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First published online 25 January 2005
doi: 10.1242/jcs.01665

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The mechanism of cell adhesion by classical cadherins: the role of domain 1

Oliver J. Harrison¹, Elaine M. Corps¹, Torunn Berge^2,* and Peter J. Kilshaw^1,

¹ The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
² Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK

View larger version (23K): [in a new window]   Fig. 1. Atomic force image of purified dimeric N-cadherin-Fc fusion protein adsorbed to a mica surface. The two curved `arms' in each molecule are the five extracellular domains of N-cadherin, which are joined to the Fc region. The thickness of the deposited molecules is reflected in their shading and approximate values were obtained; 5 nm for the more intensely white Fc region and 3 nm for the cadherin domains.

View larger version (84K): [in a new window]   Fig. 2. The two-domain structure of N-cadherin, PDB 1NCJ (Tamura et al., 1998). The location of the 13-mer peptide in domain 1, against which antiserum K7 was prepared, is shown in red. Trp2 is not integrated into the domain fold and is coloured blue. The position of the D134A mutation is shown in green. The two images show opposite faces of the domains.

View larger version (17K): [in a new window]   Fig. 3. Binding of peptide-specific antibody K7 to the wild type and mutant N-cadherin-Fc fusion protein. (a) The mutations W2G and D134A in N-cadherin Fc, dimerised via Fc, were tested singly or in combination and compared with results from the wild-type (Wt) molecule. Calcium was present in the assay at 1.25 mM. (b) N-cadherin-Fc mutant W2G was pre-equilibrated with varying levels of Ca2+, which were maintained throughout the assay. (c) Monomeric N-cadherin-Fc was tested in the presence of 1.25 mM Ca2+.

View larger version (27K): [in a new window]   Fig. 4. Epitope mapping of antibody GC4. (a) Binding of mAb GC4 to dimeric N-cadherin Fc bearing point mutations, in the presence of 1.25 mM calcium. (b) Amino acids K64, P65, D67 and Q70 (purple) are seen in relation to the position of non-intercalated Trp2 (blue) and the ßA strand (orange).

View larger version (23K): [in a new window]   Fig. 5. Binding of mAb GC4 to dimeric N-cadherin-Fc mutants in the presence or absence of calcium. (a) The wild type (Wt) and the W2G mutant are compared in the presence of 1.25 mM Ca2+. (b) The same titration was performed in the absence of Ca2+; the two titrations were performed together in the same assay plate and values can be compared directly. Results in the presence of EGTA (not shown) were almost identical to those in b. (c) Wild-type N-cadherin-Fc was tested at varying levels of calcium. (d) The hydrophobic pocket mutant A78M was compared with wild-type N-cadherin in the presence of 1 mM calcium. This comparison was also made in the absence of calcium (e). Similarly, a comparison between the N-terminal extension mutant MDP and wild type N-cadherin was made in the presence (f) or absence (g) of 1 mM calcium. Finally, coordination of calcium in the domain 1-2 junction was disrupted using the mutation D134A, while retaining calcium (1 mM) in the assay buffer. (h) The effect of this mutation, compared with the wild type. (i) The greater effect of D134A on the MDP extension mutant.

View larger version (26K): [in a new window]   Fig. 6. Modelling the formation of `reporter' disulphide bonds formed during strand exchange in domain 1. Structures are derived from PDB 1NCI (Shapiro et al., 1995). The two opposing N-cadherin domains are shown in pale blue and brown respectively. The side chain of Trp2 (brown) is shown located in the hydrophobic pocket of the blue domain. (a) The backbone of C1 is shown in red and the side chain forms a disulphide bond (yellow) with C27 (dark blue). (b) The disulphide bond is formed between C1 (red) and C25 (dark blue).

View larger version (15K): [in a new window]   Fig. 7. Comparison of monomeric and dimeric N-cadherin-Fc in supporting adhesion of DX3 melanoma cells. Cadherin preparations, shown to be monodisperse, were titrated onto an assay plate coated with goat anti-human Fc. DX3 cells were applied and adhesion was assessed as described. Dimeric N-cadherin-Fc containing the mutations W2G and D134A provided a negative control.

View larger version (22K): [in a new window]   Fig. 8. Effect of cysteine point mutations on the adhesive capacity of monomeric N-cadherin-Fc. (a) The assay was conducted in HBSS + 2%FCS (oxidising conditions). (b) Reducing conditions were established by adding 10 mM DTT. Approximately 65% of wild-type cells adhered to the plate in both assays. Reduction restored adhesive capacity of the double mutants D1C,R25C and D1C,D27C.

View larger version (21K): [in a new window]   Fig. 9. Expression of cell surface N-cadherin by K562 transfectants and by DX3 melanoma cells. (a) Stable K562 transfectants were stained with mAb NCD-2 to chicken N-cadherin. The four panels show comparable levels of expression. (b) Untransfected K562 cells were tested with mAb 8C11 to human N-cadherin and the first panel verifies that these cells do not naturally express N-cadherin. The final panel shows DX3 cells stained with 8C11, showing strong expression of human N-cadherin. (c) The molecular size of cellular N-cadherin from K562 transfectants (Wt) is compared with that of monomeric N-cadherin Fc fusion protein (Wt) bearing the mutations F405A,Y407A in the Fc region to prevent dimerization. N-cadherin extracted from normal myoblast cells is also shown for comparison. The gel was run under non-reducing conditions and blotting was conducted using a mixture of pan cadherin antibody to the cytoplasmic domain and anti-Fc.

View larger version (58K): [in a new window]   Fig. 10. Formation of `reporter' disulphide bonds during cadherin-mediated cell adhesion. K562 cells expressing wild-type or mutant N-cadherin were allowed to adhere, under reducing conditions, to a panel of monomeric N-cadherin-Fc molecules bearing the same set of mutations. Oxidising conditions were then restored and the formation of disulphide bonds was assessed by SDS-PAGE and immunoblotting as described. (a) The blot was developed with antibody to cellular cadherin cytoplasmic domain. The upper panels show gels run under non-reducing conditions. Disulphide-bonded trans-dimers form only when cadherin molecules bearing complementary cysteine point mutations were apposed. In contrast, the D1C and D27C mutations allowed formation of disulphide-bonded cis-dimers on the cell surface. With wild-type cells (Wt, right panel), no disulphide-bonded species are seen. The track labelled `uncoated' in this series reflects a small degree of background adhesion to wells lacking cadherin and shows the position of the cis-dimer. The lower panel shows the same preparations run under reducing conditions. (b) In a similar experiment, the blot was developed with anti-Fc. Again the trans-dimer can be seen when mutations D1C and R25C were apposed. As in (a), cis-dimers were formed by D1C and D27C mutants but not by R25C. Cis-dimers formed by monomeric N-cadherin-Fc are seen to run slightly below the trans-dimer in contrast to the situation in (a) where the cellular cis-dimer runs above the trans-species.

View larger version (72K): [in a new window]   Fig. 11. Isolation of disulphide-bonded trans-dimers. Magnetic beads coated with N-cadherin-Fc bearing the mutation D27C were allowed to stick to K562 cells expressing wild-type N-cadherin or the mutants D1C, R25C or D27C. After formation of disulphide bonds, the trans-dimers attached to the beads were isolated from cis-dimers and non-involved cell surface N-cadherin. The trans-dimers were detected by immunoblotting for cellular N-cadherin cytoplasmic domain. The upper panel shows that trans-dimers formed only with the combination D27C beads adhering to D1C cells. The main band at about 300 kDa represents N-cadherin-Fc (dimerised by the Fc hinge region) disulphide-bonded to one cellular cadherin molecule. Higher order assemblies are also seen. The right-hand panel shows total cellular cadherin for comparison. The lower panel shows a gel loading control.

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