PROTEIN 4.1 EXPRESSION DURING ERYTHROID DIFFERENTIATION

本建议的长远目标是制定一个详细的 对红细胞膜组装和重塑的认识 差异化，并描述 细胞外基质、基质细胞和红系前体， 触发这些与差异化相关的过程。 实现我们 为了达到既定的目标，我们提出了以下一系列的研究：1）进行 详细的80 kD红细胞的结构-功能分析4. 1个N- 末端30 kD膜结合结构域，以绘制 血型糖蛋白C、p55、带3和钙调蛋白，并鉴定 与高度保守的C-末端22-24 kD结构域相互作用。 （二） 分析血浆膜相关蛋白4.1亚型功能， 终末红细胞分化，以探讨这一假设， 将不同结构的4.1同种型组装到质膜上 在红细胞生成过程中， 膜生物物理性质的相互作用和变化。 3)定义 蛋白4.1亚型如何促进核和中心体 架构和功能。 我们将分析蛋白4.1定位 相对于充分表征的核和中心体蛋白， 细胞周期的各个阶段，并确定4.1结合伴侣。 我们 将探索核4.1的功能重要性， 转染的正常构建体或推定的显性表达水平 阴性衍生物，并通过测试研究中心体4.1功能 4.1缺失在中心体重建测定中的作用。 四、 确定成红细胞和骨之间的粘附相互作用 骨髓细胞外基质和基质细胞调控4.1基因 表情 我们将检验我们的假设， 粘附分子α 4 β 1整合素与VCAM-1或纤连蛋白改变 其与细胞骨架的细胞内结合，从而启动 信号级联到细胞核。 我们还将确定 α 4 β 1介导的培养成红细胞与纤连蛋白的粘附 肽、内皮细胞或骨髓巨噬细胞影响 4.1基因表达的分化相关变化。 我们 预计这些拟议目标的成功实现 将为膜的组装和重塑提供基本的见解 在红系分化和粘附相互作用的作用 来触发这些与差异相关的过程。 而这 反过来，应该能够更好地机械地理解 遗传性球形红细胞增多症和遗传性 椭圆形红细胞增多症，并提供进口洞察骨的作用， 骨髓微环境对供者分化的调控作用 造血干细胞移植后。

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.