STRUCTURE-FUNCTION OF THE HA RECEPTOR FOR ENDOCYTOSIS

描述（由申请人提供）：透明质酸（HA）和硫酸软骨素（CS）是脊椎动物中普遍存在的细胞外基质组分。HA是细胞行为的有力调节剂，例如在细胞迁移、发育、癌症、伤口愈合和血管生成期间。越来越多的证据表明，HA的正常合成、降解和周转的改变在发育缺陷和疾病如关节炎和癌症的发病机制中可能是至关重要的。我们最近纯化并克隆了负责从淋巴和血液中清除HA和CS的人内吞受体。这种受体被称为内吞作用HA受体（HARE）。人HARE（Human HARE，hHARE）在肝、脾和淋巴结的窦状隙细胞中以190 kD和315 kD两种亚型表达，可能具有独立的功能。该项目的长期目标是了解hHARE在正常HA/CS周转中的生理作用以及异常或有缺陷的HA/CS稳态的病理后果。HARE的两种同种型是由一个大的I型膜蛋白前体（2551个氨基酸）产生的，该前体含有四个富含Cys的结构域，一个连接结构域，跨膜结构域和一个小的胞质结构域。hHARE和大鼠HARE（rHARE）约有80%相同，并作为内吞受体发挥功能，对其他糖胺聚糖具有相似但不相同的特异性。最近的结果证实，较小的大鼠或人HARE同种型本身是一种包被的凹坑靶向的再循环受体，能够介导HA和所有测试的CS类型的内吞作用，但不是硫酸角质素、硫酸乙酰肝素或肝素。本项目将首次表征天然和重组hHARE的结构和配体结合功能。待检验的生化假设是hHARE的特定结构特征有助于多个HA和CS结合位点和多个分选信号，用于通过内吞途径运输蛋白质。我们将研究的生物学假设是，HARE可能在造血中正常发挥作用，在病理学上在转移中发挥作用。我们将利用生物化学、分子生物学、细胞生物学和发育生物学等技术对HARE的结构和生物学功能进行验证，具体目的如下：1）鉴定190 kD和315 kD hHARE的HA和CS结合结构域; 3）鉴定将hHARE靶向包被的小凹以及细胞内路由和再循环所需的序列基序和残基; 4）评估hHARE介导肿瘤细胞粘附和转移的能力; 5）研究HARE在脊椎动物发育中的作用。该项目的结果将提供确定HARE在正常人体生理学和异常或疾病过程中的作用所需的新知识和工具。

DESCRIPTION (provided by applicant): Hyaluronan (HA) and chondroitin sulfate (CS) are ubiquitous extracellular matrix components in vertebrates. HA is a powerful modulator of cell behavior, e.g. during cell migration, development, cancer, wound healing and angiogenesis. Accumulating evidence suggests that alterations in the normal synthesis, degradation and turnover of HA can be critical in the pathogenesis of developmental defects and diseases such as arthritis and cancer. We recently purified and cloned the human endocytic receptor that is responsible for the clearance of HA and CS from the lymph and blood. This receptor is called the HA Receptor for Endocytosis (HARE). Human HARE (hHARE) is expressed in the sinusoidal cells of liver, spleen and lymph node as two isoforms of 190 kD and 315 kD that can probably function independently. The long-term objective of the project is to understand the physiological role of hHARE in normal HA/CS turnover and the pathological consequences of abnormal or defective HA/CS homeostasis. Two isoforms of HARE are generated from a large type I membrane protein precursor (2551 amino acids) that contains four Cys-rich domains, a Link domain, transmembrane domain and a small cytoplasmic domain. The hHARE and rat HARE (rHARE) are about 80 percent identical and function as endocytic receptors with similar, but not identical, specificities for other glycosaminoglycans. Recent results confirm that the smaller rat or human HARE isoform is, by itself, a coated-pit targeted, recycling receptor able to mediate the endocytosis of HA and all the CS types tested, but not keratan sulfate, heparan sulfate, or heparin. This project will characterize the structure and ligand-binding functions of native and recombinant hHARE for the first time. The biochemical hypothesis to be examined is that specific structural features of hHARE contribute to multiple HA- and CS-binding sites and to multiple sorting signals for trafficking the protein through the endocytic pathway. The biological hypotheses we will examine are that HARE may function normally in hematopoiesis and pathologically in metastasis. We will employ techniques in biochemistry, molecular, cell and developmental biology to test these hypotheses about the structure and biological functions of HARE in the following specific aims: 1) To identify the HA- and CS-binding domains in the 190 kD and 315 kD hHARE; 2) To characterize the disulfide bonds and post-translational modifications of the 190 kD hHARE; 3) To identify sequence motifs and residues required for targeting hHARE to coated pits and for intracellular routing and recycling; 4) To assess the ability of hHARE to mediate adhesion to, and metastasis of, tumor cells; 5) To investigate the role of HARE in vertebrate development. Results from this project will provide new knowledge and tools needed to determine the role of HARE in normal human physiology and in abnormal or disease processes.