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

• 来源期刊: 中国药理学报(英文版)
• 关键词: tyrosine sulfation chemokine receptor interferon y-inducible protein 10 CXCR3
• 年，卷（期）: 2009,（2）
• 页码范围: 193-201
• 页数: 9页
• 语种: 英文
• DOI: 10.1038/aps.2008.24

摘要

Aim:Human CXCR3,a seven-transmembrane segment (7TMS),is predominantly expressed in Th1-mediated responses.Interferon-γ-inducible protein 10 (IP-10) is an important ligand for CXCR3.Their interaction is pivotal for leukocyte migra-tion 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 [35S]-cysteine and methionine or [35S]-Na2SO3 and then analyzed by immunoprecipitation to measure sulfation.Experi-ments with 125I-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 transduc-tion.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.Conclusion: 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.