The molecular interactions that couple the desmosomal cadherins to the intermediate filament cytoskeleton have not been well characterized. Previous studies had shown that the amino-terminal domain of desmoplakin is important for localizing desmoplakin to the cytoplasmic plaque of the desmosome (Stappenbeck et al., 1993
; Bornslaeger et al., 1996
). In the current study, we sought to determine if desmosomal cadherin–plakoglobin complexes interact with the amino-terminal domain of desmoplakin. The results indicate that the amino-terminal domain of desmoplakin clusters desmosomal cadherin–plakoglobin complexes and binds directly to plakoglobin, but not desmoglein. These results provide new insights into the hierarchy of molecular interactions that occur in the cytoplasmic plaque of the desmosome.
In L-cell lines co-expressing DP-NTP, both Dsg1- (Fig. ) and Dsc2a–plakoglobin (Fig. ) complexes are clustered into punctate regions of staining, which appear to be at the plasma membrane. It is interesting to note that the desmosomal cadherins are clustered in L-cells co-expressing DP-NTP, even though these cells do not aggregate in suspension (not shown). The fact that the desmosomal cadherins are clustered but are not engaged in adhesion in L-cells indicates that clustering can occur independent of adhesion. These observations are consistent with the finding that desmosomal complexes resembling half desmosomes are assembled in HaCaT keratinocytes grown in low calcium medium (Demlehner et al., 1995
). Clustering of desmosomal cadherins and plakoglobin represents only one aspect of desmosome assembly, and it will be interesting to determine if other desmosomal components such as pinin (Ouyang and Sugrue, 1992
), envoplakin (Ruhrberg et al., 1996
), and the plakophilins require desmosomal cadherin-mediated adhesion for assembly into the plaque.
Co-immunoprecipitation experiments demonstrated that plakoglobin and DP-NTP are present in complexes that can be extracted from L-cell lysates (Fig. ). Although we could not detect the desmosomal cadherins in these complexes, it is likely that the complexes that form between DP-NTP and plakoglobin do contain the cadherins. This is supported by the fact that the desmosomal cadherins are clustered by DP-NTP along with plakoglobin. Furthermore, in cell lines expressing the EcadDsg1 chimera, plakoglobin and DP-NTP are both redistributed to cell–cell interfaces and colocalize with the chimera. These observations strongly suggest that the desmosomal cadherins, plakoglobin, and DP-NTP exist in the same complex.
In addition to the observation that plakoglobin and DP-NTP co-immunoprecipitate, yeast two hybrid analysis demonstrated that DP-NTP binds directly to plakoglobin, but not Dsg1 (Fig. ). The lack of DP-NTP binding to Dsg1 in the two hybrid system is consistent with the observation that Dsg1 clustering did not occur in L-cell lines in the absence of plakoglobin and with the observation that DP-NTP was not recruited to COS cell–cell borders with the EcadDsg1 chimera in the absence of plakoglobin (Fig. ). These results lead us to propose that plakoglobin couples desmoplakin to the desmosomal cadherins, thereby linking these cadherins to the intermediate filament cytoskeleton. Previous studies by Troyanovsky et al. (1993
) demonstrated that a connexin-Dsc2a chimera recruited endogenous desmoplakin to the plasma membrane, even when the plakoglobin-binding region of the Dsc2a tail was deleted. The data presented here do not rule out the possibility that desmoplakin binds to desmocollin directly, or that other proteins in addition to plakoglobin can facilitate desmoplakin–desmosomal cadherin interactions. However, our data indicate that plakoglobin plays an important role in linking desmoplakin to the desmoglein cytoplasmic domain. In the desmosome, therefore, plakoglobin may play a role analogous to the adherens junction protein β-catenin, which couples the classical cadherins to α-catenin (Aberle et al., 1994
; Jou et al., 1995
; Cowin and Burke, 1996
Recently, transgenic mice with a null mutation in the plakoglobin gene were reported to have defects in desmosome assembly in the intercalated discs of the heart (Bierkamp et al., 1996
; Ruiz et al., 1996
). In these mice, Dsg2 no longer clustered into distinct junctional structures but exhibited a diffuse distribution on the cell surface. In addition, normal desmosomes were no longer detected in the intercalated discs of the plakoglobin null mice, and adherens junctions and desmosomal components appeared to be mixed into the same junctional structures. Interestingly, desmoplakin localized in these structures, suggesting that this molecule can associate with other junctional proteins in addition to plakoglobin. A family of proteins related to plakoglobin, termed plakophilins, has been identified, and the desmosomal component originally termed band 6 is now known to be a plakophilin family member (Hatzfeld et al., 1994
; Heid et al., 1994
; Hatzfeld and Nachtsheim, 1996
; Mertens et al., 1996
). It is possible that plakophilin family members or other desmosomal components also play a role in linking the desmosomal cadherins to desmoplakin.
An interesting property of plakoglobin is that it binds to both classical and desmosomal cadherins. The observation that DP-NTP binds to plakoglobin and clusters desmosomal cadherin–plakoglobin complexes implies that desmoplakin binds to plakoglobin that is associated with the desmosomal cadherin cytoplasmic domain. In contrast, plakoglobin that is associated with the desmosomal cadherin cytoplasmic domain is unable to bind to α-catenin (Fig. ; Plott et al., 1994
; Roh and Stanley, 1995a
). Recent studies have demonstrated that the α-catenin and Dsg1 binding sites on plakoglobin overlap in the amino-terminal armadillo repeats of plakoglobin, suggesting that Dsg1 and α-catenin cannot bind to the same plakoglobin molecule simultaneously (Sacco et al., 1995
; Aberle et al., 1996
; Chitaev et al., 1996
; Troyanovsky et al., 1996
; Witcher et al., 1996
). However, the classical cadherins bind to the central armadillo repeats of plakoglobin, leaving the amino-terminal armadillo repeats of plakoglobin available to bind α-catenin. In the present study, deletion mutants of plakoglobin lacking either the amino- or carboxyl-terminal domain co-immunoprecipitated with DP-NTP and were clustered in L-cells co-expressing DP-NTP. These results, along with the ability of the amino-terminal plakoglobin deletion to interact with DP-NTP in the two hybrid system, suggest that desmoplakin may bind to the central armadillo repeats of plakoglobin. In A431 cells, the expression of plakoglobin deletion mutants lacking the amino- and carboxyl-terminal end domains promoted the assembly of elongated and fused desmosomes, suggesting that the central armadillo repeats of plakoglobin can promote interactions between desmosomal proteins, which may include desmoplakin (Palka and Green, 1997
). Future studies will be directed at mapping precisely where desmoplakin binds to plakoglobin in relation to the sites on plakoglobin that bind to the desmosomal cadherins. Such studies should provide insight into the mechanisms by which the cadherin cytoplasmic tails govern the domains on plakoglobin that are available for interactions with either α-catenin or desmoplakin.