? The anchoring complex is a membrane-associated cell-matrix adhesive organelle of basal keratinocytes. Studies supported by this grant have focused on the molecular characterization of a major component of the anchoring complex, BP180, and on the role of this protein in both acquired and inherited blistering diseases. BP180 is a homotrimeric transmembrane glycoprotein with a globular N-terminal head domain that interacts with components of the hemidesmosomal plaque and a long C-terminal collagenous domain that projects into the extracellular region beneath the hemidesmosome. Using an animal model developed by our laboratory, it has been demonstrated that an autoimmune response directed against the BP180 protein is a key initiatory step in the pathogenesis of bullous pemphigoid and several other acquired blistering diseases. Genetic defects in COL17A1, the gene encoding BP180, are responsible for a subset of non-Herlitz junctional epidermolysis bullosa (JEB-nh), a subepidermal blistering disease. ? ? The first aim of this grant will define sites on BP180 that are essential for its structure, stability and function. Wild type and mutated recombinant forms of BP180 will be purified and subjected to a wide range of biochemical analyses aimed at monitoring the assembly and stability of the BP180 collagen triple helices. Wild type and mutant BP180 isoforms will be introduced into BP180-deficient keratinocytes from JEB-nh patients and other cultured cells. The genetically manipulated cells will be assayed for changes in biological properties, e.g., hemidesmosome assembly, ligand binding, cell-matrix attachment, and cell migration.
The second aim will further explore the structural and functional consequences of disease-associated mutations in BP180. Mutations associated with JEB-nh will be introduced into full-length or truncated versions of recombinant BP180 and then subjected to a wide range of biochemical and biological analyses. It is our hope that this information will lead to a better understanding of the molecular basis of this disease. The goal of the third aim is to further clarify the role of BP180 as a target of IgG and IgA autoantibodies associated with a family of sub-epithelial blistering diseases. In particular we will focus our attention on the fine specificities of these autoantibodies and on their ability to trigger a cell-signaling event. ? ?
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