STRUCTURE-FUNCTION OF THE HA RECEPTOR FOR ENDOCYTOSIS

描述(申请人提供)：透明质酸(HA)和硫酸软骨素(CS)是脊椎动物普遍存在的细胞外基质成分。HA是细胞行为的强大调节器，例如在细胞迁移、发育、癌症、伤口愈合和血管生成过程中。越来越多的证据表明，HA的正常合成、降解和周转的改变可能在发育缺陷和疾病(如关节炎和癌症)的发病机制中起关键作用。我们最近纯化并克隆了负责清除淋巴和血液中HA和CS的人内吞受体。这种受体被称为内吞HA受体(HARE)。人HARE(HHARE)在肝、脾和淋巴结的肝窦细胞中以190kD和315kD两种可能独立发挥作用的亚型表达。该项目的长期目标是了解hHARE在正常HA/CS代谢中的生理作用，以及HA/CS平衡异常或缺陷的病理后果。HARE的两种异构体是由一个大的I型膜蛋白前体(2551个氨基酸)产生的，它包含四个富含Cys的结构域，一个Link域，一个跨膜域和一个小的细胞质结构域。HHARE和大鼠HARE(RHARE)大约80%相同，功能类似于内吞受体，但与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，较小的大鼠或人的Hare亚型本身就是一个包衣坑靶向循环受体，能够介导HA和所有受试CS类型的内吞，但不能调节硫酸角蛋白、硫酸肝素或肝素。本项目将首次对天然和重组hHARE的结构和配体结合功能进行表征。需要检验的生化假说是，hHARE的特殊结构特征有助于通过内吞途径运输蛋白质的多个HA和CS结合位点和多个分选信号。我们将检验的生物学假设是，HARE可能在造血和转移中发挥正常的作用。我们将利用生物化学、分子、细胞和发育生物学技术来验证这些关于HARE结构和生物学功能的假说，具体目标如下：1)确定190 kD和315 kD hHARE中的HA和CS结合结构域；2)表征190 kD hHARE的二硫键和翻译后修饰；3)确定将hHARE靶向覆盖的凹坑以及细胞内路由和循环所需的序列基序和残基；4)评估hHARE介导肿瘤细胞黏附和转移的能力；5)研究HARE在脊椎动物发育中的作用。该项目的结果将提供所需的新知识和工具，以确定HARE在正常人体生理和异常或疾病过程中的作用。

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.