HEMIDESMOSOMAL PROTEIN LINKAGE MAP BY TWO HYBRID SYSTEM

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The investigator proposes to use a yeast two-hybrid approach with the extracellular domain of BPAG2, and the extracellular and intracellular domains of a6 and b4 integrins as "bait" to screen cDNAs libraries for interacting polypeptides. Putative interactors will be confirmed using co-immunoprecipitation analysis and binding assays. Once confirmed, cDNA clones will be further characterized and their genomic positions determined; antibodies will be generated, Northern blot analysis, in situ hybridization and immunocytochemistry will be used to characterize the expression of these proteins. The hemidesmosome is the primary site of epithelial-basal lamina attachment; defects in hemidesmosomal structure/function lead to blistering diseases in humans. A number of hemidesmosomal components have been identified, these include the integral membrane proteins BPAG2/BP180 (which has a collagen-like extracellular domain) and the integrin pair a6b4 (which interacts with laminin 5). The extracellular domains of these proteins interact with collagen VII-containing anchoring fibers to anchor the hemidesmosome to the basal lamina. In the cytoplasm BPAG1e, plectin, p2OO and IFAP300 have been proposed to mediate the anchorage of keratin-type intermediate filaments to the hemidesmosome. While some of the interactions between these proteins have been identified, the full range of their interactions remains to be defined. It is also very likely that many more hemidesmosomal proteins remain to be identified. The yeast two-hybrid system is therefore a reasonable approach to identifying new polypeptides and studying their interactions with one another. The author's preliminary results indicate that they have mastered the technical aspects of this method. Data already developed indicate that a bait derived from collagen VII interacts with thrombospondin I (an interaction already defined by 'conventional' methods). A bait derived from the cytoplasmic domain of BPAG2/BP180 suggests interactions with the cytoplasmic domain of b4 integrin, the C-terminal domain of plectin, the N-terminal domain of the src-substrate and armadillo-repeat protein p120, the C-terminal domain of the keratin 18, and a novel polypeptide related to desmoplakin and envoplakin (in addition to other novel putative interactors).