CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis

Abstract

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.Chemokines, or chemoattractant cytokines, are a family of small (8-to 10-kDa) secreted proteins that play an important role in the recruitment and activation of leukocytes (29). Approximately 50 chemokines have been described, and these chemokines interact with some redundancy with 20 G-proteincoupled chemokine receptors. The chemokine system, with its ability to control the migration of leukocytes, plays a critical role in both innate and adaptive immunity.CXCR3 is a chemokine receptor that is expressed on the surface of a number of cell types, including activated CD4 ϩ and CD8 ϩ T cells, NK and NK-T cells, plasmacytoid dendritic cells, and some B cells (26,27,38,45). CXCR3 is activated by three related chemokines: CXCL9, CXCL10, and CXCL11 (9,26,27,38,45). Each of these ligands is induced by gamma interferon and is produced in Th1-type immune responses (9, 14, 31). CXCR3 has been localized to infiltrating effector T cells in a wide variety of human inflammatory diseases, including atherosclerosis (32), rheumatoid arthritis (38), multiple sclerosis (3, 43), heart and lung transplant rejection (1, 22, 50), and psoriasis (17). The CXCR3 ligands have similarly been identified in these same lesions (1,17,20,32,36), leading to the hypothesis that this receptor-ligand system plays an important role in the recruitment of effector T cells into these lesions, resulting in T-cell-mediated inflammation. Supporting this hypothesis, CXCR3-deficient mice were protected from heart allograft transplant rejection and autoimmune type 1 diabetes mellitus in murine models (19,23). Likewise, using CXCL10-deficient mice and inhi...

Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10

“…Here we have shown not only that tyrosines 27 and 29 are sulfated but also that these sulfated moieties are critically important for binding native IP-10. Our data that double sulfated tyrosines contribute to ligand binding are inconsistent with the previous report showing that single mutation of tyrosine 27 or 29 of CXCR3 decreases its ability to bind its ligands, including IP-10 [36] . There are several possible explanations for these inconsistencies.…”

Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

“…The N terminus of the receptor is also a site for PTM providing additional negative charge such as sulfation and O-linked glycosylation, both of which have been reported to be crucial for ligand binding to the receptors CCR5, CX3CR1 and CXCR3 [16][17][18][19]. The activation event following ligand tethering is postulated to occur in close proximity to the cell membrane and involve the disruption of interactions between side chains of neighbouring TM helices by the chemokine N terminus [13,14].…”

“…2). Coupled with this, we also explored the role of posttranslational modifications (PTM) of this receptor region, since studies of CCR5, CX3CR1 and CXCR3 have shown that O-linked oligosaccharides and sulfated tyrosine residues within the N terminus play an important role in chemokine binding [16][17][18][19]. The impact of PTM on CXCR6 function was probed using both site-directed mutagenesis and pharmacological inhibitors of glycosylation and sulfation.…”

Gliadin Induces an Increase in Intestinal Permeability and Zonulin Release by Binding to the Chemokine Receptor CXCR3

“…13,20 In contrast, the glutamate receptor 21 and heat shock proteins 22 are involved in other cellular functions. Based on the observation that CXCR3 activities are potentially relevant to CD pathogenesis, we pursued the possible role of CXCR3 as a receptor for PT-gliadin.…”