HEMIDESMOSOMAL PROTEIN LINKAGE MAP BY TWO-HYBRID SYSTEM

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The present proposal is based on the discoveries which were made while utilizing the yeast two-hybrid system to reveal the hemidesmosomal network of proteins Centering on the 180-kD bullous pemphigoid antigen (BPAG2/BP180), a transmembrane molecule also known as a type XVI Collagen. An intracellular domain of BP180, when used as a bait, showed interaction with beta4 integrin intracellular domain, with cytokeratin 18, with p120 molecule, and with the specific domain, called linker-region within the carboxy-terminal globular domains of plectin and periplakin. The plakin family of proteins, currently consisting of five members (plectin, desmoplakin, BP230, envoplakin and periplakin) gives integrity to cells and tissues as revealed through the studies on the function of normal proteins, phenotype of transgenic mice, or patients with gene mutations or autoimmune diseases. The overall goal of the current study is to disclose the hemidesmosomal protein linkage map using yeast two- hybrid system. The main focus of the proposed work is to reveal the biological significance of the two-hybrid interaction between the amino- terminal globular domain of type XVII collagen/BPAG2/BP180 molecule and the carboxy-terminal globular tail domain "linker" of periplakin. Towards this goal, specific antibodies recognizing epitopes within the domains of interest in periplakin and BP180 will be developed, to be used as specific tools for 1) the detection and localization of these proteins in cells and tissues and to study their co-localization by immunofluorescence with specific emphasis on tissues identified by the preliminary results; 2) In vitro binding assays will be conducted to confirm the yeast two-hybrid interactions; 3) The most relevant biological data will be obtained from the "making a connection/breaking the connection" -type experiments. The expression of selected proteins and protein domains in the transfected cells will be used to reveal the in vivo interaction or the loss of such interaction; 4) The yeast two-hybrid system will be used to further characterize the interaction between BP180 and the plakin family of proteins. It is expected that these studies will provide critical information on BP180/periplakin interactions towards the overall goal of understanding the protein-protein network providing stability to the cutaneous basement membrane zone.