Adhesion Among Cadherin Cell Dissertation Heterophilic Homophilic Interaction Molecule

Abstract

Cadherins constitute a superfamily of cell–cell adhesion molecules expressed in many different cell types that are required for proper cellular function and maintenance of tissue architecture. Classical cadherins are the best understood class of cadherins. They are single membrane spanning proteins with a divergent extracellular domain of five repeats and a conserved cytoplasmic domain. Binding between cadherin extracellular domains is weak, but strong cell–cell adhesion develops during lateral clustering of cadherins by proteins that link the cadherin cytoplasmic domain to the actin cytoskeleton. Understanding how different regions of cadherins regulate cell–cell adhesion has been a major focus of study. Here, we examine evidence of the structure and function of the extracellular domain of classical cadherins in regard to the control of recognition and adhesive contacts between cadherins on opposing cell surfaces. Early experiments that focused on understanding the homotypic, Ca++-dependent characteristics of cadherin adhesion are discussed, and data supporting the widely accepted cis- and trans-dimer models of cadherins are analyzed.

Keywords: Cadherin, Cell–cell adhesion, Calcium, Structure, Dimer

1 Introduction

Cadherins are an important superfamily of cell–cell adhesion proteins comprising over 40 members (Takeichi 1990), and perhaps even more when the proto-cadherin family is included (Frank and Kemler 2002). Cadherins are expressed in many cells and tissues, and are evolutionarily conserved in vertebrates and invertebrates (Kemler 1992; Takeichi 1995; Gumbiner 1996). Here we focus on a subset of cadherins, the classical cadherins, that are the best described and understood. We focus on the structure and function of the extracellular domain that controls recognition and adhesive contacts between cadherins on opposing cell surfaces.

Classical cadherins, of which E-, P-, N-, and R-cadherin are members, were shown to mediate segregation of different cell populations in early studies. Segregation was based on homophilic adhesion in which cells expressing one cadherin subtype (E-cadherin for example) segregated in suspension from cell expressing a different cadherin subtype (P-cadherin). Cadherin-mediated homotypic adhesion appears to depend on binding specificity, Ca++-dependence, and molecular contacts of cadherins and cadherin–cadherin adhesion. Sequence analysis of classical cadherins reveals that they have five tandemly repeated domains in the extracellular domain, termed extracellular cadherin repeats 1–5 (EC1–5) (Fig. 1) with EC1 at the amino terminus (Hatta et al. 1988). When synthesized, cadherins contain signal and precursor peptides that are cleaved during processing and maturation of the protein in the endoplasmic reticulum (ER) and Golgi. The precursor peptide is cleaved before cell surface presentation of the cadherin, and cleavage is required for adhesive function (Ozawa and Kemler 1990).

Fig. 1

Schematic of a classical cadherin. The schematic depicts the domain organization of a classical cadherin. Five extracellular repeats (EC1–5) are preceded by a precursor peptide which is cleaved during maturation. Ca++-binding sites (+ marks) are...

How do cadherin extracellular domains interact to form cell–cell adhesions? The most prevalent models describe two cadherins within the same membrane forming a lateral or cis-dimer and that this dimer promotes adhesive dimerization (trans-dimer) of cadherins on adjacent cells (Fig. 2). The models differ in their description of the organization and mechanism of the extracellular domains in the trans-dimers. Two models describe trans-dimerization through EC1 alone while a third describes complete intercalation of cadherin extracellular domains. Each model further depicts that both cis- and trans-dimerization depend on Ca++ to bind between each EC repeat to stabilize and order extracellular domains. We will analyze the data supporting these conclusions.

Fig. 2A–C

General methods to examine cadherin adhesion functions. A Schematic of cadherin distribution during epithelial cell–cell adhesion as imaged by live cell microscopy of Madin-Darby canine kidney (MDCK) cells expressing green fluorescent protein...

2 Ca++ and Cadherin Adhesion

Understanding the Ca++-dependence of cadherin adhesion was addressed in early experiments. Ca++ was shown to protect the cadherin extracellular domain from degradation by trypsin, suggesting a role in structural organization of the domain. However, the mechanism remains unclear. After solving the protein sequence of uvomorulin (E-cadherin), Ringwald et al. (1987) identified three internal repeats in the extracellular domain. Within these domains they identified two distinct putative Ca++-binding loops based on sequence homology to known Ca++-binding regions. Although the analysis of the extracellular domain was not exactly correct, the authors first demonstrated the basic structure of the cadherin extracellular domain, a functional domain composed of distinct, repeated units each of which are likely able to bind Ca++. A year later with the sequencing of N-cadherin and use of a different alignment program, the five internal cadherin repeats were identified (Hatta et al. 1988). The authors, however, did not comment on Ca++ binding sites. Nevertheless, sequence alignment of the known cadherins shows conservation in each of the five internal repeats of the putative Ca++-binding sites proposed by Ringwald et al. (1987).

Evidence of the functional role of Ca++ in cadherin adhesion came initially from studies on the recombinant extracellular domain of E-cadherin (Pokutta et al. 1994). Electron microscopy of the recombinant domain directly showed dependence on Ca++ for elongation and maintenance of a rigid bent rod-like structure, but the curvature was not commented upon. In the absence of Ca++, the domain was apparently disordered and not elongated, and resembled a globular structure. Ca++ binding was reversible. These conformational changes were confirmed by circular dichroism spectroscopy. A large change in the ellipticity of the CD spectrum was observed between 202–215 nm after removal of Ca++. The change in CD spectra was used to examine conformational changes at different Ca++ concentrations, and an average Kd of 42–45 μM was determined. As there were multiple Ca++ ions known to bind to the cadherin extracellular domain, it was not possible to determine whether all sites have the same Kd or the value obtained was an average. The authors also noticed an increase in the fluorescence of tryptophan upon Ca++ binding. They used this characteristic to titrate Ca++ as well and determine two different Kd values, one at 130 μM and the second at 210 μM. The results suggested that the Kd obtained from titration based on CD spectrum changes was an average, and the change in tryptophan fluorescence measured a Ca++ binding site of low affinity. Finally, Pokutta et al. (1994) measured Ca++ affinities by protection against tryptic digestion. From these experiments they observed a Kd of 24 μM, but there appeared to be cooperative binding here as well. Though unable to satisfactorily measure exact dissociation constants of the different Ca++ binding sites, the authors were able to prove the existence of high and low affinity binding sites and first observed and modeled the mechanism of Ca++-dependent adhesion whereby Ca++ acted to rigidify the extracellular domain.

The dissociation constants of Ca++ at the various binding sites were also determined using a fragment of the first two EC repeats of E-cadherin (ECAD12) (Koch et al. 1997). By monitoring the CD spectra change of ECAD12, an average Kd of 360 μM for the Ca++ binding sites between EC1 and EC2 was recorded. ECAD12 bound three Ca++ ions, and using equilibrium dialysis two Kd values of 330 μM and one Kd value of 2 mM were determined. Interestingly, when the Ca++ binding sites of the full E-cadherin extracellular domain were analyzed by equilibrium dialysis, an average Kd of only 30 μM was calculated for a total of nine Ca++ ions bound to the protein fragment.

3 Cadherin Adhesion Structure: Trans-Dimer

3.1 Early Studies

Initial studies of cadherin extracellular domain adhesion focused on determining which EC repeats are involved in adhesion and specificity. Nose et al. (1990) used a domain swapping strategy to determine the location of sites controlling cadherin subtype specificity (i.e., which domains controlled E↔E, P↔P specificity). When expressed in cadherin-deficient fibroblast L cells, wildtype E- and P-cadherins mediated sorting and segregation of cells expressing the same cadherin (Fig. 3). However, when E-cadherin EC1 was switched with EC1 of P-cadherin to generate a chimera of P-cadherin EC1 and E-cadherin EC2–5, the chimeric protein now mediated adhesion with L cells expressing P-cadherin. The authors concluded that EC1 contained sites that determine cadherin adhesion specificity. They further narrowed the region to between residues 61 and 113. However, it is noteworthy that swapping sub-regions of EC1 (residues 1–31 and 1–67) did not provide a complete switch in adhesion specificity. Rather, there was a decrease in specificity; chimeric proteins were able to mediate some adhesion between either E-cadherin- or P-cadherin-expressing cells. Thus, adhesion specificity is located in the amino terminal EC1 repeat, but it cannot be excluded that other sites outside EC1 are also involved. Nevertheless, a role for EC1 was also supported by early studies using monoclonal antibodies directed against EC1 that block the adhesion function of cadherins (Yoshida-Noro et al. 1984; Behrens et al. 1985; Gumbiner and Simons 1986; Hatta and Takeichi 1986; Nose and Takeichi 1986). However, the residues recognized by these antibodies and involved in adhesion remain unknown.

Fig. 3A–C

Cadherin adhesion models. ATrans-dimerization model of interdigitated cadherin domains by Sivasankar et al. (2001). Cis-dimerized cadherins in opposing membranes are able to bind through several different mechanisms. One mechanism involves complete interdigitation...

Tripeptides based on protein–protein interfaces have been useful in examining adhesion of integrins to extracellular matrix, and a similar strategy has been used with cadherins. Blashuck et al. (1990) hypothesized a tripeptide adhesion sequence was present in cadherins. Sequence analysis identified several potential tripeptide adhesion sequences, and they tested whether these peptides blocked normal cadherin–cadherin adhesion, aggregation, and blastocyst compaction. The authors showed that only the histidine-alanine-valine (HAV) sequence from the EC1 repeat of all classical cadherins is likely important in adhesion. Mouse blastocysts incubated with a decapeptide derived from N-cadherin containing the HAV sequence failed to compact. Furthermore, rat dorsal root ganglia did not extend neurites over astrocytes in the presence of the HAV-containing peptide. Both blastocyst compaction and neurite extension are mediated by E-cadherin and N-cadherin, respectively. Because the HAV sequence is found in the EC1 of all classical cadherins, this evidence gives further support to the developing model of EC1 involvement in mediating both adhesion and specificity of cadherins.

The HAV sequence has continued to be studied as a potential mediator and recognition sequence of cadherin adhesion. Based on the inhibitory aspect of the HAV-containing decapeptide, Williams et al. (2000) showed that cyclic peptides containing HAV were better than linear peptides in inhibiting N-cadherin-mediated neurite extension of cerebellar neurons over N-cadherin-expressing 3T3 cells. In addition, they found that incorporation into the cyclic peptides of specific residues flanking the HAV sequence in N-cadherin increased their inhibitory activity. Interestingly, if they incorporated flanking residues from E-cadherin rather than N-cadherin, the cyclic peptides did not inhibit N-cadherin adhesion. It should be noted that several of the peptides derived from N-cadherin sequence failed to inhibit N-cadherin adhesion. It would have been more informative if the E-cadherin-derived cyclic peptides were shown to inhibit E-cadherin, but not N-cadherin adhesion. Further evidence for the involvement of EC1 in cadherin adhesion was revealed in a study from the same group on small peptide agonists of cadherin adhesion (Williams et al. 2002). The authors demonstrated that a recombinant N-cadherin EC1 domain was able to inhibit neurite outgrowth from cerebellar neurons over 3T3 cells expressing N-cadherin. Together, these results demonstrate that residues flanking the HAV sequence are potential mediators of cadherin adhesion specificity.

Despite strong evidence, using a variety of experimental approaches, for a role of EC1 in adhesion, some evidence points to a model in which additional EC repeats are required for adhesion. The monoclonal antibody DECMA-1 blocks E-cadherin adhesion (Vestweber and Kemler 1985). Mapping of the epitope showed that it is directed against the EC4/EC5 boundary rather than EC1, as shown for other inhibitory antibodies (see above and Fig. 1) (Ozawa et al. 1990). Ozawa et al. (1990) showed also that E-cadherin contains at least one disulfide bond, and that this bond is important, but not required, for cadherin–cadherin adhesion. Sequence analysis showed only EC5 to have potential disulfide bonds [confirmed by crystal structure (Boggon et al. 2002)] adding further support to some role of EC4–5 in cadherin adhesion.

Analysis of crystal structures of cadherin extracellular domains provided new information on molecular interactions between cadherin EC domains. Because EC1 was suspect in adhesive interactions, the initial focus of crystal structure studies was EC1. The first crystal of cadherin EC1 was from N-cadherin (Shapiro et al. 1995). It raised more questions than answers about past evidence of EC1. First, three different crystal forms were observed, one contained a single molecule in the asymmetric unit of the crystal lattice, and the other two contained two molecules per asymmetric unit in different orientations. The molecular interactions described (see below) were observed in all the crystal forms, but mostly only as crystal packing interactions. Second, the structure revealed that the HAV tripeptide, though on the surface, is partially buried and obscured, and a potential dimer could not be assigned to correlate with the HAV sequence (Fig. 4A and 4A′). The structure did help explain why a single linear sequence of the protein could not be identified as the determinant of cadherin specificity.

Fig. 4A–C

Structure of EC1 and strand dimer. A N-cadherin EC1 is shown in a ribbon with the HAV sequence and W2 side chains. A′ Ninety degree rotation of the structure in A. B Close-up of W2 docking in the hydrophobic pocket in the strand dimer. W2 of one...

The shape of the cadherin repeat is a β-barrel structure, and residues in close proximity on the surface of the protein are not necessarily close in the primary structure of the protein. An intriguing interaction is between two domains with their long axes aligned in a roughly parallel, not antiparallel, orientation. The authors suggested that this interaction might be a putative lateral or cis-dimer (see below for discussion of cis-dimers). Additionally, tryptophan 2 (W2) of each domain was inserted into a hydrophobic pocket of the adjacent cadherin in what was termed a “strand dimer” (Fig. 4). The strand dimer was the major characteristic of lateral cadherin dimers. Interestingly, the hydrophobic pocket accepting the side chain of W2 is composed, in part, of the alanine from the HAV tripeptide.

3.2 Recent Studies, Evolving Models

The first crystal structure of the full cadherin extracellular domain showed that the strand dimer, inferred from early structures to represent a cis-dimer, may in fact be representative of the association of cadherins on neighboring cells (Boggon et al. 2002). The structure shows the characteristic strand dimer with the EC1 domains of the two molecules in a roughly parallel orientation; however, the rest of the extracellular domain adopts a curved structure rather than the assumed rigid straight structure (Fig. 4C). The curvature of the structure is such that the long axis of EC1 is roughly perpendicular to the long axis of EC5. Based on this new evidence, the authors proposed a model that the strand dimer mediated trans-dimerization and that cis-dimerization occurred through a previously undescribed interaction.

The key characteristic of the strand dimer is intercalation of W2 into the hydrophobic pocket of an opposing cadherin. Experiments focusing on W2 and the hydrophobic pocket demonstrate their importance (Tamura et al. 1998; Pertz et al. 1999; Ahrens et al. 2002; Perret et al. 2002). Mutation of W2, A78, or A80 (the alanines comprising parts of the hydrophobic pocket) inhibits cell aggregation, bead aggregation, and cell-bead binding. Of particular note are the studies by Pertz et al. (1999) which used a chimeric protein of the E-cadherin extracellular domain fused to the coiled-coil pentamerization domain of cartilage oligomatrix protein (ECADCOMP). Using electron microscopy to examine structure and interactions, Pertz et al. (1999) showed that ECADCOMP forms a pentamer in solution. In the presence of Ca++, the E-cadherin extracellular domain adopts a bent rod structure, two of which in a pentamer can form a ring-like structure, inferred as a putative cis-interaction (Fig. 5). There are instances in which two ring-like structures are in contact in a putative trans-interaction. An ECADCOMP carrying the W2A mutation, while still able to adopt a ring structure, is never seen in association with a second ring. While all other data cannot distinguish between a role in cis- or trans-dimerization, these electron microscopy studies support a model in which W2 docking in the hydrophobic pocket is required for trans-dimerization.

Fig. 5

ECADCOMP conformations. ECADCOMP visualized by rotary shadowing electron microscopy. The top row shows non-dimerized pentamers in a star-like pattern. The middle row shows pentamers in which two or four E-cadherin extracellular domains have formed a ring-like...

A second model has been proposed in which the strand dimer is not directly involved in trans-dimerization (Koch et al. 1999; Pertz et al. 1999). Crystal studies of EC1 and 2 of N-cadherin and E-cadherin showed an apparent cis-dimer lacking the strand dimer. In two of the crystals, the amino terminus was disordered and could not be resolved (Nagar et al. 1996; Tamura et al. 1998). The third crystal showed W2 docking into the hydrophobic pocket of its own protein (Pertz et al. 1999). Pertz et al. (1999) proposed that W2 is not involved in strand exchange and direct intermolecular interactions, but rather is required as an allosteric activator for trans-dimerization. The conclusion was based on the assumption that W2 is required for cis-dimerization, an assumption which the authors proved wrong. Note that ECADCOMP bearing a W2A mutation was seen by electron microscopy to form ring-like structures in an apparent cis-dimerization but was unable to oligomerize into concentric ring structures.

Other studies continue to support a model of W2 as an allosteric effector rather than a direct mediator of trans-adhesion. Nuclear magnetic resonance (NMR) analysis of Ca++-dependent dimers and oligomers of E-cadherin EC1 and 2 [the same construct used by Pertz et al. (1999) for crystallographic studies] suggested that W2 is buried in the hydrophobic pocket of its own molecule (Haussinger et al. 2002). Analysis of {1H} (15N) shifts of W2 indole group revealed no significant change in the orientation of W2 during Ca++-dependent dimerization and oligomerization of E-cadherin EC1 and 2. Additionally, the {1H}-15N nuclear Overhauser enhancements (NOEs) of the W2 indole group in the presence and absence of 600 μM Ca++ indicate considerable flexibility of the residue in both monomeric and aggregated states that would not be expected if it were fixed in the hydrophobic pocket of a neighboring molecule as depicted in the strand dimer model. One caveat of this study, though, is the presence of an N-terminal methionine as a cloning and protein expression artifact. Previous studies showed that precise cleavage of the precursor peptide of cadherins is required for full adhesive functionality (Ozawa and Kemler 1990).

3.3 Further Functional Studies of EC Domains in Adhesion

EC1 was first modeled as the adhesive domain because it was found to be involved in specificity, contained the HAV adhesion sequence, and N-cadherin EC1 packed in an antiparallel orientation as if in a trans-dimer in one crystal form (Blaschuk et al. 1990; Nose et al. 1990; Shapiro et al. 1995). However, the precise orientation of EC1 in the trans-dimer remains unclear. Nevertheless, adhesion through EC1 remains the model best fitting all data, and additional studies have been presented which strengthen this view.

First, further work on ECADCOMP showed that adhesive pentamers oligomerized through the N-terminal regions of E-cadherin (Tomschy et al. 1996; Koch et al. 1999; Pertz et al. 1999). It is unclear, however, exactly which parts of the N-terminus are involved in adhesion. Second, the studies of Perret et al. (2002) demonstrated adhesive events between cadherin fragments of just EC1 and 2. To investigate kinetics of cadherin trans adhesion, they constructed an E-cadherin extracellular fragment consisting of EC1 and 2 with a C-terminal His tag (E-cad1/2). Beads were coated with an antibody against the His tag, and E-cad1/2 was added to them. A mica surface was prepared by adsorbing Ni++ to the surface and then chelating the E-cad1/2 His tag directly. The authors visualized the cadherin-coated beads as they rolled across the cadherin-coated surface and measured the duration and frequency of stop events interpreted as cadherin adhesion between the bead and surface (Fig. 3). An approximately fivefold decrease in the frequency of binding events was observed when the tryptophan analog I3A or an E-cad1/2 with the W2A mutation was used, thereby further supporting a role of EC1 and W2 in adhesion.

These results, however, are different from those of Chappuis-Flament et al. (2001). In similar adhesion flow experiments, the authors used various C-cadherin fragments, missing one or several of the EC repeats, fused to the Fc domain of IgG. The cadherin fragments were bound directly to protein A-coated beads through the Fc domain, and their function was tested by Ca++-dependent bead aggregation. In these experiments, at least three cadherin EC repeats were required for strong Ca++-dependent adhesion. The authors were unable to measure adhesion by bead aggregation or in a flow assay of a cadherin fragment of EC1 and 2 alone. However, EC1 and 2 were shown to be required in all the assays performed, while the additional repeat could be either EC3, EC4, or EC5. The third domain had to be an EC repeat, though, as fibronectin repeats fused to only EC1 and 2 failed to mediate bead aggregation. The differences in experimental design and aims between these experiments and those of Perret et al. (2002), specifically attachment of the cadherin fragment to the beads and analysis of single adhesion events versus bulk adhesion properties, could explain the different conclusions arrived by the two sets of experiments. However, it is clear from both studies that EC1 and 2 are required for adhesion.

A different experimental design has been used to examine a role of additional EC repeats, i.e., other than just EC1 in adhesion. Sivasankar and colleagues, using a surface force apparatus, demonstrated that the strongest adhesive interaction between cadherins on two surfaces occurred at a minimum distance of about 25 nm (Sivasankar et al. 1999; Leckband and Sivasankar 2000; Sivasankar et al. 2001). Interestingly, this is the approximate length of a cadherin extracellular domain if all the repeats would be in a straight line, one after the other. However, as noted earlier, electron microscopy of the extracellular domain of E-cadherin and the crystal structure of the full-length extracellular domain of C-cadherin depict the cadherin extracellular domain as a bent rod (Pokutta et al. 1994; Tomschy et al. 1996; Ahrens et al. 2002; Boggon et al. 2002). Due to the curve of the extracellular domain, the trans-adhesion dimer proposed by Boggon et al. (2002) would occur between surfaces approximately 25 nm apart. Additionally, analysis of oligomerized dimers of E-cadherin, using the c-Jun/c-Fos dimerization system, reveals a measurement of adhesive dimerization between EC1 of approximately 25 nm. If EC5 of each extracellular domain is oriented perpendicular to the surface and each cadherin is interacting with the cadherin on the opposite side (left–right, right–left), then the calculated distance between two theoretical surfaces bearing the E-cadherin c-Jun/c-Fos dimers is approximately 28 nm. In each of these models, however, a single cadherin itself would stand only 10–15 nm from the plasma membrane surface. This conclusion conflicts with measurements of the cadherin extracellular domain height analyzed with the surface force apparatus (Sivasankar et al. 2001). These measurements showed that at least some cadherins can extend up to 20 nm from a surface. Furthermore, adhesive interactions were detected with the surface force apparatus even if the cadherin surfaces were only allowed to approach to 30 and 40 nm of each other. This result suggests that adhesive interactions can occur between cadherins that are not in a bent configuration. Since the structure of the cadherin extracellular domain is potentially a bent rod, measurements with the surface force apparatus do support a model in which the N-terminal EC1 repeat is solely involved in trans-adhesion if the cadherin extracellular domain is able to adopt multiple rigid orientations, but they also support a model in which multiple EC repeats intercalate during cadherin adhesion.

Taken together, we know that EC1 is required for adhesion and at least partly is responsible for homotypic specificity. Multiple EC repeats are likely involved in adhesion as well, but it is unclear whether they directly form adhesive contacts with an opposing cadherin or simply correctly present EC1 to an opposing cadherin. All evidence supports a model in which EC1 alone interacts in adhesion, but further experiments are required: functional adhesion must be verified while identifying or knowing the orientation of the cadherin extracellular domains. The W2 residue plays a role in adhesion, but the exact mechanism remains to be determined. Experiments to date have not been able to distinguish between W2 as an allosteric activator of trans-adhesion or as a direct mediator of trans-adhesion through the strand dimer observed in crystal structures. Finally, though the HAV sequence does appear to have a function in adhesion (mutation of the alanine and use of HAV-containing peptides), the mechanism is unknown. Mutation studies of the other residues in the tripeptide may help to explain the function of HAV. Additionally, structural studies with HAV peptides may help to explain their ability to inhibit adhesion. The precise determination of all residues involved and required for adhesion would help in determining the true molecular interactions of cadherin adhesion.

4 Cadherin Adhesion Structure: Cis-Dimer

Formation of lateral cadherin dimers, referred to as cis-dimers, was first proposed based on crystal structures of EC1 of N-cadherin (Shapiro et al. 1995). This conclusion was based on the observation that individual EC1 domains packed in a parallel orientation, representative of proteins that had originated from the same cell membrane.

The first functional evidence for cis-dimerization of cadherins came from studies using purified C-cadherin extracellular domain (Brieher et al. 1996). C-cadherin extracellular domain separated into two peaks through a gel filtration column. Crosslinking of protein fractions showed that the higher molecular weight peak corresponded to dimer and the lower molecular weight peak corresponded to monomer. The putative dimer from the high molecular weight fractions was shown to have a higher adhesive potential in a cell adhesion assay than the low molecular weight monomer fractions. The authors concluded that these results confirm lateral dimerization of the cadherin extracellular domain-promoted adhesive dimerization. However, whether the extracellular domains in the dimer fractions were in a parallel (cis-dimer) or antiparallel (trans-dimer) orientation was not determined.

A key experiment on lateral dimerization of cadherins was performed in vivo (Takeda et al. 1999). Using cadherin-deficient L cells, full-length, wildtype E-cadherin or a chimeric E-cadherin fused directly to the actin-binding domain of α-catenin (Eαcat) was ectopically expressed. When cadherin-expressing cells were treated with the crosslinking agent 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP) and solubilized, monomers and dimers of E-cadherin were identified. These dimers could arise from either lateral or adhesive trans-interactions. To test this, cells expressing E-cadherin and cells expressing Eαcat were cocultured. The cocultures were crosslinked and then analyzed by immunoblot for α-catenin. Only monomers and homodimers of Eαcat were observed. If the dimers were adhesive dimers, heterodimers of E-cadherin and Eαcat would also be expected. The authors additionally showed that these lateral dimers were only found in adherent cells; if cells were grown in the presence of ethyleneglycoltetraacetic acid (EGTA), low-Ca++ medium, or cadherin-inhibiting antibodies, lateral dimers could not be crosslinked. The conclusion drawn was that lateral cadherin dimers are a functional unit for cadherin adhesion. However, a second conclusion is also supported by the data, that cadherins are only able to be crosslinked into lateral dimers because of the increase in local concentration of cadherins on the cell surface during cadherin-mediated adhesion.

Cis-dimer formation has been further investigated by immunoprecipitation experiments without first crosslinking the proteins (Chitaev and Troyanovsky 1998; Klingelhofer et al. 2000; Shan et al. 2000). Immunoprecipitations showed complexes of cadherins believed to represent cis-dimers isolated from cell lysates. Chitaev and Troyanovsky (1998) provide evidence that E-cadherin forms lateral dimers through a Ca++-independent/W2-dependent mechanism. However, in a following study from the same group, Klingelhofer et al. (2000) suggest E-cadherin and P-cadherin can form hetero cis-dimers through multiple mechanisms. They present data suggesting heterocomplex formation in the presence or absence of Ca++ and also dependent and independent of W2. The W2-independent mechanism required the absence of Ca++. Additionally, Shan et al. (2000) found R-cadherin/N-cadherin heterocomplexes could be coimmunoprecipitated from cells expressing both cadherin subtypes. They showed also that the R-cadherin/N-cadherin interaction appears to be real because E-cadherin, if coexpressed with R-cadherin, is not coimmunoprecipitated with R-cadherin. The fact that these lateral cadherin interactions are Ca++-independent is in direct conflict with the data of Takeda et al. (1999) in which the lateral cadherin dimers they could crosslink were Ca++-dependent. The data from these immunoprecipitation experiments are very difficult to interpret in light of the various models of W2 involvement in trans- and cis-dimerization, and different methods are required to fully address the formation of lateral cadherin complexes in vivo.

Cis-dimerization has also been examined using peptides (Williams et al. 2002). Short peptide HAV-containing antagonists were dimerized so that two adhesion sites were on the same molecule. These peptides were found to act as agonists for neurite outgrowth, a cadherin-mediated adhesion response, on 3T3 cells lacking N-cadherin expression. Dimeric peptides of a second putative adhesion site containing INPISG also activated neurite outgrowth in the cerebellar neuron system. Cyclic monomeric peptides were able to block the activation by these dimeric peptides. It can be concluded that lateral dimerization or clustering of cadherins is able to mediate cellular response. It is not clear, however, if the dimeric peptides promoted adhesion (no assays were performed to test this), and they could act independently of adhesion since N-cadherin binds and activates fibroblast growth factor (FGF) receptors in the neurons (Williams et al. 1994; Saffell et al. 1997; Williams et al. 2001).

Crystal structures of the first two EC repeats show possible lateral dimer organization. Two different crystals have been presented of E-cadherin EC1 and EC2 (Nagar et al. 1996; Pertz et al. 1999). Each shows the two domain fragments aligned lengthwise in a parallel orientation. The region of closest contact is around the Ca++ binding sites between EC1 and 2 such that the proteins together adopt a sort of twisted “X” configuration (Fig. 6). The first crystal structure showed a less likely dimerization interaction as it was mediated by several water molecules and the interface was relatively small (Nagar et al. 1996). The second published structure was able to resolve more of the protein to show W2 docking into the hydrophobic pocket of its own molecule (Pertz et al. 1999). Additionally, the authors observed a lateral dimer they believed to be more stable also in the shape of an intertwisted “X”. There have been no biological assays done to conclusively confirm either interaction. The N-cadherin EC1/2 structure showed what appeared to be a lateral dimer, but no strand dimer was detected as previously described for the N-cadherin EC1 domain alone (Tamura et al. 1998). Tamura et al. (1998) mention a crystal packing interface possibly involved in trans adhesion, but they allude to results suggesting it is not a real interface and the one described in earlier work of N-cadherin EC1 (Shapiro et al. 1995) is more likely correct.

Fig. 6

Structures of the cadherin cis-dimer. A Structure from the E-cadherin crystal by Pertz et al (1999). The overall alignment of the two Ecad12 fragments is a slightly twisted “X”. A close view of W2 in each EC1 reveals it is docking in the...

With the crystallization of the complete cadherin extracellular domain and the discovery that the strand dimer possibly mediated trans-, not cis-, dimerization, Boggon et al. (2002) had to formulate a new molecular model for the cis-dimer. The authors described a putative cis-dimer interaction based on the crystal packing interactions between a groove in EC1 of one cadherin and a bulge on EC2 of a second cadherin (Fig. 6). The proposed interaction showed two adjacent cadherins aligned with their N-termini pointed in the same direction. This interaction would allow cadherins within the same membrane to organize in a continuous linear array. Interestingly, the surface of the groove in EC1 is composed in small part by the histidine and valine of the HAV tripeptide. Functional significance has not been verified.

Electron microscopy analysis of ECADCOMP supports a different cis-dimer model. In their studies on Ca++-dependent adhesion, Tomschy et al. (1996) and Pertz et al. (1999) describe association of E-cadherin extracellular domains within a single pentamer before extracellular domains in different pentamers adhere. They base this on the observation that ring-like structures are found in some isolated pentamers at high Ca++ concentrations, but they are always seen as the adherent cadherins when two or more pentamers associate. In contrast to the model proposed by Boggon et al. (2002), the cadherins in this cis-dimer would bend toward each other and the N-termini of the extracellular domains would point in opposite directions. The conclusion that these data represent a real cis interaction and that this interaction is required before trans adhesion is valid. However, there is another equally possible explanation: Because the extracellular domain is a curved rod and the extracellular domains are constrained by the pentamerization domain, they simply lay on the surface in an orientation that appears like a cis interaction. When two pentamers associate through a single extracellular domain, a second is brought into the structure quickly because the effective concentration of extracellular domains is very high, and the two adherent pairs simply lay flat to appear as two adjacent ring-like structures, again because of the constraint imposed by the pentamerization domain.

The cis-dimer model requires more conclusive validation. The fact that lateral complexes of cadherin can form in vivo is certain, but the question remains concerning whether these complexes serve a specific role in adhesion or if they are simply the result of cadherin clustering by its anchor to the actin cytoskeleton. Data should be obtained that show a distinct difference in the adhesive properties of known cadherin cis-dimers versus single cadherin extracellular domains. All experiments to date have either not shown a difference in adhesion due to cis-dimerization or have not determined the specific orientation of the dimers. In addition, the molecular interactions of cadherin cis-dimers must be determined. Models based on the packing in crystal structures continue to change and offer conflicting views. As remains with the trans-dimer model, residues involved in cis-dimerization must be determined.

Acknowledgments

Work from the Nelson Laboratory is supported by NIH GM55227, and T.D.P. is also supported by a Howard Hughes Medical Institute Predoctoral Fellowship.

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To form an anchoring junction, cells must first adhere. A bulky cytoskeletal apparatus must then be assembled around the molecules that directly mediate the adhesion. The result is a well-defined structure—a desmosome, a hemidesmosome, a focal adhesion, or an adherens junction—that is easily identified in the electron microscope. Indeed, electron microscopy provided the basis for the original classification of cell junctions. In the early stages of cell junctiondevelopment, however, before the cytoskeletal apparatus has assembled, cells often adhere to one another without clearly displaying these characteristic structures; in the electron microscope, one may simply see two plasma membranes separated by a small gap of a definite width. Functional tests show, nevertheless, that the two cells are stuck to each other, and biochemical analysis can reveal the molecules responsible for the adhesion.

The study of cell-cell junctions and the study of cell-cell adhesion were once quite distinct endeavors, originating from two different experimental approaches—junctions through electron microscopic description, and adhesion through functional tests and biochemistry. Only in recent years have these two approaches begun to converge in a unified view of the molecular basis of cell junctions and cell adhesion. In the previous section, we concentrated on the structures of mature cell junctions. In this section, we turn to functional and biochemical studies of the cell-cell adhesion mechanisms that operate when cells migrate over other cells and when they assemble into tissues—mechanisms that precede the construction of mature cell-cell anchoring junctions. We begin with a critical question for embryonic development: what mechanisms ensure that a cell attaches to appropriate neighbors at the right time?

Animal Cells Can Assemble into Tissues Either in Place or After They Migrate

Many simple tissues, including most epithelial tissues, derive from precursor cells whose progeny are prevented from wandering away by being attached to the extracellular matrix, to other cells, or to both (Figure 19-22). But the accumulating cells do not simply remain passively stuck together; instead, the tissue architecture is generated and actively maintained by selective adhesions that the cells make and progressively adjust.

Figure 19-22

The simplest mechanism by which cells assemble to form a tissue. The progeny of the founder cell are retained in the epithelium by the basal lamina and by cell-cell adhesion mechanisms, including the formation of intercellular junctions.

Selective adhesion is even more essential for the development of tissues that have more complex origins involving cell migration. In these tissues, one population of cells invades another and assembles with it, and perhaps with other migrant cells, to form an orderly structure. In vertebrate embryos, for example, cells from the neural crest break away from the epithelial neural tube, of which they are initially a part, and migrate along specific paths to many other regions (discussed in Chapter 21). There they assemble with other cells and with one another to differentiate into a variety of tissues, including those of the peripheral nervous system (Figure 19-23).

Figure 19-23

An example of a more complex mechanism by which cells assemble to form a tissue. Some cells that are initially part of the epithelial neural tube alter their adhesive properties and disengage from the epithelium to form the neural crest on the upper surface (more...)

Cell motility and cell adhesion combine to bring about these kinds of morphogenetic events. The process requires some mechanism for directing the cells to their final destination. This may involve chemotaxis or chemorepulsion, the secretion of a soluble chemical that attracts or repels migrating cells, respectively, or pathway guidance, the laying down of adhesive or repellent molecules in the extracellular matrix or on cell surfaces to guide the migrating cells along the right paths. Then, once a migrating cell has reached its destination, it must recognize and join other cells of the appropriate type to assemble into a tissue. How this latter process occurs can be studied if cells of different embryonic tissues are artificially mingled, after which they often spontaneously sort out to restore a more normal arrangement, as we discuss next.

Dissociated Vertebrate Cells Can Reassemble into Organized Tissues Through Selective Cell-Cell Adhesion

Unlike adult vertebrate tissues, which are difficult to dissociate, embryonic vertebrate tissues are easily dissociated. This is usually done by treating the tissue with low concentrations of a proteolytic enzyme such as trypsin, sometimes combined with the removal of extracellular Ca2+ and Mg2+ with a divalent-cation chelator (such as EDTA). These reagents disrupt the protein-protein interactions (many of which are divalent-cation-dependent) that hold cells together. Remarkably, the dissociated cells often reassemble in vitro into structures that resemble the original tissue. Such findings reveal that tissue structure is not just a product of history; it is actively maintained and stabilized by the system of affinities that cells have for one another and for the extracellular matrix.

A striking example of this phenomenon is seen when dissociated cells from two embryonic vertebrate organs, such as the liver and the retina, are mixed together and artificially formed into a pellet: the mixed aggregates gradually sort out according to their organ of origin. More generally, disaggregated cells are found to adhere more readily to aggregates of their own organ than to aggregates of other organs. Evidently there are cell-cell recognition systems that make cells of the same differentiated tissue preferentially adhere to one another; these adhesive preferences are presumably important in stabilizing tissue architecture.

Cells adhere to each other and to the extracellular matrix through cell-surface proteins called cell adhesion molecules (CAMs)—a category that includes the transmembrane adhesion proteins we have already discussed. CAMs can be cell-cell adhesion molecules or cell-matrix adhesion molecules. Some CAMs are Ca2+-dependent, whereas others are Ca2+-independent. The Ca2+-dependent CAMs seem to be primarily responsible for the tissue-specific cell-cell adhesion seen in early vertebrate embryos, explaining why these cells can be disaggregated with Ca2+-chelating agents.

CAMs were initially identified by making antibodies against cell-surface molecules and then testing the antibodies for their ability to inhibit cell-cell adhesion in a test tube. Those rare antibodies that inhibit the adhesion were then used to characterize and isolate the adhesion molecule recognized by the antibodies.

Cadherins Mediate Ca2+-dependent Cell-Cell Adhesion

The cadherins are the major CAMs responsible for Ca2+-dependent cell-cell adhesion in vertebrate tissues. The first three cadherins that were discovered were named according to the main tissues in which they were found: E-cadherin is present on many types of epithelial cells; N-cadherin on nerve, muscle, and lens cells; and P-cadherin on cells in the placenta and epidermis. All are also found in various other tissues; N-cadherin, for example, is expressed in fibroblasts, and E-cadherin is expressed in parts of the brain. These and other classical cadherins are related in sequence throughout their extracellular and intracellular domains. There are also a large number of nonclassical cadherins, with more than 50 expressed in the brain alone. The nonclassical cadherins include proteins with known adhesive function, such as the desmosomal cadherins discussed earlier and the diverse protocadherins found in the brain. They also include proteins that appear to have nonadhesive functions, such as T-cadherin, which lacks a transmembrane domain and is attached to the plasma membrane of nerve and muscle cells by a glycosylphosphatidylinositol (GPI) anchor, and the Fat protein, which was first identified as the product of a tumor-suppressor gene in Drosophila. Together, the classical and nonclassical cadherin proteins constitute the cadherin superfamily (Table 19-3).

Table 19-3

Some Members of the Cadherin Superfamily.

Cadherins are expressed in both invertebrates and vertebrates. Virtually all vertebrate cells seem to express one or more cadherins, according to the cell type. They are the main adhesion molecules holding cells together in early embryonic tissues. In culture, the removal of extracellular Ca2+ or treatment with anti-cadherin antibodies disrupts embryonic tissues, and, if cadherin-mediated adhesion is left intact, antibodies against other adhesion molecules have little effect. Mutations that inactivate the function of E-cadherin cause mouse embryos to fall apart and die early in development.

Most cadherins are single-pass transmembrane glycoproteins about 700–750 amino acids long. Structural studies suggest that they associate in the plasma membrane to form dimers or larger oligomers. The large extracellular part of the polypeptide chain is usually folded into five or six cadherin repeats, which are structurally related to immunoglobulin (Ig) domains (Figure 19-24A and B). The crystal structures of E- and N-cadherin have helped to explain the importance of Ca2+ binding for cadherin function. The Ca2+ ions are positioned between each pair of cadherin repeats, locking the repeats together to form a stiff, rodlike structure: the more Ca2+ ions that are bound, the more rigid the structure is. If Ca2+ is removed, the extracellular part of the protein becomes floppy and is rapidly degraded by proteolytic enzymes (Figure 19-24C).

Figure 19-24

The structure and function of cadherins. (A) A classical cadherin molecule. The protein is a homodimer, with the extracellular part of each polypeptide folded into five cadherin repeats. There are Ca2+-binding sites between each pair of repeats. (B) The (more...)

Cadherins Have Crucial Roles in Development

E-cadherin is the best-characterized cadherin. It is usually concentrated in adherens junctions in mature epithelial cells, where it helps connect the cortical actin cytoskeletons of the cells it holds together (see Figure 19-9B). E-cadherin is also the first cadherin expressed during mammalian development. It helps cause compaction, an important morphological change that occurs at the eight-cell stage of mouse embryo development. During compaction, the loosely attached cells, called blastomeres, become tightly packed together and joined by intercellular junctions. Antibodies against E-cadherin block blastomere compaction, whereas antibodies that react with various other cell-surface molecules on these cells do not.

It seems likely that cadherins are also crucial in later stages of vertebrate development, since their appearance and disappearance correlate with major morphogenetic events in which tissues segregate from one another. As the neural tube forms and pinches off from the overlying ectoderm, for example, neural tube cells lose E-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to express E-cadherin (Figure 19-25). Then, when the neural crest cells migrate away from the neural tube, these cadherins become scarcely detectable, and another cadherin (cadherin-7) appears that helps hold the migrating cells together as loosely associated cell groups. Finally, when the cells aggregate to form a ganglion, they re-express N-cadherin (see Figure 19-23).

Figure 19-25

The distribution of E-cadherin and N-cadherin in the developing nervous system. Immunofluorescence micrographs of a cross section of a chick embryo showing the developing neural tube labeled with antibodies against (A) E-cadherin and (B) N-cadherin. Note (more...)

If N-cadherin is overexpressed in the emerging neural crest cells, the cells fail to escape from the neural tube. Thus, not only do cell groups that originate from one cell layer exhibit distinct patterns of cadherin expression when separating from one another, but these switches in cadherin expression seem to be intimately involved in the separation process.

Cadherins Mediate Cell-Cell Adhesion by a Homophilic Mechanism

How do cell-cell adhesion molecules such as the cadherins bind cells together? Three possibilities are illustrated in Figure 19-26: (1) in homophilic binding, molecules on one cell bind to other molecules of the same kind on adjacent cells; (2) in heterophilic binding, the molecules on one cell bind to molecules of a different kind on adjacent cells; (3) in linker-dependent binding, cell-surface receptors on adjacent cells are linked to one another by secreted multivalent linker molecules. Although all three mechanisms have been found to operate in animals, cadherins usually link cells by the homophilic mechanism. In a line of cultured fibroblasts called L cells, for example, the cells neither express cadherins nor adhere to one another. When L cells are transfected with DNA encoding E-cadherin, the transfected cells become adherent to one another by a Ca2+-dependent mechanism, and the adhesion is inhibited by anti-E-cadherin antibodies. Since cadherin proteins can bind directly to one another and the transfected cells do not bind to untransfected L cells, one can conclude that E-cadherin binds cells together through the interaction of two E-cadherin molecules on different cells.

Figure 19-26

Three mechanisms by which cell-surface molecules can mediate cell-cell adhesion. Although all of these mechanisms can operate in animals, the one that depends on an extracellular linker molecule seems to be the least common.

If L cells expressing different cadherins are mixed together, they sort out and aggregate separately, indicating that different cadherins preferentially bind to their own type (Figure 19-27A), mimicking what happens when cells derived from tissues that express different cadherins are mixed together. A similar segregation of cells occurs if L cells expressing different amounts of the same cadherin are mixed together (Figure 19-27B). It therefore seems likely that both qualitative and quantitative differences in the expression of cadherins have a role in organizing tissues.

Figure 19-27

Cadherin-dependent cell sorting. Cells in culture can sort themselves out according to the type and level of cadherins they express. This can be visualized by labeling different populations of cells with dyes of different colors. (A) Cells expressing (more...)

In the nervous system especially, there are many different cadherins, each with a distinct but overlapping pattern of expression (Figure 19-28A). As they are concentrated at synapses, they are thought to have a role in synapse formation and stabilization. Some of the nonclassical cadherins, such as the protocadherins, are strong candidates for helping to determine the specificity of synaptic connections. Like antibodies, they differ in their N-terminal (variable) regions but are identical in their C-terminal (constant) regions. The extracellular variable region and intracellular constant region are encoded by separate exons, with the variable-region exons arranged in tandem arrays upstream of the constant-region exons (Figure 19-28B). The diversity of protocadherins is generated by a combination of differential promoter usage and alternative RNA splicing, rather than by site-specific recombination as occurs in antibody diversification (discussed in Chapter 24).

Figure 19-28

Cadherin diversity in the central nervous system. (A) Expression patterns for three classical cadherins in the embryonic mouse brain. (B) The arrangement of exons that encode the members of one of the three known protocadherin families of nonclassical (more...)

Cadherins Are Linked to the Actin Cytoskeleton by Catenins

Most cadherins, including all classical and some nonclassical ones, function as transmembrane adhesion proteins that indirectly link the actin cytoskeletons of the cells they join together. This arrangement occurs in adherens junctions (see Figure 19-9B). The highly conserved cytoplasmic tail of these cadherins interacts indirectly with actin filaments by means of a group of intracellular anchor proteins called catenins (Figure 19-29). This interaction is essential for efficient cell-cell adhesion, as classical cadherins that lack their cytoplasmic domain cannot hold cells strongly together.

Figure 19-29

The linkage of classical cadherins to actin filaments. The cadherins are coupled indirectly to actin filaments by the anchor proteins α-catenin and β-catenin. A third intracellular protein, called p120, also binds to the cadherin cytoplasmic (more...)

As discussed earlier, the nonclassical cadherins that form desmosomes interact with intermediate filaments, rather than with actin filaments. Their cytoplasmic domain binds to a different set of intracellular anchor proteins, which in turn bind to intermediate filaments (see Figure 19-11D).

Some cells can regulate the adhesive activity of their cadherins. This regulation may be important for the cellular rearrangements that occur within epithelia when these cell sheets change their shape and organization during animal development (see Figure 19-10). The molecular basis of this regulation is uncertain but may involve the phosphorylation of anchor proteins attached to the cytoplasmic tail of the cadherins.

Some cadherins can help transmit signals to the cell interior. Vascular endothelial cadherin (VE-cadherin), for example, not only mediates adhesion between endothelial cells but also is required for endothelial cell survival. Although endothelial cells that do not express VE-cadherin still adhere to one another via N-cadherin, they do not survive (see Table 19-3, p. 1082). Their survival depends on an extracellular signal protein called vascular endothelial growth factor (VEGF), which binds to a receptor tyrosine kinase (discussed in Chapter 15) that uses VE-cadherin as a co-receptor.

Selectins Mediate Transient Cell-Cell Adhesions in the Bloodstream

White blood cells lead a nomadic life, moving to and fro between the bloodstream and the tissues, and this necessitates special adhesive properties. These properties depend on selectins. Selectins are cell-surface carbohydrate-binding proteins (lectins) that mediate a variety of transient, Ca2+-dependent, cell-cell adhesion interactions in the bloodstream. There are at least three types: L-selectin on white blood cells, P-selectin on blood platelets and on endothelial cells that have been locally activated by an inflammatory response, and E-selectin on activated endothelial cells. Each selectin is a transmembrane protein with a highly conserved lectindomain that binds to a specific oligosaccharide on another cell (Figure 19-30A).

Figure 19-30

The structure and function of selectins. (A) The structure of P-selectin. The selectin attaches to the actin cytoskeleton through anchor proteins that are still poorly characterized. (B) How selectins and integrins mediate the cell-cell adhesions required (more...)

Selectins have an important role in binding white blood cells to endothelial cells lining blood vessels, thereby enabling the blood cells to migrate out of the bloodstream into a tissue. In a lymphoid organ, the endothelial cells express oligosaccharides that are recognized by L-selectin on lymphocytes, causing the lymphocytes to loiter and become trapped. Conversely, at sites of inflammation, the endothelial cells switch on expression of selectins, which recognize the oligosaccharides on white blood cells and platelets, flagging the cells down to help deal with the local emergency. Selectins do not act alone, however; they collaborate with integrins, which strengthen the binding of the blood cells to the endothelium. The cell-cell adhesions mediated by both selectins and integrins are heterophilic (see Figure 19-26): selectins bind to specific oligosaccharides on glycoproteins and glycolipids, while integrins bind to specific proteins.

Selectins and integrins act in sequence to let white blood cells leave the bloodstream and enter tissues. The selectins mediate a weak adhesion because the binding of the lectindomain of the selectin to its carbohydrateligand is of low affinity. This allows the white blood cell to adhere weakly and reversibly to the endothelium, rolling along the surface of the blood vessel propelled by the flow of blood. The rolling continues until the blood cell activates its integrins (discussed later), now causing the cell to bind strongly to the endothelial cell surface and to crawl out of the blood vessel between adjacent endothelial cells (Figure 19-30B).

Members of the Immunoglobulin Superfamily of Proteins Mediate Ca2+-independent Cell-Cell Adhesion

Cadherins, selectins, and integrins all depend on extracellular Ca2+ (or Mg2+ for some integrins) to function in cell adhesion. The molecules responsible for Ca2+-independent cell-cell adhesion belong mainly to the large and ancient immunoglobulin (Ig) superfamily of proteins. These proteins contain one or more Ig-like domains that are characteristic of antibody molecules (discussed in Chapter 24). One of the best-studied examples is the neural cell adhesion molecule (N-CAM), which is expressed by a variety of cell types, including most nerve cells. N-CAM is the most prevalent of the Ca2+-independent cell-cell adhesion molecules in vertebrates, and, like cadherins, it is thought to bind cells together by a homophilic mechanism (between N-CAM molecules on adjacent cells). Some Ig-like cell-cell adhesion proteins, however, use a heterophilic mechanism. Intercellular adhesion molecules (ICAMs) on endothelial cells, for example, bind to integrins on blood cells when blood cells migrate out of the bloodstream, as just discussed.

There are at least 20 forms of N-CAM, all generated by alternative splicing of an RNAtranscript produced from a single gene. In all forms, the large extracellular part of the polypeptide chain is folded into five Ig-like domains (Figure 19-31). Some forms of N-CAM carry an unusually large quantity of sialic acid (with chains containing hundreds of repeating sialic acid units). By virtue of their negative charge, these long polysialic acid chains hinder cell adhesion, and there is increasing evidence that N-CAM heavily loaded with sialic acid serves to prevent adhesion, rather than cause it.

Figure 19-31

The cell adhesion protein N-CAM. (A) Four forms of N-CAM. The extracellular part of the polypeptide chain in each case is folded into five Ig-like domains (and one or two other domains called fibronectin type III repeats). Disulfide bonds (red) connect (more...)

Although cadherins and Ig family members are frequently expressed on the same cells, the adhesions mediated by cadherins are much stronger, and they are largely responsible for holding cells together, segregating cell collectives into discrete tissues, and maintaining tissue integrity. N-CAM and other members of the Ig family seem to contribute more to the fine-tuning of these adhesive interactions during development and regeneration. In the developing rodent pancreas, for example, the formation of the islets of Langerhans requires cell aggregation, followed by cell sorting. Whereas inhibition of cadherin function prevents cell aggregation and islet formation, loss of N-CAM only impairs the cell sorting process, so that disorganized islets form.

Similarly, whereas mutant mice that lack N-cadherin die early in development, mutant mice that lack N-CAM develop relatively normally, although they do have some defects in neural development. Mutations in other genes that encode Ig-like cell adhesion proteins, however, can cause more severe neural defects. L1gene mutations in humans, for example, cause mental retardation and other neurological defects resulting from abnormalities in the migration of nerve cells and their axons.

The importance of Ig-like cell adhesion proteins in connecting the neurons of the developing nervous system has been demonstrated dramatically in Drosophila. An N-CAM-like protein called fasciclin III (FAS3) is expressed transiently on some motor neurons, as well as on the muscle cells they normally innervate. If FAS3 is genetically removed from these neurons, they fail to recognize their muscle targets and do not make synapses with them. Conversely, if motor neurons that normally do not express FAS3 are made to express this protein, they now synapse with FAS3-expressing muscle cells to which they normally do not connect. It seems that FAS3 mediates these synaptic connections by a homophilic “matchmaking” mechanism.

Like the cadherins, some Ig-like proteins do more than just bind cells together. They can also transmit signals to the cell interior. Some forms of N-CAM in nerve cells, for example, associate with Src family cytoplasmic tyrosine kinases (discussed in Chapter 15), which relay signals onward by phosphorylating intracellular proteins on tyrosines. Other Ig family members are transmembrane tyrosine phosphatases (discussed in Chapter 15) that help guide growing axons to their target cells, presumably by dephosphorylating specific intracellular proteins.

Multiple Types of Cell-Surface Molecules Act in Parallel to Mediate Selective Cell-Cell Adhesion

A single type of cell utilizes multiple molecular mechanisms in adhering to other cells. Some of these mechanisms involve organized cell junctions, while others do not (Figure 19-32). Each cell in a multicellular animal contains an assortment of cell-surface receptors that enables the cell to respond specifically to a complementary set of soluble extracellular signal molecules, such as hormones and growth factors. Likewise, each cell in a tissue has a particular combination (and concentration) of cell-surface adhesion molecules that enables it to bind in its own characteristic way to other cells and to the extracellular matrix. And just as receptors for soluble extracellular signal molecules generate intracellular signals that alter the cell's behavior, so too can cell adhesion molecules, although the signaling mechanisms they use are generally not as well understood.

Figure 19-32

A summary of the junctional and nonjunctional adhesive mechanisms used by animal cells in binding to one another and to the extracellular matrix. The junctional mechanisms are shown in epithelial cells, while the nonjunctional mechanisms are shown in (more...)

Unlike receptors for soluble signal molecules, which bind their specific ligand with high affinity, the receptors that bind to molecules on cell surfaces or in the extracellular matrix usually do so with relatively low affinity. These low-affinity receptors rely on the enormous increase in binding strength gained through the simultaneous binding of multiple receptors to multiple ligands on an opposing cell or in the adjacent matrix. One could call this the “Velcro principle.”

We have seen, however, that the interaction of the extracellular binding domains of these cell-surface molecules is not enough to ensure cell adhesion. At least in the case of cadherins and, as we shall see, integrins, the adhesion molecules must also attach (via anchor proteins) to the cytoskeleton inside the cell. The cytoskeleton is thought to assist and stabilize the lateral clustering of the adhesion molecules to facilitate multipoint binding. The cytoskeleton is also required to enable the adhering cell to exert traction on the adjacent cell or matrix (and vice versa). Thus, the mixture of specific types of cell-cell adhesion molecules present on any two cells, as well as their concentration, cytoskeletal linkages, and distribution on the cell surface, determine the total affinity with which the two cells bind to each other.

Nonjunctional Contacts May Initiate Cell-Cell Adhesions That Junctional Contacts Then Orient and Stabilize

We have seen that adhesive contacts between cells play a crucial part in organizing the formation of tissues and organs in developing embryos or in adult tissues undergoing repair after injury. Most often, these contacts do not involve the formation of organized intercellular junctions that show up as specialized structures in the electron microscope. The interacting plasma membranes are simply seen to come close together and run parallel, separated by a space of 10–20 nm. This type of “nonjunctional” contact may be optimal for cell locomotion—close enough to provide traction and to allow transmembrane adhesion proteins to interact, but not so tight, or so solidly anchored to the cytoskeleton, as to immobilize the cell.

A reasonable hypothesis is that nonjunctional cell-cell adhesion proteins initiate cell-cell adhesions, which are then oriented and stabilized by the assembly of full-blown intercellular junctions. Many of the transmembrane proteins involved can diffuse in the plane of the plasma membrane and, in this or other ways, can be recruited to sites of cell-cell (and cell-matrix) contact, enabling nonjunctional adhesions to enlarge and mature into junctional adhesions. This has been demonstrated for some integrins and cadherins, which help initiate cell adhesion and then later become integral parts of cell junctions. The migrating tip of an axon, for example, has an even distribution of cadherins on its surface, which helps it adhere to other cells along the migration pathway. It also has an intracellular pool of cadherins in vesicles just under the plasma membrane. When the axon reaches its target cell, it is thought to release the intracellular cadherin molecules onto the cell surface, where they help form a stable contact, which matures into a chemical synapse.

As discussed earlier, antibodies against adherens junction proteins block the formation of tight junctions, as well as adherens junctions, suggesting that the assembly of one type of junction can be a prerequisite for the formation of another. An increasing number of monoclonal antibodies and peptide fragments have been produced that can block a single type of cell adhesion molecule. Moreover, an increasing number of genes encoding these cell-surface proteins have been identified, creating new opportunities for manipulating the adhesive machinery of cells in culture and in experimental animals. It is now possible, therefore, to inactivate the various cell-cell adhesion proteins in combinations—a requirement for deciphering the rules of cell-cell recognition and binding used to build complex tissues.

Summary

Cells dissociated from various tissues of vertebrate embryos preferentially reassociate with cells from the same tissue when they are mixed together. This tissue-specific recognition process in vertebrates is mediated mainly by a family of Ca2+-dependent cell-cell adhesion proteins called cadherins, which hold cells together by a homophilic interaction between these transmembrane proteins on adjacent cells. For this interaction to be effective, the cytoplasmic part of the cadherins must be linked to the cytoskeleton by cytoplasmic anchor proteins called catenins.

Two other families of transmembrane adhesion proteins have major roles in cell-cell adhesion. Selectins function in transient Ca2+-dependent cell-cell adhesions in the bloodstream by binding to specific oligosaccharides on the surface of another cell. Members of the immunoglobulin superfamily, including N-CAM, mediate Ca2+-independent cell-cell adhesion processes that are especially important during neural development.

Even a single cell type uses multiple molecular mechanisms in adhering to other cells (and to the extracellular matrix). Thus, the specificity of cell-cell (and cell-matrix) adhesion seen in embryonic development must result from the integration of several different adhesion systems, of which some are associated with specialized cell junctions, while others are not.

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