Classical cadherins

seminars in CEll BIOLOGY, Vol 3, 1992: pp 149-155

Classical cadherins Rolf Kemler Cadherins represent a gene family of Ca2 + -dependent cell adhesion molecules (CAMs) identified duringdevelopment and in adultorgans. Theygenerally mediate cell-cell adhesion by homotypic interaction, although heterotypic binding between diJIerent cadhain molecules is possible. Molecular cloning and sequence comparison has ledto thecharacterization ofa highly homologousgroup of 'classical' cadherins and more distantly related members, together composing a gene superfamily. The classical cadherins are transmembraneglycoproteins which exhibit, in addition to thestructuralhomologies, a very similar overall protein topology. Protein sequence comparison has led to the identification of domains of common functional importance. The cytoplasmic domains ofcadherins associate with peripheral cytoplasmic proteins termed catenin ex, {3 and 'Ywith molecularweights of1 02, 88 and 80 kDa respectively. This complexformationseems to regulate theadhesioefunction ojcadherins, most likely byconnectingcadherins with actin microjilaments. Possible implications ofcateninsfor cadherin function arediscussed. Key words: morphogenesis I epithelia I adhesion I membrane proteinsI cytoplasmic anchorage

DEPENDING on their requirement of Ca2 + for promoting adhesiveness, cell adhesion molecules (CAMs) have been grouped in two major classes. This operational classification, first made by Takeichi.! has not only helped to distinguish between different CAMs, but it has also led to the identification of the Ca2 + -dependent CAM family termed cadherins. Specifically, in contrast to other Ca2 + -dependent CAMs, cadherins show a remarkable resistance against proteolytic degradationin the presence of Ca2 + .2-4 Until three or four years ago it was thought that the cadherin gene family is composed of a rather limited number of members.vf These by now 'classical'cadherinswere all initially identified in a functionalcell adhesion assay. How they were related to each other was not fully understood at first. From the Max-Planck-Institutfiir lmmunblologie, Stiibeuieg 51, D-7800 Freiburg, Germany ©1992 Academic Press Ltd 1043-4682/92/030147 + 07$5. 00/0

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Subsequently, molecular cloning and sequence analysis has revealed their close structural resemblance. Taking advantageof these homologies, application of cross-hybridizations and PCR technology has allowed the identification and characterization of an increasing number of new members. Despite their clear structural homology to cadherins, it remains to be shown for many of these new members whether they actually exhibit' adhesion properties. The currentlist of members of the cadherin gene superfamily is given in Table 1.

The cadherin gene superfamily The way cadherins are grouped in Table 1 is preliminary and is largely due to historical reasons, i.e. which tissues they were first found in, since complete structural information on many new members listed is lacking. However, it already seems evident thatcadherinsrepresentthe productsofa gene superfamilyin which several subgroupscan be defined, andit will be extremely interestingto establishanevolutionarytree with allmembers. The presentsubgroups ofepithelial, neuronalanddesmosomal cadherinsmay need further subdivisions, once more detailed informationis available. Forexample, P-cad18 might be a member of an independent subgroup although it is more homologous to Evcad/uvomorulin/J'than to N-cad;23 or B-cad21 (also called K-CAM22), originally isolated from brain tissue, is also ex-. pressed in epithelial tissues. The most studied group consists of the epithelial cadherins5.40.41 with mouse uvomorulin/Eccad.Zf human CAM 120/80,9.10 canine rr-l 11 and Arc-Jl2 and chicken L-CAM,20 where some listed members are identical (rr-llArc-I and uvomorulin/E-cad)and others represent interspecies homologs of uvomorulin (CAM 120/80) or closely related molecules (L-CAM). Another group consists of the neuronal cadherins, with N-cad23 (also called A-CAM38 and N-Cal-CAM24) as the most prominent member and T-, R·, M-, and Bcad21.24.27.28 as more recent examples. Unlike the epithelial cadherins, members from this group are known to be expressed in multiple cell types of neural and mesodermal origin.

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R. Kemler Table 1. List of known cadherin gene of super-family members. The classification into subgroups is incomplete and has to be taken as preliminary since detailed information on all members is not yet available. Synonyms for the same cadherin in the same organism are separated by a slash (I); the brackets group identical cadherins from different organisms.

Embryonal and epithelial cadherins

Cadherin

Species

T

F

L

Refs

Uvomorulinl E-Cad CAM 120/80 Arc-I n-1 gp 140 KI E-cad XB-cad

Mouse

+ + + + + +

8

Human Dog Dog Xenopus

7 8 9,10 11 12 13 14 15

U-cad

Xenopus

EP-cad

Xenopus

Early embryo and epithelia MCF-7 MDCK MDCK Early embryo and epithelia Early embryo and epithelia Early embryo and epithelia Early embryo and epithelia Liver Neuronal cells

+

• •

20 21,22

Chicken

Neuronal and mesodermal cells

+ +

18

23 38

Human Xenopus

+

Bovine Mouse Chicken Chicken Rat

Neuronal and mesodermal cells Neuronal and mesodermal cells Endothelial Myoblast Neuronal cells Retina Brain tissue

Human

Epithelial cells

+

Human

Epithelial cells

18

34

,Human

Epidermis

18

35

8 16

18 19 36 30 37

Xenopus

L-CAM Chicken B-cad/K-CAM Chicken Neuronal and meso- N-cadi dermal cadherins A-CAM I N-Cal-CAM N-cad N-cad N-cad M-cad T-cad R-cad Cad 4 Desmosomal cadherins

Unclassified cadherins

Desmocollin DG WIll Desmoglein DGI PVA P-cad V-cad Cad 5-11 Fat protein

Mouse Human Bovine Rat Drosophila

16

16

+

17

24 25 39 26 27 28 29 30

+

iPlacenta + Placenta + Endothelial cells + Brain tissue Larval imaginal discs

9

31-33

T, tissue or cell type where the cadherinwas identified; F, the adhesive function has been demonstrated ( + ), L, chromosomal localization of the gene; asterisks indicate a tandem arrangementof these genes.22

The mouse uvomorulin gene is localized on chromosome 8 (Urn-Locus),42 and interestingly the P-cad gene was also mapped to the same chromosome.P In humans, the genes for both uvomorulin/CAM 120/80 10 and Pvcad have been localized to chromosome 16. This and the recent published clustered localization of chicken L-CAM and K-CAM ( = B-cad)22 genes are the first examples for physical linkage of differentcadheringenes. Once more genes are chromosomally mapped, one could address the questionwhetherthe clusteredoccurrenceofthese

genes coincides with some regulatoryorevolutionary subgroup arrangement. The genes of mouse uvomorulin and P-cad, and chicken L-CAM have nearly identical exon structure.v' This high degree of conservation of the genomic organization could indicate that these cadherins have been generated by gene duplication from a common ancestor.v' However, the origin of the divergence of cadherins may be much more complex. Recent analysis indicates, for example, that the domain structureis not generated simply by exon duplication but rather

Ca2 ~ -dependent CAMs

by a more complex process in which gene recombination and/orintegrationof introns could also have been involved.vi A more distantly related subgroup of cadherinlike proteins is represented by some desmosomal glycoproteins31-34- and the Pemphigus vulgaris disease antigen,35 which share additional structuralfeatures besides the cadherin domain (see following review) . The primary structure of V -ca d 36 has not yet been determined, and how the Drosophila fat tumour suppressor gene-? product and cad 5-11 30 fit into this framework still needs to be investigated.

Structure-functionanalysis Most of the information about structural features which might be of functional importancecomes from work with the ' classical' cadherins uvomorulinl E-cad,7 ,8 N-cad,23 P-cad,18 and L-CAM20 (see also Buxton and Magee, this issue). The establishment of the primary structure of several cadherins revealed that these molecules are integral membrane glycoproteins with a single membrane-spanningdomain (ref 5 and Figure 1 of Buxton and Magee). The truncated T-cad28 with only an extracellular domain is an exception in this respect, and if more examples like it can be found, this could turn out to be an important mechanism for regulatingadhesiveness. The extracellular part of cadherins is largely composed of a series of domains each of around 110 amino acids and with internal homology. As shown for uvomorulin, each domain contains two putative Ca2 + -binding sites,7,45 and a peptide with the sequence of one Ca2 + -binding motif can indeed form a complex with Ca2 + .45 This suggests that these motifs mediate the Ca2 + -protein interaction and as part of the repeating domains confer the Ca2 + -dependent protein conformation. The domain structure including the Ca2 + -binding motifs is a general characteristicof all members of the cadherin gene family from Drosophila to man. A single amino acid substitution in one Ca2 + -binding motif of uvomorulin completely inactivates the adhesive properties and renders the protein susceptible to proteolytic degradation at or close to the site of the substiturion.t- Whether this Ca2 + -binding site is directly involved in the adhesive binding remains to be determined. Mutant proteins with similar substitutions in other Ca2 + -binding motifs should be compared in this respect.

151

Cadherins seem to interact stronger in a homotypic manner.f! but weaker heterotypic interactions are also possible.t'' The molecular mechanism of adhesive binding between cadherin molecules is still not completely understood, although some progress has been reported recently. The amino terminal domain of 113 amino acid residues seems to mediate the selectivity of binding, as has been convincingly shown with the use of chimeric proteins between E- and Pvcadherin.t? The importance of the amino terminal region for adhesive binding has also been reported by analysing the proteolytic processing of the uvomorulin precursorpolypeptide.tf However, these studies do not clarify whether the' interaction of cadherins from neighbouring cells involves only the respective amino terminal domains or whether the molecules interact all .along their length in a head-to-tail manner (Figure la,b), although the latter possibility seems more likely. As already mentioned, a single amino acid substitution at one Ca2 + -binding motif, which is not located in the amino terminal domain, completely abolishes the adhesive function.P Also, structural changes close to the membrane proximal part of the extracellularregion affect adhesive binding.j? These findings, together with the fact that the epitopes of five blocking monoclonal antibodies against different cadherinsare localized either at the amino terminal region or close to the membrane spanning domain,41,49 might indicate that the entire protein conformation is crucial for adhesive functions (see Figure l c). If so, the overall protein topology would then guaranteethe best fit for the homotypic interaction. Minor changes in the protein conformation can result in loss of the specificity of binding, or induce even more drastic effects, as already shown for the single amino acid substitution. Comparing all the classical cadherins, the cytoplasmic region exhibits the strongest degree of homology between different mernbers.s-t! The analysis of deletion mutants of uvomorulin led to the identification of three interacting proteins, termed catenins.50,51 Catenin a, {3 and 'Y with molecular weights of 102, 88 and 80 kDa respectively were found to be complexed with a defined domain of the cytoplasmic region (Figure 1).52 Most of the present information about catenins comes from studies on their association with uvomorulinlE-cad. Pulse-chase labelling experiments revealed that the uvomorulin precursor polypeptide is already associated with {3 eatenin and that a and 'Y catenin are assembled into the complex aroundthe time of proteolytic processing

152

R. Kemler

Figure1. Schematic model to explain the major properties of classical cadherins. The amino terminal region is of major importancefor the homotypic interactions, but different interactions are possible as depicted in (a) and (b). The cadherindomain structurewith the Ca2+-binding motifs (black fans) is thought to stabilize the protein conformation. The carboxy terminal region in the cytoplasm is complexed with a, {3 and 'Y catenins, which regulate the connection to actin microfilaments (Ac).

of this propeptide. 53-55 The uvomorulin of a cell is totally complexed with catenins, and it has been suggested that this association is fundamental for uvomorulin function.56,57 Complex formation may be important for the intracellular transport and membrane assembly of uvomorulin. In cell aggregation assays, only uvomorulins able to associate with catenins express full adhesive capacity.52,58 This indicates a very remarkable regulative interference between the association of catenins and the extracellular-mediatedadhesiveness. Most likely, this regulation of adhesiveness occurs by linking the uvomorulin-catenincomplex to the actin microfilament network. Depending on the cell type, some 15-35 % of all cellular uvomorulin is linked to actin bundles,52.54 and a catenin appears to playa key role in this cytoplasmic anchorage (Figure 1).52 Catenins are not just involved in linking uvomorulin to actin; moreover, they are part of a multicomponent submembranous network which connects uvomorulin to other

integral membrane proteins.59 This argues for a much wider biological role of catenins. Catenins are found more or less ubiquitously distributed, being identified in chicken, mouse and human in various cell types most of which are known to be negative for uvornorulin.P? This suggests that a similar complex formation also holds for most of the other cadherins. Indeed, or-catenin has been described to be associated in vivo with mouse N- and P-cad, chicken A-CAM and Xenopus U-cad. 53,60,61 Even more importantly, in cells expressing two cadherins (uvomorulin and N-cad) catenins preferentially bind to uvomorulin.v! It is tempting to speculate that in this way a cell which expresses more than one cadherin at a time could regulate selective binding via a particularcadherin. Recently, the molecular cloning and sequencing of the gene and the inferred primary structureof a catenin were reported.61,62 Sequence comparison revealed homology with vinculin. This homology is restricted to defined regions of both molecules,

153

Ca2 ~ -dependent CAMs

possibly reflecting the location of shared biological functions. Vinculin is localized in adherens-type junctions and in focal contactss'' and has been reported to be involved in the cytoplasmic anchorage of receptors for extracellular matrix proteins. The homology between vinculin and o-catenin opens the possibility of a new protein family of peripheral cytoplasmic proteins mediating the anchorage of transmembranereceptors. The molecularcharacterization of {3 and 'Y eatenin is less advanced than that of a catenin. In particular,detailed knowledge about {3 catenin would be helpful toward establishing the molecular link between cadherins and the actin network. The structure of Xenopus {3 catenin has recently been reported.v! and it exhibits homology to human plakoglobin and the product of the Drosophila armadillo segment polarity gene. Whether this homology is indicative of anotherprotein family remains elusive. Additional information is needed, but the present data about eatenins already suggests that these proteins will exhibit basic cellular functions.

biologists and could be addressed with the use of various in vitro cell culture systems. However, answers to these questions are less likely to give ideas about the morphoregulatoryrole that developmental biologists would like to consider. This arearequires functional analysis in vivo to study cell-cell interaction and differentiation in space and time. Gene targeting experiments in mouse embryonic stem cells and the subsequent generation of chimeric or transgenic mice might be the method of choice to alter specific adhesion mechanisms duringdevelopment. Another very interesting system to study is Xenopus development. The expression of a mutant cadherinby mRNA injection into fertilized Xenopus. eggs or the heterotypic overexpression of a cadherin mRNA might lead to mutant phenotypes caused by altered cell adhesion phenomena. These and other attempts should allow one to answer more precise questions about the functional role of CAMs during development.

Acknowledgements Future prospects The initial period of cadherin work focused on the identification and structural comparison of the molecules and their genes . Due to detailed structure! function analysis this field has meanwhile entered an exciting new phase. The combinatorial use of molecularand cell biological techniques should lead to a comprehensive picture about the biological roles of cadherins. To this end there are still some large problems to solve such as to establish the evolutionary tree of the gene family, or to gain precise knowledge about the intermolecularinteractionwhich triggers adhesion. The latter should preferably include X-ray crystallographic or 2-D NMR analysis. From what has been learnedso far about cadherins the cytoplasmic domain seems to play a central functional role. The identification of catenins as cytoplasmic anchorageproteins opens up many new possibilities. Forexample, one would like to ask, how is the interaction of catenins with a particular cadherin regulated? Are there more catenin homologues, or other cytoplasmic proteins associated with the complex? Can catenins form homo- and!or heterodimers and what might be the partner molecules? Also, could catenins be part of a possible adhesion-dependentsignal transductionmechanism? These and similar questions are of interest for cell

I thank Randy Cassada for critically reading the manuscript, Helga Kochanowski for the graphics and Rosemary Schneider for secretarial work.

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