The purpose of the planned research is to investigate assembly of desmosomes. The major desmosome proteins, desmoplakin I and desmoplakin II, have been purified from pig tongue epithelium after extraction in buffers containing 4 M urea. The investigators will use the purified proteins labelled with 125I-labelled Bolton-Hunter reagent in binding experiments with purified desmosomes depleted of desmoplakins to try to determine physiologically significant binding proteins for desmoplakins. Desmosome proteins transferred to nitrocellulose will be used to detect the identity of specific proteins to which desmosome bind. The ability of purified desmoplakins to bind to desmosomes and to participate in desmosome assembly will also be examined in cultured cells using fluorophore- derivatized desmoplakins injected into living cells. The authors have determined the structure of the desmoplakin I dimer, but it is not clear how the two monomers associate. Monomers will therefore be derivatized under dissociating conditions in order to produce a stable monomer which can be examined under electron microscopy. Antibodies to the unique region of desmoplakin I will be used to determine the location of this region on the dimer by electron microscopy. Fragments of desmoplakin I will be produced by limited proteolysis, and fragments containing the unique region will be identified by immunoblotting with this antibody. Since desmoplakins are phosphorylated and since evidence suggests that phosphorylation may regulate interaction of some cytoskeletal proteins, the ability of protein kinase C or Ca++calmodulin dependent kinase to phosphorylate desmoplakins will be studied, and phosphorylation of immunoprecipitated desmoplakins will be examined after exposure of keratinocytes to the calcium switch or TPA. The association of desmoplakin I with keratin filaments will be assessed in a keratin binding assay, and the ability of a purified 200 kd doublet to enhance binding of desmoplakin I to purified keratin filaments will be tested. These studies should increase our knowledge of the role of specific protein interactions in desmosome assembly.
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