Journal of Molecular Biology
Heterotypic trans-Interaction of LI- and E-Cadherin and Their Localization in Plasmalemmal Microdomains☆
Introduction
Cadherins are a structurally diverse superfamily of transmembrane glycoproteins mediating Ca2+-dependent adhesion between adjacent cells.1, 2, 3 They play a crucial role in embryogenesis, tissue morphogenesis, and the maintenance of the tissue-specific differentiation state.4, 5, 6, 7 Classical cadherins—the most extensively studied cadherin family—are composed of a highly conserved cytoplasmic region, a single transmembrane domain, and an extracellular part that is organized in a series of five homologous subdomains denoted as cadherin repeats.8, 9 The cadherin ectodomain is maintained in a rodlike conformation due to the stabilization by Ca2+ ions.10, 11 Under physiological Ca2+ concentration, cadherin molecules were found to associate into parallel cis-dimers representing the functional unit required for trans-interactions with cadherin cis-dimers of adjoining cells.12, 13
E-cadherin (uvomorulin, cadherin-1) is the prototype of classical cadherins and an integral part of the adherens-type junctions of epithelial cells.3, 14 In enterocytes, E-cadherin is coexpressed with the structurally divergent LI-cadherin15, 16 but localizes to different membrane regions. LI-cadherin (cadherin-17) is evenly distributed along the lateral contact zones of enterocytes but is excluded from adherens junctions,15 which are formed by E-cadherin and the cytoplasmic proteins β-catenin, p120-catenin, and α-catenin.14, 17 LI-cadherin belongs to a distinct group within the cadherin superfamily denoted as 7D-cadherins (seven-domain cadherins18, 19). In contrast to classical cadherins, LI-cadherin is composed of seven extracellular cadherin repeats and a cytoplasmic domain of only 25 amino acids that shares no similarity to the highly conserved cytoplasmic region of classical cadherins necessary for the interaction with β- and p120-catenin.20, 21, 22 Nevertheless, LI-cadherin acts as a functional Ca2+-dependent adhesion molecule when expressed in Drosophila S2 cells.15 Interestingly, its basic adhesive function seems to be independent of any interaction with cytoplasmic components since a chimeric construct composed of the complete LI-cadherin ectodomain fused to a glycosylphosphatidylinositol anchor is still capable of mediating cell–cell adhesion.23
In recent years, structural investigations of the cadherin repeats have provided important clues about the molecular interface of the homotypic bond.10, 24, 25, 26, 27 However, the molecular mechanism underlying the cadherin-mediated homotypic adhesion and the regulation of its adhesive state independent of cytoplasmic interactions are still not well understood. The regulation of the homotypic binding via the cadherin ectodomain can be achieved by the modulation of its binding strength as well as by its clustering.3, 28 Given the large number of cadherins with structurally distinct ectodomains, it is of major interest to reveal whether a difference in the ectodomain structure results in a different biochemistry of cadherin interaction.
With the exception of LI-cadherin,29 only classical cadherins have been studied with respect to the binding parameters of their extracellular homotypic interactions30, 31, 32 and only little is known about heterotypic interactions among different cadherins.33, 34 In the present study, we characterized a specific heterotypic trans-interaction of LI- with E-cadherin on the single-molecule level as well as on the cellular level and revealed a striking difference in their plasma membrane distribution.
Section snippets
LI-cadherin interacts heterotypically with E-cadherin on the single-molecule level
To test the specificity of LI-cadherin interactions on the single-molecule level, we used chimeric recombinant proteins composed of the complete cadherin ectodomain of LI-, E-, or VE-cadherin fused to the Fc part of human IgG1 (LI-Fc, E-Fc, VE-Fc). LI-Fc and VE-Fc have been described and characterized previously.29, 30, 35 trans-Interaction of single cadherin molecules was analyzed by atomic force microscopy (AFM) as described in detail for VE- and N-cadherin30, 36 as well as for desmoglein 1.37
Discussion
The great variety of members of the cadherin family of adhesion molecules expressed in vertebrates raises the question as to what extent their structural divergence implies differences in their binding characteristics. So far, mostly classical cadherins have been analyzed, that is, with regard to the binding parameters of their homotypic trans-interaction,12, 30, 31, 32, 36, 37, 41 and only few studies deal with the heterotypic interaction among classical cadherins.33, 34 Recently, we reported
Recombinant proteins
The generation of recombinant chimeric proteins composed of either the complete LI- or VE-cadherin ectodomain fused to the Fc part of human IgG1 (LI-Fc and VE-Fc) is described elsewhere.29, 35 Recombinant E-Fc was constructed and purified similar to the LI- and VE-Fc constructs. The complete murine E-cadherin ectodomain was PCR amplified and inserted into the CMV5-IgG expression plasmid (a kind gift from D. Vestweber, Münster, Germany), which codes for the Fc part of human IgG1, including the
Acknowledgements
This work was financially supported by the Deutsche Forschungsgemeinschaft through grants SFB487/B5 and SFB366/C2
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2014, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :To clarify this point, we transfected MDCK-cells, which endogenously express E-cadherin with a full length VE-cadherin. We performed this experiment as we could recently show that E-cadherin is not localised in cholesterol rich membrane domains [9]. Fractioning with the sucrose gradient method revealed that again VE-cadherin is mainly localised in low density, i.e. cholesterol rich membrane domains (Fig. 3A) whereas endogenous E-cadherin in these cells is effectively excluded from these membranes and is mainly found in the high-density fractions (Fig. 3B).
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2014, Trends in Cell BiologyNew insights into the evolution of metazoan cadherins and catenins
2013, Progress in Molecular Biology and Translational ScienceCitation Excerpt :Cadherin 16 and 17, however, might use the single conserved tryptophan in the beginning of their third EC repeat because the first two domains are duplications. A heterophilic trans interaction is possible between LI-cadherin (CDH17) and E-cadherin (CDH1).4 Heterophilic interactions have also been described between the members of the type-II subfamily.5
Desmosome assembly and cell-cell adhesion are membrane raft-dependent processes
2011, Journal of Biological ChemistryCitation Excerpt :However, in general, it is still believed that cell membranes are divided into high cholesterol raft domains and low cholesterol nonraft domains. The transmembrane proteins of cell-cell junctions, such as N-cadherins of adherens junctions (11, 12) and occludin and specific claudins (4, 7) of tight junctions (13–15), have recently been found in MRs, whereas E-cadherins of adherens junctions (16) and β1 integrins (12, 15) of focal contacts are excluded from these membrane domains. MRs contain various connexins but not complexes of gap junctions, showing that sometimes only individual junctional proteins cosediment with rafts (17).
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W.B. and M.W.W. contributed equally to this work.
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W.B. and M.W.W. contributed equally to this work.
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R. Koob died before submission of the manuscript.
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Present address: M. W. Wendeler, Novartis Pharma AG, Basel, Switzerland.