Four major components of the erythrocyte cortical cytoskeleton (spectrin, actin, protein 4.1, and ankyrin) are now understood in considerable detail. Nearly a score of inherited diseases with erythrocyte instability have also been linked to specific molecular defects in one of these proteins. However, our understanding of the role of other erythrocyte cytoskeletal proteins, or even how many additional proteins there are that interact with the spectrin skeleton, or what diseases result from their dysfunction, remains rudimentary. Building on our premise that the spectrin cytoskeleton bestows stability on the plasma membrane by controlling membrane protein and lipid organization, both during erythrocyte maturation and in the mature cell, the proposed studies continue our long-term focus on spectrin as the central component of the cytoskeleton, and our goal of fully understanding the molecular basis of erythrocyte membrane organization and dynamics. Three complementary approaches will be pursued: 1) identification of novel proteins that interact with spectrin, and delineation of their sites of interaction within spectrin using deletional analysis and in vitro binding assays. Novel proteins that bind spectrin will be identified and characterized by genetic selection and protein interaction screening techniques. Initial efforts will focus on identifying the interaction site of SLP2, a novel red cell skeletal protein we have recently identified, and establishing the identity of six novel ligands that we have documented to bind spectrin's SH3 domain by co-precipitation and in vitro binding assays. 2) Resolve the three-dimensional structure of three discrete protein-protein interaction domains in spectrin, or as appropriate, in its ligands. These studies will employ multidimensional NMR and X-ray crystallography. Motifs to be scrutinized include the ankyrin-independent membrane binding domain in beta-spectrin's repeats unit 1 (MAD1), the two-repeat unit ankyrin-binding domain (repeats 14-15), and the structure of the polyphosphorylated COOH terminus of BIE1 spectrin. Finally, in Aim 3), we develop massively-parallel screening methodologies to facilitate the rapid evaluation of protein and protein phosphorylation status in normal and abnormal red cells, using tissue micro arrays and ligand capture micro arrays. Collectively, these studies will enhance our understanding of the spectrin cytoskeleton under in vivo conditions, identify the molecular basis of new inherited diseases, and extend the generality and significance of the erythrocyte paradigm for the study of more complex cells.
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