Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10.
OBJECTIVE
Human CXCR3, a seven-transmembrane segment (7TMS), is predominantly expressed in Th1-mediated responses. Interferon-gamma-inducible protein 10 (IP-10) is an important ligand for CXCR3. Their interaction is pivotal for leukocyte migration and activation. Tyrosine sulfation in 7TMS is a posttranslational modification that contributes substantially to ligand binding. We aimed to study the role of tyrosine sulfation of CXCR3 in the protein's binding to IP-10.


METHODS
Plasmids encoding CXCR3 and its mutants were prepared by PCR and site-directed mutagenesis. HEK 293T cells were transfected with plasmids encoding CXCR3 or its variants using calcium phosphate. Transfected cells were labeled with [(35)S]-cysteine and methionine or [(35)S]-Na(2)SO(3) and then analyzed by immunoprecipitation to measure sulfation. Experiments with (125)I-labeled IP-10 were carried out to evaluate the affinity of CXCR3 for its ligand. Calcium influx assays were used to measure intercellular signal transduction.


RESULTS
Our data show that sulfate moieties are added to tyrosines 27 and 29 of CXCR3. Mutation of these two tyrosines to phenylalanines substantially decreases binding of CXCR3 to IP-10 and appears to eliminate the associated signal transduction. Tyrosine sulfation of CXCR3 is enhanced by tyrosyl protein sulfotransferases (TPSTs), and it is weakened by shRNA constructs. The binding ability of CXCR3 to IP-10 is increased by TPSTs and decreased by shRNAs.


CONCLUSIONS
This study identifies two sulfated tyrosines in the N-terminus of CXCR3 as part of the binding site for IP-10, and it underscores the fact that tyrosine sulfation in the N-termini of 7TMS receptors is functionally important for ligand interactions. Our study suggests a molecular target for inhibiting this ligand-receptor interaction.
Gao
Jin-Ming
JM
Department of Respiratory Disease, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China. gaojm@pumch.cn
Xiang
Ruo-Lan
RL
Jiang
Lei
L
Li
Wen-Hui
WH
Feng
Qi-Ping
QP
Guo
Zi-Jiang
ZJ
Sun
Qi
Q
Zeng
Zheng-Pei
ZP
Fang
Fu-de
FD
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D054367:Receptors, CXCR3 / Q000235:genetics / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
C114753:CXCL10 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054367:Receptors, CXCR3; D013431:Sulfates; D014443:Tyrosine; D002118:Calcium