The intercellular junctions of epithelial and endothelial cells are specializations of the cell membrane that physically integrate individual cells into tissues. These junctions consist of distinct substructures visible via ultrastructural analysis, such as tight junctions (TJs), adherens junctions (AJs), or desmosomes, with different functions at the level of the individual cell, of the organ, and of the entire organism, which, for example, include apical-basal polarity, barrier formation, resistance to the mechanical strain and propagation of mechanical forces, and morphogenesis ^{[1][2][3][4][5]}[1,2,3,4,5]. The integrity of these junctions is necessary for the maintenance of functional tissues. Abnormalities in the organization or function of the junctions are frequently associated with disorders like inflammation and malignant transformation ^[6][7][8][6,7,8]. Consistent with the necessity to develop adhesive systems as a prerequisite for the development of multicellular organisms, the origination of proteins mediating cell–cell adhesion dates back to the closest extant unicellular relative of multicellular organisms ^[9][10][9,10]. Interestingly, while AJs are used to mediate adhesion in the earliest metazoa, both desmosomes and TJs exist only in vertebrates ^[11][12][11,12]. Desmosomes have probably evolved from AJs as they use similar molecular components to link adjacent cells [12], whereas TJs have probably evolved from the Septate Junctions (SJs) of invertebrates, which are morphologically different from TJs but are functionally homologous to TJs and contain molecular components that are related to the components found in vertebrate TJs [13].

All three types of junctions are composed of several multiprotein complexes. These complexes consist of at least one integral membrane protein that interacts with a cytoplasmic protein which, together with other scaffolding proteins, forms a junctional plaque that is linked to the actin cytoskeleton, the intermediate filament system, or the microtubule system ^[14][15][16][14,15,16]. In addition to their role in connecting the cellular filamental systems to the cell–cell junctions, all three types of cell junctions serve as hubs for signaling events that regulate proliferation, differentiation, or cell migration ^[17][18][19][17,18,19]. The integral membrane proteins localized at the different types of junctions thus not only physically link adjacent cells to form a structural continuum of epithelial cells, but also regulate the specific localization of signaling hubs along the intercellular junctions.

Interestingly, the most diverse repertoire of adhesion molecules and integral membrane proteins is localized at the TJs. As opposed to AJs and desmosomes, which use cadherins and members of the nectin family of the immunoglobulin superfamily (IgSF) to interact with adjacent cells, TJs make use of various members of the IgSF, members of the claudin family, members of the TJ-associated Marvel protein (TAMP) family, and members of the Crumbs family of integral membrane proteins to regulate their function.

Integral membrane proteins localized at the TJs can be classified into four major groups: Crumbs homolog 3 (CRB3), a member of the Crumbs family of proteins; claudins; TAMPs; and members of the IgSF of adhesion molecules (Figure 1).

2. CRB3

CRB3 is a member of the vertebrate Crumbs family of proteins, which are homologous to Drosophila Crumbs. CRB3 and has a very short extracellular region that consists of 33 amino acids (AA), a single transmembrane region, and a short cytoplasmic region of 40 AA ^[20][26]. In addition to its localization at TJs, CRB3 is also localized at the apical membrane domain of epithelial cells ^[21][27]. As opposed to Drosophila Crumbs, the vertebrate CRB3 isoform localized at the TJs is most likely not involved in homophilic or heterophilic interactions ^[22][28].

3. Claudins

Claudins are a family of tetraspan transmembrane proteins comprising 27 members ^[23][24][29,30]. Notably, claudins support cell aggregation when expressed in fibroblasts, indicating that their adhesive activity not only regulates their localization at homotypic cell–cell contacts but also contributes to the physical cell–cell adhesion at the TJs ^[25][31]. A central property of claudins is their ability to multimerize by interacting both in cis and in trans with either the same or a different claudin family member to form strands that are visible via freeze-fracture electron microscopy (EM) ^[26][27][32,33]. Claudin-based strands can act as occluding barriers for water and small solutes, as well as anion- or cation-selective paracellular channels, and are the principal paracellular permeability regulators of epithelial and endothelial barriers ^[28][34].

4. TAMPs

Members of the TAMP family include occludin, tricellulin/MarvelD2, and MarvelD3 ^[29][35]. Similar to claudins, TAMPs are tetraspan transmembrane proteins, but based on their sequence homologies, they are not related to claudins. Their characteristic feature is a conserved four-transmembrane “MAL and related proteins for vesicle trafficking and membrane link” (Marvel) domain ^[30][36]. Of the three TAMPs, tricellulin is unique as it is enriched at sites of contact between three cells (tricellular TJs, tTJs) ^[31][37]. Heterotypic interactions between tricellulin and MarvelD3, as well as between occludin and MarvelD3, have been described via co-immunoprecipitation (CoIP) and via Förster resonance energy transfer (FRET) experiments. These interactions most likely occur in cis ^[29][32][35,38]. A homophilic interaction in trans has been found for occludin but not for tricellulin or MarvelD3 ^[32][33][38,39]. While several studies have suggested that TAMPs per se are not essential for the development of an epithelial barrier function ^[34][35][36][40,41,42], recent findings indicate that tricellulin is required for the establishment of the barrier function in mammary gland-derived epithelial cells ^[37][43].

5. IgSF Proteins

IgSF members localized at the TJs include the junctional adhesion molecule (JAM) family members JAM-A and JAM-C ^[38][44], and the JAM-related adhesion molecules Coxsackie- and Adenovirus-Receptor (CAR), JAM4, CAR-like membrane protein (CLMP), and Endothelial Cell-Selective Adhesion Molecule (ESAM, in endothelial cells) ^{[39][40][41][42]}[45,46,47,48]. All these IgSF proteins can undergo trans-homophilic interactions which stabilize their localization at cell–cell contacts. In addition, the trans-homophilic activities of CAR, JAM4, CLMP, and ESAM support cell aggregation after transfection in cells ^{[39][40][41][43]}[45,46,47,49], suggesting that their adhesive activities contribute to the strength of the physical interaction at the TJs. Angulins (Angulin-1, -2, -3) are IgSF proteins with a single N-terminal Ig-like domain ^[44][50]. Among all other IgSF proteins localized at the TJs, angulins are unique in that they are enriched at the tTJs ^[45][51]. A main function of all three angulins is to recruit tricellulin to the tTJs ^[45][46][51,52]. The ectopic expression of angulin-1 in L cell fibroblasts resulted in angulin-1 enrichment at cell–cell contacts, suggesting that angulin-1 is engaged in homophilic or heterophilic trans interactions ^[46][52]. Strikingly, many of the integral membrane proteins localized at the TJs contain a C-terminal PDZ domain-binding motif (PBM), through which they can interact with the PDZ domains present in many TJ-localized scaffolding proteins (Table 1).

Integral Membrane Protein	Size (AA)	Cytoplasmic Region (AA)	COOH-Terminal Residues (PBM)
CRB3	120	40	- EERLI (+)
JAM-A	299	40	- SSFLV (+)
JAM-C	310	48	- SSFVI (+)
CAR	365	107	- DGSIV (+)
JAM4	407	122	- NTTVV (+)
ESAM	390	121	- AGSLV (+)
Angulin-1/LSR	649	369	- ESLVV (+)
Angulin-2/ILDR1	546	358	- RSVVI (+)
Angulin-3/ILDR2	639	432	- MSLVV (+)
Claudins (1–26)	207–305	27–66	- XXXYF (+)
Occludin	522	257	- DRQKT (−)
Tricellulin	558	196	- VQGYS (−)
MarvelD3	401	20	- EMFEF (+)

The presence of multiple PDZ domains in many scaffolding proteins localized at the TJs (Figure 1), combined with the promiscuity of PDZ domains in ligand binding, allows for the incorporation of several integral membrane proteins in a single scaffolding protein-organized complex. Vice versa, individual membrane proteins can recruit and assemble distinct protein complexes at the TJ. These biochemical properties may, in part, explain the high complexity and dynamics of protein complexes at the TJs ^[21][27].