CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis.
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.
Colvin
Richard A
RA
Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, 149 Thirteenth Street, Room 8031, Charlestown, MA 02129, USA.
Campanella
Gabriele S V
GS
Manice
Lindsay A
LA
Luster
Andrew D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
C114753:CXCL10 protein, human; C515202:CXCL11 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D019743:Chemokines, CXC; C515203:Cxcl11 protein, mouse; C515188:Cxcr3 protein, mouse; D008024:Ligands; D010455:Peptides; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine; D011994:Recombinant Proteins; D014443:Tyrosine; D001120:Arginine