Abstract. We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209. These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed fulllength DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.

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Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a fulllength 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major a-helical-rich bundles.Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-Ispecific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.Desmosomes are major sites of intercellular contact found in a variety of cells-i.e., epithelial cells, cardiac myocytes, arachnoidal cells of the meninges, and dendritic reticulum cells of germinal centers in lymph nodes (1-3). The desmosomal plaques serve as specific attachment points for intermediate filaments (IFs), which form a structural framework for the cytoplasm. Of the proteins comprising the desmosomal plaques, two closely related major components, desmoplakins (DPs) I and II [reported sizes vary from 240 to 285 kDa and from 210 to 225 kDa, respectively (4-6)], are among the candidate linkers for mediating IF anchorage to the cell surface. These two proteins have been localized to the fibrous, less electron-dense portion of the plaque through which IFs appear to loop and are associated with the IF cytoskeleton during desmosome assembly (7).Until recently (8, 9), relatively little was known about the molecular structure of DP-I and -II (DNA or protein level), the regulatory mechanisms governing DP expression, the functional roles of the DPs in desmosome assembly, or their relationships with other desmosomal components and cytoskeletal elements, such as IFs. In a previous study (9), computer-aided analysis carried out on a partial predicted DP-I amino acid sequence identified two major structural domains within DP-I: a rod-like central domain and a globular C terminus. Here we report the sequence and predicted structure of the DP N terminus. § Using this information, we propose a model for the entire DP-I polypeptide that is consistent with rotary shadowed electron microscope images showing DP-I to be a dumbbell-shaped molecule (6). We have also examined in more detail the mechanism by which the highly related DP-I and -II mRNAs are generated. Northern blot mapping and sequence analysis of a DP genomic clone were performed to confirm the boundaries of the DP-Ispecific region an...

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Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)