Usher syndrome is the leading hereditary cause of combined deafness and blindness, accounting for over half of the 20,000 deaf and blind people in the United States. Of the 10 known genetic loci associated with Usher syndrome, Usher type Ha (USH2A) is the most common. People with USH2A have congenital high frequency sensorineural hearing loss associated with progressive retinitis pigmentosa. The gene responsible for USH2A has recently been identified, and shown to encode a novel protein. Conceptual translation of the USH2A gene predicted either an extracellular matrix protein or cell surface receptor. In the preliminary results section of this proposal we show that the USH2A protein, which we call usherin, is an abundant basement membrane protein with widespread, but not ubiquitous, tissue distribution. Usherin is very abundant in both cochlear and retinal basement membranes. Understanding how the usherin protein contributes to the structural architecture and the functional dynamics of the basement membranes where it is found will help us decipher the mechanism of pathogenesis in people who lack this protein.
The aims of this proposal are designed to identify the structural and functional properties of the usherin protein. Fusion peptides comprising the functional domains of the protein as well as an expressed full-length recombinant protein will be employed to identify what basement membrane proteins interact with usherin, and what domain(s) of the usherin molecule they interact with. We present data illustrating a type IV collagen/usherin interaction defined using this approach. We will employ immortomouse-derived strial marginal cells, retinal pigment epithelial (RPE) cells, and endothelial cells to define usherin/cell surface receptor interactions, and identify the specific receptors involved. Using this approach, we demonstrate domain-specific usherin receptor binding on RPE cells. Fusion peptides will be engineered that harbor amino acid substitutions resulting from missense mutations found in families with Usher syndrome type ila. These peptides will be employed in established competitive binding assays to determine the consequence of these mutations on usherin function. Combined, these studies will provide a basic understanding of how this new class of basement membrane protein contributes to the structural and functional properties of the basement membranes where it is found. Furthermore, these studies will provide a molecular basis for understanding the specific changes associated with USH2A pathology.
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