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

Abstract

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 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. 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.

Tyrosine sulfation in N-terminal domain of human C5a receptor is necessary for binding of chemotaxis inhibitory protein of Staphylococcus aureus

“…We cannot exclude the possibility that the expression of phenylalanine at this site changed receptor conformation due to the increase in hydrophobicity caused by the lack of the hydroxyl group [14,26] . We assessed these possibilities by using tyrosine protein sulfotransferases 1 and 2 (TPSTs), which have been shown to increase the extent of tyrosine sulfation of GPCRs for ligand binding [14,18,19] . We co-transfected the plasmid encoding C5aR variant YFY with constructs for TPST1 and 2.…”

“…C5aR has been shown to be modified by N-linked glycosylation. We treated C5aR-transfected-cells with various concentrations of sodium chlorate, a nontoxic inhibitor of ATP sulfrylase activity [14,26] , for at least 48 h in sulfate-free media. We identified a direct inhibition of the binding of CHIPS to wild-type C5aR by sodium chlorate in a concentration-dependent manner.…”

“…Binding experiments were performed with HEK 293T cells npg transfected with wild type C5aR or its variants using calcium phosphate [14,17,18] . One day later, cells were washed with PBS, and split into two separate aliquots, one used for binding assay and one for receptor expression analyses.…”

“…Transfected cells were cultured in medium containing the various concentrations of sodium chlorate for at least 48 h at 37 o C [14,24] .…”

“…This modification tends to occur in acidic regions of proteins, usually containing multiple tyrosines [9,11] . Several receptors for chemokines and hormones, including CCR5, CXCR3, C3XCR1, and thyroid-stimulating hormone receptor, have been shown to be sulfated on tyrosines in their amino terminal extracellular domains and some of this sulfation is critical for ligand binding [12][13][14][15][16] . Amino-terminal sulfation of tyrosines in the chemotactic receptor for C5a also contributes to formation of the docking site for the C5a anaphylatoxin [17] .…”