The Journal of Dermatology Vol. 19: 765-769, 1992


Structure of Desmoplakin and Its Association with Intermediate Filaments Kathleen J. Green, Thaddeus S. Stappenbeck, David AD. Parry* and M. Luisa A Virata Abstract 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 dumbbellshaped homodimer, with a central a-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 ofDPI. 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 a-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 ofthe 95 kDa rod domain-of DPI did not visibly alter IF networks. Immunogold localization of C0S-7 cells transfected with constructs including the carboxyl terminus ofDP demonstrated an accumulation of mutant protein within which IF subunits were sequestered. These results suggest a role for'the DP carboxyl terminus in the attachment of IF to the desmosomal plaque in either a direct or indirect manner, Introduction Desmosomes are intercellularjunctions that serve as cell surface attachment points for intermediate filaments (IF), creating a continuous network throughout a tissue by aligning the IF cytoskeleton in all neighboring cells (1-4). Interestingly, intermediate filaments of the epithelial type (Type I and II) as well as the mesenchymal and muscle type (Type III) have been observed to associate with desmosomes (3). However, these intercellular junctions are particularly abundant in tissues that experience mechanical stress, such as epidermis. The major high molecular weight desmosome proteins, desmoplakins (DP) I and II, have been Department of Pathology and the Cancer Center, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611, U.SA *Department of Physics and Biophysics, Massey University, Palmerston North, New Zealand.

localized to the fibrous, less electron-dense portion of the cytoplasmic plaque in the region where IF appears to associate (2, 5, 6). Although DPs are primary candidates for linking the IF cytoskeleton to the inner cell surface at desmosomes, .so far in vitro techniques haveffu1ed to demonstrate such an interaction (7, 8). It is possible that the denaturing conditions used to extract DP from desmosomes during purification might affect its in vitro binding capabilities, or that DP may interact with IF only in the presence of accessory or linking proteins that stabilize or mediate the association. To avoid difficulties inherent in in vitro binding experiments, we have chosen a molecular genetic approach to test the possible interaction between various domains of DP and IF in cultured cells. To this end, cDNA clones encoding the entire predicted coding sequence ofDPI and II have been obtained and a model of the DP molecule has been proposed (9, 10). Using this information, constructs encoding the


Green et al Desmoplakin I





Structure of desmoplakin and its association with intermediate filaments.

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 ...
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