STRUCTURE-FUNCTION OF THE HA RECEPTOR FOR ENDOCYTOSIS

HA 受体内吞作用的结构-功能

• 批准号: 6915188
• 负责人: PAUL H WEIGEL
• 金额: $ 28.25万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6915188
• 关键词: CD44 molecule; SCID mouse; binding sites; carbohydrate receptor; cell migration; chondroitin; clinical research; endocytosis; extracellular matrix; human tissue; hyaluronate; laboratory rabbit; laboratory rat; metastasis; molecular cloning; posttranslational modifications; protein isoforms; protein purification; protein sequence; protein structure function; receptor binding; receptor expression; transfection /expression vector

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.

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成分。 HA 是细胞行为的强大调节剂，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 正常合成、降解和周转的改变对于发育缺陷和关节炎和癌症等疾病的发病机制至关重要。我们最近纯化并克隆了负责从淋巴和血液中清除 HA 和 CS 的人类内吞受体。该受体称为 HA 内吞作用受体 (HARE)。人 HARE (hHARE) 在肝、脾和淋巴结的窦细胞中表达为 190 kD 和 315 kD 的两种亚型，可能可以独立发挥功能。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 的两种亚型由大型 I 型膜蛋白前体（2551 个氨基酸）产生，该前体包含四个富含 Cys 的结构域、一个 Link 结构域、跨膜结构域和一个小的细胞质结构域。 hHARE 和大鼠 HARE (rHARE) 大约 80% 相同，并且作为内吞受体发挥作用，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，较小的大鼠或人类 HARE 亚型本身就是一种涂层坑靶向回收受体，能够介导 HA 和所有测试的 CS 类型的内吞作用，但不能介导硫酸角质素、硫酸乙酰肝素或肝素。该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 结合位点以及通过内吞途径运输蛋白质的多个分选信号。我们将检验的生物学假设是，HARE 可能在造血过程中正常发挥作用，并在转移过程中发挥病理作用。我们将采用生物化学、分子、细胞和发育生物学技术来测试有关 HARE 结构和生物学功能的假设，具体目标如下： 1) 鉴定 190 kD 和 315 kD 中的 HA 和 CS 结合域哈雷； 2) 表征190 kD hHARE的二硫键和翻译后修饰； 3) 鉴定将 hHARE 靶向包被凹坑以及细胞内路由和回收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 在正常人体生理和异常或疾病过程中的作用所需的新知识和工具。