The p55 protein appears to stabilize the interaction of protein 4.1 and glycophorin C in mature red blood cells. Previous studies of hereditary elliptocytosis patients have shown that p55-protein 4.1-glycophorin C complexes maintain stability and mechanical properties of red cells. Despite this finding, virtually nothing is known about p55 function in early erythroid precursors or non-erythroid cells. This proposal is based on our hypothesis that p55 plays an important structural and signaling role in pathways of cell proliferation and differentiation. This assumption is buttressed by the high degree of homology of the p55 primary structure to the Drosophila disc's large tumor suppressor, as well as to other signaling proteins of the rapidly growing MAGUK family. The investigators will test their hypothesis in vivo using gene targeting in embryonic stem cells to """"""""knock out"""""""" X-linked p55 gene expression and mutate individual p55 protein domains. Specific goals: (a) Generate targeting constructs that """"""""shut off"""""""" systemic expression of p55 in vivo. They already have one such construct, which introduces a disruption after exon 6 (SH3 domain) of the p55 gene. (b) Investigate the physiological roles of p55 protein domains. Initial focus will be on three well-defined domains: protein 4.1-binding domain, PDZ domain, and SH3 domain. Mice generated from these constructs will facilitate study of the more specialized functions of individual p55 domains in vivo. (c) Analyze the function of p55 during cellular development and growth. They will determine whether p55 plays a key role in mammalian erythropoiesis via interaction with components of the cytoskeleton and signaling pathways by examining the influence of p55 on synthesis, membrane assembly, and turnover of protein 4.1 and glycophorin C. The role of p55 in signaling at the membrane-cytoskeleton interface will be investigated by correlating development of phenotype with defects in apoptosis, p21ras signaling, and the actin cytoskeleton. To assess the role of p55 in cell proliferation and differentiation, they will conduct extensive pathological analyses of lesions developed in p55 mutant mice. In addition, they plan to cross-breed p55-null or -mutant mice with mice containing mutations in the p53, Rb, NF1, and Brca1 tumor suppressors. Analysis of double mutants will elucidate the in vivo combined effect of p55 deficiency and deficiencies of known tumor suppressors.
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