Pemphigus is a class of devastating epidermal blistering diseases in which autoantibodies are generated against cell-cell adhesion molecules present in the skin and mucous membranes. Pemphigus IgG target desmosomes, a structure that couples the keratin intermediate filament network to regions of strong cell-cell adhesion. In pemphigus vulgaris (PV), the primary target of the autoantibodies is desmoglein-3 (Dsg3), a member of the desmosomal cadherin subfamily of adhesion molecules. The work outlined in this proposal investigates the fundamental mechanisms of desmosome assembly and disassembly in human keratinocytes and how these processes are disrupted by PV patient IgG. A major focus of the proposal is how desmosomal proteins are targeted to lipid raft membrane microdomains, and how the association of desmosomal proteins with lipid rafts regulates desmosome formation and disassembly in response to pemphigus IgG. We hypothesize that desmosomal protein targeting to lipid rafts is essential for desmosomal protein clustering during desmosome assembly, and for endocytosis of desmosomal cadherins during disassembly in response to PV IgG. We will utilize PV patient samples, including skin biopsies and purified PV IgG, to determine how PV IgG affect desmosomes in human skin and to determine how PV IgG impact Dsg3 adhesive function using cell biological and single molecular biophysical approaches. Additionally, we will define the amino-acid determinants in Dsg3 that target the protein to lipid rafts and, using mutants deficient in raft targeting, determine how association with lipid rafts impacts desmoglein function. Similar studies will be performed with desmosomal plaque proteins such as the plakophilins and desmoplakin to determine the hierarchy of desmosome protein association with lipid raft membrane domains. These experimental approaches will be integrated to advance our understanding of basic cellular mechanisms of keratinocyte adhesion and to reveal how these mechanism are compromised in a serious epidermal blistering disorder.