In ELISA-like assays with purified s190-HARE, 10 nM B-Hep without (black bar) or with (gray bars) 100 nM unlabeled competitor GAGs was incubated for 2 h at 37C. AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. TCS 21311 125I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both TCS 21311 Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE. Keywords: chondroitin TCS 21311 sulfate, endocytosis, glycosaminoglycan turnover, heparin, Stabilin-2 Introduction Scavenger receptors are present in a variety of tissues, particularly the reticuloendothelial systems that clear the lymph and plasma of metabolic waste products and macromolecular debris. Currently, scavenger receptors are organized into five classes and one unclassified group (Yuasa and Watanabe 2003). Scavenger receptors can be broadly expressed or tissue specific, e.g., ranging from MSR1, which is found in macrophages in diverse tissues (Ashkenas et al. 1993), to Macrosialin/CD68, which is expressed only on macrophages found in the liver and peritoneum (Yoshida et al. 1998). The one common feature among scavenger receptors of the various classes is that they all bind low-density lipoprotein (LDL) and/or one or more naturally occurring LDL derivatives: acetylated LDL (AcLDL) and oxidized LDL (oxLDL). Most of these receptors also bind other classical polyanionic ligands such as maleylated bovine serum albumin (BSA), dextran sulfate, chondroitin sulfate (CS), fucoidan, and polyribonucleotides (e.g., poly I, poly G). Although not all of these latter ligands occur naturally, they all share features with natural analogs that facilitate high affinity and specific binding to their respective receptors (Krieger et al. 1993). In this report, we characterize the ligand-binding profile of the hyaluronic acid (HA) receptor for endocytosis (HARE), also known as Stabilin-2/FEEL-2, which is a scavenger receptor that is highly expressed in the sinusoidal, or noncontiguous, endothelium of liver, lymph node, and spleen (Laurent and Fraser 1992). Fraser et al. (1983) first identified a scavenger clearance activity for HA when they injected mice with 14C-HA and found it sequestered in these organs. In Rabbit Polyclonal to NFE2L3 addition, HARE is found in oviduct, corneal and lens epithelium, heart valve mesenchymal cells, ependymal cells lining ventricles in brain, and epithelial cells covering renal papillae (Falkowski et al. 2003). The rat receptor was identified, purified, and characterized first (Yannariello-Brown et al. 1997; Zhou et al. 1999). Human HARE was later cloned and purified (Politz et al. 2002; Zhou et al. 2003), and then characterized in stable cell lines (Harris et al. 2004, 2007). The same protein was initially identified in the human genome database as a Stabilin-1 analog of unknown function (Politz et al. 2002). Human HARE is a 2551-amino-acid, 315 kDa, type-1 transmembrane receptor expressed on the cell surface and intracellular (e.g., endocytic) compartments. Two HARE isoforms (not splice variants) of different mass (e.g., 190 kDa and 315 kDa in human) are found in native tissues (Zhou et al. 1999, 2003; Weigel et al. 2002). During biosynthesis and exocytosis in cells stably expressing full-length human HARE cDNA, a minor subset of the 315 kDa receptor pool is proteolytically cleaved to create the 190 kDa HARE, the C-terminal 1416 aa of the 315-HARE protein (Harris et al. 2007). Although the mechanism and biological TCS 21311 reasons for this protein processing are not yet known, both forms of the receptor are functional (Harris et al. 2004, 2007) and occur in the same tissues. HARE recognizes a host of different ligands, including HA (Yannariello-Brown et al..