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. 2011 Dec 26;195(7):1115-21.
doi: 10.1083/jcb.201108115. Epub 2011 Dec 19.

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

Michelle R Emond  1 , Sayantanee Biswas, Cheasequah J Blevins, James D Jontes
Affiliations

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

Michelle R Emond et al. J Cell Biol. .

Abstract

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.

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Figures

Figure 1.
Figure 1.
Pcdh19 and Ncad form an adhesive complex. (A) A schematic representation of fusion proteins. Pcdh19 and Ncad ECs were each tagged with either the Fc region of human IgG or with 6xHis. The proposed complexes used in bead aggregation assays are also shown. (B) HEK293 cells were cotransfected with the Fc and 6xHis fusions, which are efficiently produced and secreted into the culture medium. Pcdh19EC and NcadEC form a stable complex, as they can be coisolated using pull-downs of the 6xHis-tagged proteins. (C) Protein A beads were recovered from an aggregation assay, and protein was run on an SDS-PAGE gel and silver stained. In the left lane, aggregation was performed with Pcdh19EC-Fc only, and beads used in the right lane were coated with Pcdh19EC-Fc and NcadEC-6xHis. (D and E) Protein A beads were coated with either Pcdh19EC-Fc or with a complex of Pcdh19EC-Fc and NcadEC-6xHis and allowed to aggregate in the presence of 2 mM CaCl2 or 2 mM EDTA. As previously shown, Pcdh19EC-Fc does not exhibit calcium-dependent homophilic adhesion. However, the complex of Pcdh19EC-Fc and NcadEC-6xHis does mediate bead aggregation. The time course of Pcdh19EC-NcadEC bead aggregation (n = 3) shows robust adhesive activity compared with Pcdh19EC alone (n = 3). (F and G) Although beads coated with NcadEC-Fc (n = 4) aggregate in the presence of calcium, the size of the aggregates is significantly larger for the complex of NcadEC-Fc with Pcdh19EC-6xHis (n = 4). Error bars represent SEM. Bars, 50 µm.
Figure 2.
Figure 2.
Ncad function is not required for Pcdh19–Ncad adhesion. (A) We generated point mutants in NcadEC-Fc that are known to impair adhesion of classical cadherins. The W2A mutation interferes with the formation of the strand dimer, and R14E disrupts the X dimer. Protein A beads coated with the Fc fusions of W2A, R14E, or W2A/R14E mutants do not exhibit aggregation. However, the complexes of Pcdh19EC-Fc with the 6xHis-tagged mutants mediate robust calcium-dependent aggregation. Bar, 50 µm. (B) The formation of the Pcdh19EC-Fc–mutant NcadEC-6xHis complexes was verified by Western blotting after an aggregation experiment. (C) Quantitative characterization of aggregate sizes reveals that complexes formed with the Ncad mutants are as effective as wild-type Ncad in mediating homophilic adhesion (n = 3 for each experiment). Error bars are ±SEM.
Figure 3.
Figure 3.
The EFMR mutation 135–137dup disrupts homophilic adhesion by the Pcdh19–Ncad complex. (A) A schematic showing the missense mutations found in human pcdh19. Cyto, cytodomain; S, signal peptide; T, transmembrane domain. (B) Pull-downs showing the association of Fc fusions of selected EFMR mutants with NcadEC-6xHis. (C) Bead aggregation assays showing that selected EFMR mutations (L23P, L77R, and D117N) support calcium-dependent adhesion when coupled with NcadEC-6xHis, whereas the S135-N137 duplication does not. Bar, 50 µm.
Figure 4.
Figure 4.
Pcdh19–Ncad adhesion is not compatible with Ncad homophilic binding. (A) Red or green fluorescent protein A beads were coated with NcadEC-Fc and mixed before the addition of calcium. After 30 min, mixed aggregates were observed. (B) Red or green fluorescent protein A beads were coated with the complex of Pcdh19EC-Fc–NcadEC-6xHis. As in A, mixed aggregates were observed after 30 min. (C) Green fluorescent protein A beads were coated with Pcdh19EC-Fc–NcadEC-6xHis and mixed with red fluorescent protein A beads coated with NcadEC-Fc. After mixing, calcium was added, and the beads were allowed to aggregate for 30 min. The beads did not form mixed aggregates, indicating that Pcdh19–Ncad does not cross-adhere with Ncad. (D) Pcdh17EC forms an adhesive complex with NcadEC. Mixed aggregates of red and green fluorescent protein A beads coated with the Pcdh17EC-Fc–NcadEC-6xHis complex are shown. (E) Aggregates of beads coated with Pcdh17EC-Fc–NcadEC-6xHis (green) segregate from beads coated with NcadEC-Fc (red). (F) Bead aggregation of Pcdh19EC–NcadEC (red) and Pcdh17EC–NcadEC (green) complexes. The distinct protocadherin complexes exhibit homophilic specificity, as they form distinct aggregates. However, there may be weak heterophilic association, as red and green clusters appear to have some ability to associate even though the beads do not exhibit extensive intermixing. Bar, 25 µm.
Figure 5.
Figure 5.
Ncad enhances Pcdh19 adhesion in cell aggregation assay. (A) CHO cells do not exhibit calcium-dependent cell aggregation. (B) When expressing Pcdh19-p2a-GFP, CHO cells form small aggregates, indicating low levels of adhesion. Expression of Pcdh19 is verified by GFP fluorescence. (C) CHO cells transfected with the Ncad(W2A/R14E) double mutant do not aggregate in the presence of calcium. Expression of the Ncad mutant is verified by RFP fluorescence. (D) CHO cells that express both Pcdh19 and Ncad(W2A/R14E) aggregate more robustly than cells that express only Pcdh19. Coexpression of Pcdh19 and Ncad is demonstrated by the presence of both GFP and RFP fluorescence. Bar, 100 µm.
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