: with claudin by its first PDZ module (Itoh et al., 1999a), with occludin by its GK
region (Itoh et al., 1999b), and with cingulin (Cordenonsi et al., 1999a; DAtri et al., 2002) and
a-catenin (Itoh et al., 1999b). The proline-rich domain of ZO-2, located at the carboxyl terminal
end of the protein binds to actin (Wittchen et al., 1999) and to protein 4.1 (Mattagajasingh et al.,
2000).
The sequence of ZO-2 contains NLS (Gonzalez-Mariscal et al., 1999) and NES (Islas et al.,2002). In sparse monolayers ZO-2 is conspicuously present at the nucleus in speckles where it co-
localizes with splicing factor SC35 (Islas et al., 2002). Recent evidence has indicated that ZO-2
associates both at the nuclei and TJ with transcription factors Fos, Jun and C/EBP (Betanzos
et al., 2001). These results thus suggest a role for ZO-2 in signaling to the nucleus the adhesion
state of the monolayer.
Although tyrosine phosphorylation of ZO-2 has been reported in v-src transfected epithelial
cells (Takeda and Tsukita, 1995), and six putative tyrosine phosphorylation sites are present
in ZO-2 sequence, the two-dimensional phosphoaminoacid analysis of native ZO-2 does
not reveal tyrosine phosphorylated residues in confluent monolayers nor in those with
disassembled junctions due to calcium chelation (Avila-Flores et al., 2001). Instead ZO-2 is
significantly phosphorylated in serine and threonine residues specially when TJs are eitherabsent or disassembled due to Ca2 removal. This increased phosphorylation is due to the action
of both cAMP-dependent protein kinase (PKA) and PKC, particularly by the atypical isoforms l
and z:ZO-2 has recently been identified as a candidate tumor suppressor protein. This assertion
responds to the observation that ZO-2 expression is either lost or significantly decreased in the
majority of breast cancer lines and adenocarcinomas, although it is mostly present in colon
cancers and prostate carcinomas (Chlenski et al., 2000). The ZO-2 gene employs two alternative
promoters that give rise to two ZO-2 isoforms that differ at their amino terminal portion by 23
amino acids. Although both isoforms are present in normal tissues, the longer one is absent in
most pancreatic cancers (Chlenski et al., 1999a, b). Moreover, over-expression of ZO-2 suppressesthe neoplastic growth of cells activated by Ras V12, polyomavirus middle T protein and
adenovirus type 9 oncogenic determinant E4. The mechanism underlying this tumor growth arrest
is still poorly understood, however, sequestration of ZO-2 in the cytoplasm with tumorigenic
proteins is observed (Glaunsinger et al., 2001).
3.1.1.3. ZO-3. ZO-3 was originally identified as a 130 kDa phosphoprotein which co-
immunoprecipitates with the ZO-1/ZO-2 complex (Balda et al., 1993). Further studies however
demonstrated that this interaction proceeds through association between the second PDZ
domains of ZO-1 and ZO-3, while no direct binding appears to take place, at least under low
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stringency conditions, between ZO-3 and ZO-2 (Wittchen et al., 1999). ZO-3 associates by its first
PDZ to claudins (Itoh et al., 1999a) and via both its amino and carboxyl terminal halves to
occludin (Haskins et al., 1998) and cingulin (Cordenonsi et al., 1999a; Wittchen et al., 2000). The
carboxyl terminal end of ZO-3 contains the class I PDZ binding motif TDL, that binds to the 6thPDZ domain of PATJ (Roh et al., 2002a). In contrast to ZO-1 and ZO-2, the amino terminal half
of ZO-3 associates to actin (Wittchen et al., 2000). ZO-3 associates to the PDZ binding motif
present in Connexin 45 (Kausalya et al., 2001), suggesting it might have a role in the targeting or
localization of gap junctions to specialized domains of the plasma membrane. ZO-3 does not
posses the long carboxyl tail that characterizes ZO-1 and ZO-2. Instead in ZO-3 the proline-rich
region typical of ZO proteins is located between the second and third PDZ domains (Haskins
et al., 1998).
The sequence of ZO-3 contains two putative bipartite NLS (Gonzalez-Mariscal et al., 1999)
and one NES (Islas et al., 2002), although no studies have yet reported its presence at the
nuclei.Transfection with the amino terminal half of ZO-3 (13 PDZ domains) delays the assembly of
tight and adherens junction (Wittchen et al., 2000), suggesting that the carboxyl terminal half that
associates with occludin and cingulin is crucial for TJ to assemble.
3.1.1.4. Pals1. InCaenorhabditis elegans, three PDZ containing proteins Lin-2, Lin-7 and Lin-10,
are necessary for the basolateral targeting of the Let-23 growth factor receptor (Kaech et al.,
1998). Pals1 is a recently discovered protein associated with Lin-7, that localizes at epithelial TJ
(Kamberov et al., 2000; Roh et al., 2002b). It is a MAGUK protein that contains one PDZ
module, SH3 and GK regions. Between the latter two a 4.1 binding domain is found. Pals1 is
different from other TJ MAGUKs, as it lacks the acidic and proline-rich domains present in ZO
proteins, and instead contains two Lin-7 binding modules termed L27 domains. The L27Cmodule binds Lin-7, while the L27N domain targets Pals1 to TJ by binding to the MRE region of
PATJ and MUPP1, both recently discovered PDZ-containing proteins of the TJ. The extreme
amino terminal region of Pals1 contains a 125 amino acid domain that bears no similarity to other
proteins and is thus referred to as unknown 1 (U1).
At the TJ, Pals1 exists in a ternary complex with PATJ and the human Crumbs homo-
logue CRB1 (Roh et al., 2002b). Crumbs together with Disc lost (Dlt) functions as an apical
polarity determinant in Drosophila. In mammalian epithelia, Pals1 interacts directly with CRB1
and thus serves as an adaptor protein, mediating the indirect interaction between CRB1 and
PATJ.
3.1.2. MAGI, the MAGUK inverted proteins of the TJ
TheMAGUK inverted proteins named MAGI have three unique structural features: (A) they
contain six PDZ domains, one located at the amino terminus and the rest at the carboxyl terminal
domain, (B) the SH3 region is replaced by two WW domains, and (C) the GK domain is found
after the first PDZ module at the amino terminus of the protein. Three members of this family
have been so far described.
3.1.2.1. MAGI-1/BAP-1. This protein co-localizes with ZO-1 at the TJ of epithelial cells (Ide
et al., 1999b). MAGI-1 appears to be a tumor suppressor, since the tumorigenic potential of the
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viral oncoproteins of the human adenovirus type 9 (E4-ORF1) and the high-risk human
papillomaviruses (E6) depends on their ability to sequester or to target MAGI-1 for degradation
(Glaunsinger et al., 2000). MAGI-1 interacts at the TJ with the signaling molecule GEP. The
latter is aGDP/GTPexchangeprotein specific for the small G protein RAP (Mino et al., 2000). Atthe glomerular podocytes MAGI-1 associates with the transmembrane glycoprotein megalin
(Patrie et al., 2001). Three splicing variants of MAGI-1 have been characterized: MAGI-1a, -1b
and -1c. MAGI-1a is found in soluble and insoluble cellular fractions. MAGI-1b localizes to the
basolateral membrane of epithelial cells and forms complexes with b-catenin and E-cadherin
during junction formation (Dobrosotskaya et al., 1997). MAGI-1c contains three bipartite
nuclear localization signals and is predominantly found at the nucleus of epithelial cells
(Dobrosotskaya and James, 2000). At the neuromuscular junction MAGI-1c interacts with
MuSK, a tyrosine kinase receptor active in differentiation (Strochlic et al., 2001).
3.1.2.2. MAGI-2. MAGI-2 is found at synaptic junctions where it functions as a scaffoldingprotein (MAGI-2/S-SCAM,synapticscaffoldingmolecule). The identification of MAGI-2 ligands
is important for the elucidation of the structure of synaptic junctions. It interacts through its GK
region, with SAPAP (SAP90associatedprotein), and by its PDZ domains with NMDA receptors,
neuroligins, MAGUIN (membrane associated guanylate kinase-interacting protein-1), b1-
adrenergic receptor, and GEP (Hirao et al., 1998; Ohtsuka et al., 1999; Xu et al., 2001; Yao
et al., 1999). MAGI-2 associates with atrophin-1 (MAGI-2/AIP1, atrophininteractingprotein), a
protein with a polyglutamine repeat expansion, which is responsible for dentatorubral and
pallidoluysian atrophy (Wood et al., 1998). In epithelial cells MAGI-2 localizes the TJ, where it
forms a complex through its second PDZ repeat with the carboxyl terminal end of PTEN. The
latter is a tumor suppressor that functions as a catalyst for the removal of 3-phosphate from
phosphatidylinoditol 3,4,5,-triphosphate. This phospholipid is a product of PI3-kinase, and isinvolved in the activation of the protooncogene AKT/PKB that suppresses apoptosis (Wu et al.,
2000a). Phosphorylation of PTEN tail causes a conformational change that results in the masking
of the PDZ binding domain (Vazquez et al., 2001). MAGI-2 also bindsb and d catenin (Ide et al.,
1999a; Kawajiri et al., 2000). Three isoforms of MAGI-2, a; b and g have been characterized(Hirao et al., 2000).
3.1.2.3. MAGI-3. MAGI-3 localizes in epithelial cells at the TJs where it binds through its second
PDZ domains to the tumor suppressor PTEN. In the synaptic junction MAGI-3 associates
through its fifth PDZ repeat to the NMDA receptor (Wu et al., 2000b).
3.1.3. PAR proteins of the TJ
PAR are partitioning-defective proteins, required for embryonic polarity. In C. elegans for
example, PAR-3 localizes asymmetrically at the anterior periphery of one cell embryos. Mutations
in PAR-3 alter the polarized distribution of other proteins involved in cell fate determination and
the orientation of the mitotic spindles in successive cell cycles (Bowerman et al., 1997; Ebnet et al.,
2001; Guo and Kemphues, 1996).
In epithelial cells PAR-3, a three PDZ containing protein, localizes at TJs, where it directly
binds through its second PDZ domain to the carboxyl terminus of JAM (Itoh et al., 2001). PAR-3
forms a complex with PAR-6, a one PDZ possessing molecule with a CRIB domain (Johansson
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et al., 2000), and atypical PKCs l and z (PAR-3/ASIP, atypical PKC isotype-specific
interacting protein) (Izumi et al., 1998). Expression of a dominant negative mutant atypical
PKC causes mislocalization of PAR-3 and cell surface polarity impairments, suggesting a role
for the PAR protein complex in the apico-basal polarization of epithelial cells (Suzuki et al.,2001).
Par-6 inhibits TJ reassembly after junctional disruption induced by Ca2 depletion, but does
not inhibit adherens junction formation. The amino terminal fragment of PKC z;which binds toPAR-6, also inhibits TJ assembly (Gao et al., 2002). In accordance, we have observed that the
MAGUK protein ZO-2 is a phosphorylation target for atypical PKCs, and that the
phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex (Avila-
Flores et al., 2001).
PAR-6 is a binding partner for the Rho GTPases Cdc42-GTP and Rac1 (Johansson et al.,
2000). Hence PAR-6 is a key adaptor that links Rac1, Cdc42 and atypical PKCs to PAR-3.
Binding of Cdc42-GTP to PAR-6 enhances the activity of the atypical PKCs (Yamanaka et al.,2001), and thus the activated Cdc42 disrupts TJs (Gao et al., 2002).
Recruitment of the PAR-3/PAR-6/CDC-42/aPKC complex to the TJs appears to be mediated
by the tethering of PAR-3 to JAM (Itoh et al., 2001).
3.1.4. MUPP1
MUPP1 is a protein that contains 13 PDZ domains (multi-PDZ domainprotein 1); therefore it
is suggested to function as a multivalent scaffold protein. It is exclusively concentrated at TJs
where it interacts through its PDZ 10 with claudins and by its PDZ 9 with JAM (Hamazaki et al.,
2002). Thus, MUPP1 functions as a cross linker between claudin-based TJ strands and JAM
oligomers in TJs. Other integral proteins might also be tethered to the claudin-based TJ strands
through MUPP1 molecules. In fact, a serotonin receptor (Ullmer et al., 1998), the protooncogenethat encodes the receptor for the stem cell factor named c-Kit (Mancini et al., 2000) and the
membrane spanning proteoglycan NG2 (Barritt et al., 2000) respectively bind to MUPP1s PDZ
domains 10, 10 and 1, although their recruitment to TJs remains unclear.
Within the amino terminus of MUPP1 a novel proteinprotein interaction domain has been
found. Since this domain has the ability to bind and recruit MAGUK protein Pals1 to TJ, it has
been named MAGUK recruitment domain (MRE) (Roh et al., 2002b).
The major oncogenic determinant for human adenovirus type 9 (E4-ORF1) aberrantly
sequesters MUPP1 within the cellular cytoplasm, whereas the high-risk human papilloma viruses
determinant HPV-18 E6 targets MUPP1 for degradation. Consequently MUPP1 is proposed to
be negatively involved in regulating cellular proliferation (Lee et al., 2000).
3.1.5. AF-6/Afadin
AF-6 is the ALL-1 fusion partner at chromosome 6. The ALL-1/AF6 chimeric protein is a
critical product associated with acute human leukemia (Prasad et al., 1993).
AF-6 is a 205 kDa multidomain protein that contains two Ras-binding domains within the
amino terminus, followed by kinesin and myosin like domains, and a PDZ module at the middle
of the protein. At the carboxyl terminal AF-6 contains three proline-rich domains followed by a
F-actin binding region. Af-6 is a component of tight (Yamamoto et al., 1997) and adherens
(Mandai et al., 1997) junctions.
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AF-6 is a target of Ras and of several related proteins (Rap1A, Rit, Rin and M-Ras) (Boettner
et al., 2000; Quilliam et al., 1999; Shao et al., 1999; Yamamoto et al., 1997). ZO-1 interacts with
the Ras binding domains of AF-6 and this association is inhibited by activated Ras. The over
expression of activated Ras perturbs cellcell contacts and decreases the accumulation of AF-6and ZO-1 at the cell borders (Yamamoto et al., 1997). AF-6 also interacts with cingulin, another
TJ plaque protein (Cordenonsi et al., 1999a).
AF-6 can directly associate through its PDZ domain with the TJ integral protein JAM. Since
both AF-6 and ZO-1 associate to the PDZ type II binding motif of JAM (Ebnet et al., 2000), these
complexes are mutually exclusive and should therefore be involved in different functions. The fact
that AF-6 is transiently expressed at the cell contacts of epithelial lines suggests, that this protein
might be more important for the formation of junctions that for the maintenance of stable
junctional complexes.
AF-6 is also a constituent of a novel cellcell adhesion system named NAP, which localizes at
adherens junctions (Asakura et al., 1999). The NAP complex is composed ofnectin, afadin andponsin. Nectin, whose name derives from the Latin word necto meaning to connect is a
molecule member of the immunoglobulin superfamily, previously identified as a poliovirus and
alpha herpes virus receptor. It associates to afadin through its carboxyl terminal PDZ binding
motif (Takahashi et al., 1999). Ponsin, whose name derives from the Latin word pons, meaning
bridge, is capable of binding to the proline-rich regions of AF-6 through its SH3 domain, and to
vinculin. Thus ponsin connects the NAP system with the cadherincatenin adhesion junctions
(Mandai et al., 1999).
AF-6 associates with profilin, a protein that activates monomeric actin units for subsequent
polymerization and participates in cortical actin assembly (Boettner et al., 2000). Thus Af-6
through its interaction with profilin could modulate actin modeling at the adhesion complexes.
AF-6 forms a complex with and serves as a substrate for Fam, a deubiquitinating enzymeproduct of thefat facetsgene (Taya et al., 1998). Fam probably maintains the stability of cellcell
contacts by deubiquitinating the components of intercellular adhesions, therefore its recruitment
to tight and adherens junctions through AF-6 might be crucial.
AF-6 is a critical regulator of cellcell junctions during development. Thus a null mutation in
the Af6 locus disrupts epithelial intercellular junctions and cell polarity during mouse
development (Zhadanov et al., 1999). Furthermore, during Drosophila embryogenesis, the
reduced expression of ZO-1 and the AF-6 homologue, Canoe, generates a failure in the embryonic
dorsal closure (Takahashi et al., 1998).
AF-6 has a splicing variant of 190 kDa (s-AF-6) that lacks the F-actin binding domain and the
third proline-rich domains, and is abundantly expressed in neuronal tissues (Mandai et al., 1997).AF-6 localizes at post-synaptic densities where it interacts and clusters with some Ephrin receptor
tyrosine kinases (RTKs) (Buchert et al., 1999). AF-6 is a phosphorylation substrate of the Eph
receptor. Since in neural tissues AF-6 accumulates in post-synaptic densities whereas ZO-1
localizes at pre-synaptic terminals, it is assumed that in this system they fulfill different roles.
3.1.6. PATJ
PATJ contains 10 PDZ domains and concentrates at the TJ of epithelial cells (Roh et al.,
2002b), although it is also found at the apical plasma membrane. Over expression of PATJ
disrupts the TJ localization of ZO-1 and ZO-3, thus suggesting that it might be involved in
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regulating the integrity of TJs. In epithelia antibodies against PATJ recognize proteins of 230,
200, 135 and 75 kDa bands (Lemmers et al., 2002).
PATJ was cloned by homology to Drosophila INAD and thus named human INAD-like
protein (hINADl) (Philipp and Flockerzi, 1997). INAD is a protein with 5 PDZ domains thatparticipates in photo-transduction in Drosophila. Recent findings however, have shown that its
similarity is stronger for Dlt. The latter is a 4 PDZ containing protein that co-localizes at the
apical region with dCrumbs, and together play a crucial role in regulating cell polarity in
Drosophila. The organization of PATJ is similar to MUPP1s, thus it has been suggested that they
are paralogues.
PATJ recruits Pals1 to TJ (Pals1 associated tight junctions protein) through the specific
interaction between the L27N domain of latter and the MRE domain of PATJ. Pals1/PATJ
interaction is not crucial for PATJ targeting to TJs. The 6th and 8th PDZ modules of PATJ
interact with ZO-3 and claudin-1, respectively, through the type I PDZ binding domains present
in the carboxyl terminal ends of these proteins. While the interaction with the former appears tobe crucial for PATJ targeting to TJ, deleting the 8th domain has little effect on PATJ localization.
Therefore PATJ is proposed to be recruited to TJ by its association to ZO-3 (Roh et al., 2002a).
3.2. TJ proteins lacking PDZ domains
Several submembranous proteins of the TJ do not contain PDZ domains. Some are involved in
vesicular trafficking to the TJ, other bridge integral TJ proteins to the actin myosin cytoskeleton,
certain are transcription factors or proteins with nuclear functions, while the task of others still
remains unclear (Table 3).
3.2.1. CingulinCingulin was named from the Latin word cingere which means to encircle. It is a 140
160 kDa protein that localizes at the TJ submembranous region of epithelial and endothelial cells
(Citi et al., 1988). Cingulin has globular head and tail domains and a central a-helical rod region.
The latter is responsible for the formation of coiled-coil parallel dimmers which can further
aggregate though intermolecular interactions. The globular head of cingulin interacts with ZO-2
(residues 150295 ofXenopuscingulin), ZO-3, AF-6, JAM, F-actin and myosin. ZO-3 and myosin
are also capable of interaction with the rest of the cingulin molecule (Bazzoni et al., 2000;
Cordenonsi et al., 1999a; DAtri and Citi, 2001; DAtri et al., 2002). The interaction with ZO-1 is
complex, since a sequence remarkably conserved at the head ofXenopus(residues 4155), mouse
(residues 4357) and human (residues 4862) cingulin denoted as ZIM, is required for ZO-1binding in pulldown experiments (DAtri et al., 2002). Yet thisZO-1interactionmotif appears not
to be sufficient for ZO-1 binding, as GST fusion proteins containing only such cingulin residues
fail to interact with ZO-1. The amino terminal region of cingulin, containing the ZIM domain, is
capable of targeting transfected cingulin to the junctions only when fused to rod-tail sequences.
Furthermore, deletion of ZIM does not abolish junctional recruitment of cingulin. These results
therefore suggest the requirement of multiple protein interactions for junctional localization to
proceed. In fibroblasts that lack the molecular context of TJ (e.g. occludin and claudins) but
contain cadherin-based cellcell adhesion sites with ZO-1, the ZIM domain of cingulin is required
for recruitment to cellcell adhesion sites. The interaction between cingulin and ZO-1 might be
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3.2.2. Symplekin
The name symplekin derives from Greek and means to weave or tie together. This 127 kDa
protein, is found at the TJ of epithelial cells, but is absent from endothelia. Symplekin can also be
detected in the nucleoplasm of a wide range of cells including those devoid of any stable cellcellcontact (Keon et al., 1996). In Xenopus oocytes symplekin has been located at Cajal bodies.
Symplekin contains 4 putative NLS and interacts at the nuclei with CstF and CPSF, both
constituents of the 30-end cleavage and polyadenylation complex of mRNA precursors. Symplekin
also has a significant similarity with the yeast protein PTA1, that is another component of the
polyadenylation machinery (Hofmann et al., 2002; Takagaki and Manley, 2000). Surprisingly,
symplekin is found in the cytoplasm associated to CPSF proteins, suggesting their involvement in
cytoplasmic polyadenylation and in the regulation of translation.
3.2.3. 7H6 antigen/barmotin
A monoclonal antibody generated against a rat bile canaliculus rich membrane fraction,recognized a novel TJ cortically associated protein named 7H6 antigen (Zhong et al., 1993). This
155 kDa protein shows homology to the SMC family. These are alpha helical coiled-coil proteins
with a putative ATPase domain (Ezoe et al., 1995). Due to these structural characteristics antigen
7H6 was also named barmotin (Muto et al., 2000). Phosphorylation of 7H6/barmotin is closely
related to its localization at the TJs, thus cellular ATP depletion reversibly dissociates 7H6/
barmotin from the junction (Zhong et al., 1994a). 7H6/barmotin appearance at TJs correlates
with the maintenance of the paracellular barrier function in epithelial (Zhong et al., 1993),
endothelial (Satoh et al., 1996) and mesothelial (Tobioka et al., 1996) cells. Furthermore, in HGF-
induced cell spreading (Muto et al., 2000), liver carcinogenesis (Zhong et al., 1994b), primary
biliary cirrhosis (Sakisaka et al., 2001), under treatment with a bacterial lipopolysacharide
(Kimura et al., 1997) and upon exposure to Helicobacter pylori(Suzuki et al., 2002), a reducedexpression of 7H6/barmotin is found that correlates with disruption of cellular polarity,
adhesiveness and an increased paracellular permeability.
The appearance of 7H6/barmotin at developing TJs suffers a transition from a dotted
arrangement to a continuous honeycomb linear appearance only after ZO-1 and occludin have
completely surrounded the cellular borders (Kimura et al., 1996). Therefore instead of being a
constituting protein of the TJ, 7H6/barmotin might play a role in TJ maintenance and
maturation.
3.2.4. Rab proteins
Rab proteins are monomeric molecules that bind GDP/GTP and exhibit an intrinsic GTPaseactivity. They constitute a branch of the Ras superfamily of G proteins and are closely related to
the yeast Ypt1 and Sec4 gene products. Rab proteins are involved in vesicle trafficking and thus
cycle between cytosolic and membrane bound forms (Novick and Zerial, 1997).
3.2.4.1. Rab13. In fibroblasts Rab13 associates with vesicles throughout the cytoplasm, while in
epithelial cells it accumulates at the TJ (Zahr
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