The long term objective of this proposal is to develop a detailed understanding of membrane assembly and remodeling during erythroid differentiation and to characterize the interactions between extracellular matrix, stromal cells and erythroid precursors which trigger these differentiation-associated processes. To achieve our stated objective we propose the following series of studies: 1) Perform detailed structure-function analysis of the 80 kD erythroid 4. 1 N- terminal 30 kD ~membrane binding~ domain to map the binding site(s) for glycophorin C, p55, band 3 and calmodulin and identify proteins which interact with the highly conserved C-terminal 22-24 kD domain. 2) Analyze plasma membrane-associated protein 4.1 isoform function during terminal erythroid differentiation to explore the hypothesis that assembly of 4.1 isoforms of varying structure onto the plasma membrane during erythropoiesis results in dynamic reorganization of protein interactions and changes in membrane biophysical properties. 3) Define how protein 4.1 isoforms contribute to nuclear and centrosomal architecture and function. We will analyze protein 4.1 localization relative to well characterized nuclear and centrosomal proteins at various stages of the cell cycle and identify 4.1 binding partners. We will explore the functional importance of nuclear 4.1 by manipulating expression levels of transfected normal constructs or putative dominant negative derivatives and investigate centrosomal 4.1 function by testing the effects of 4.1 depletion in centrosome reconstitution assays. 4) Determine whether adhesive interactions between erythroblasts and bone marrow extracellular matrix and stromal cells regulate 4.1 gene expression. We will test our hypothesis that liganding the erythroblast adhesion molecule alpha4beta1 integrin to VCAM-1 or fibronectin alters its intracellular association with the cytoskeleton thereby initiating a signaling cascade to the nucleus. We will also ascertain whether alpha4beta1 -mediated adhesion of cultured erythroblasts to fibronectin peptides, endothelial cells, or bone marrow macrophages influences differentiation-associated changes in 4.1 gene expression. We anticipate that the successful accomplishment of these proposed aims will provide fundamental insights into membrane assembly and remodeling during erythroid differentiation and the role of adhesive interactions in triggering these differentiation-associated processes. This, in turn, should enable a better mechanistic understanding of the pathophysiology of hereditary spherocytosis and hereditary elliptocytosis and provide import insights into the role of the bone marrow microenvironment in regulating differentiation of donor hematopoietic cells following transplantation.
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