PROTEIN 4.1 EXPRESSION DURING ERYTHROID DIFFERENTIATION

这项提议的长期目标是制定一项详细的 对红系细胞膜组装和重塑的认识 区分和刻画它们之间的相互作用 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。为了实现我们的目标 既定目标我们建议进行以下一系列研究：1)执行 80kD红系4.1N-2的详细结构与功能分析 末端30kD~膜结合~结构域映射结合位点(S)为 血糖素C、P55、带3和钙调蛋白，并鉴定 与高度保守的C-末端22-24kD结构域相互作用。2) 质膜相关蛋白4.1亚型功能分析 探讨红系终末分化的假说 不同结构的4.1异构体在质膜上的组装 在红细胞生成过程中会导致蛋白质的动态重组 膜生物物理性质的相互作用和变化。3)定义 蛋白质4.1亚型对核和中心体的作用 架构和功能。我们将分析蛋白质4.1的定位 相对于特性良好的核和中心体蛋白，在 在细胞周期的不同阶段，确定4.1个结合伙伴。我们 将通过操纵来探索核4.1的功能重要性 转基因正常结构物或假定为显性的表达水平 负衍生物和中心体4.1功能的检测 中心体重组分析中4.1缺失的影响。4) 确定红细胞与骨之间的粘附性相互作用 骨髓细胞外基质和基质细胞对4.1基因的调控 表情。我们将测试我们的假设，即连接红细胞 黏附分子α4β1整合素与血管细胞黏附分子VCAM-1或纤维连接蛋白改变 它与细胞骨架的细胞内联系从而启动了一种 信号级联到细胞核。我们还将确定是否 α4β1介导的红细胞与纤维连接蛋白的黏附 多肽、内皮细胞或骨髓巨噬细胞影响 4.1基因表达与分化相关的变化。我们 预期这些拟议目标的成功实现 将提供对膜组装和重塑的基本见解 红系分化过程中黏附相互作用的作用 在触发这些分化相关的过程中。这个，在 反过来，应该能够更好地从机械上理解 遗传性球形细胞增多症的病理生理学与遗传性 椭圆形红细胞增多症并提供对骨骼的作用的重要见解 骨髓微环境在调节供者分化中的作用 移植后的造血细胞。

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.