title: A hyaluronan receptor for endocytosis (HARE) link domain N-glycan is required for extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) signaling in response to the uptake of hyaluronan but not heparin, dermatan sulfate, or acetylated low density lipoprotein (LDL).
abstract: The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn(2280)), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991-1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756-1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center and The Oklahoma Center for Medical Glycobiology, Oklahoma City, Oklahoma 73104.
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Pandey
authors.affiliation: From the Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center and The Oklahoma Center for Medical Glycobiology, Oklahoma City, Oklahoma 73104 paul-weigel@ouhsc.edu.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry; D001693:Biological Transport, Active; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000378:metabolism; D004705:Endocytosis; D015870:Gene Expression; D057809:HEK293 Cells; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D008024:Ligands; D008077:Lipoproteins, LDL; D016328:NF-kappa B; D011134:Polysaccharides; D011994:Recombinant Proteins; C449927:STAB2 protein, human; C032629:acetyl-LDL; D003871:Dermatan Sulfate; D001216:Asparagine; D006820:Hyaluronic Acid; D006493:Heparin
keywords: Endocytosis; Extracellular Matrix; Luciferase; Matrix Turnover; Multiple Ligands; Receptor Recycling; Scavenger Receptor; Signal Transduction; Stabilin-2