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.
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