Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases.
CXC-chemokine receptor 4 (CXCR4) is a G protein-coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). SDF-1-induced CXCR4 signaling is indispensable for embryonic development and crucial for immune cell homing and has been implicated in metastasis of numerous types of cancer. CXCR4 also serves as the major coreceptor for cellular entry of T-cell line-tropic (X4) HIV-1 strains. Tyrosine residues in the N-terminal tail of CXCR4, which are post-translationally sulfated, are implicated in the high-affinity binding of SDF-1 to CXCR4. However, the specific roles of three potential tyrosine sulfation sites are not well understood. We investigated the pattern and sequence of CXCR4 sulfation by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a peptide that corresponds to amino acids 1-38 of the receptor (CXCR4 1-38). We analyzed the reaction products with a combination of reversed-phase HPLC, proteolytic cleavage, and mass spectrometry. We found that CXCR4 1-38 is sulfated efficiently by both TPST enzymes, leading to a final product with three sulfotyrosine residues. Sulfates were added stepwise to the peptide, producing specific intermediates with one or two sulfotyrosines. The pattern of sulfation in these intermediates indicates that with both enzymes Tyr-21 is sulfated first, followed by Tyr-12 or Tyr-7. Using heteronuclear NMR spectroscopy, we demonstrated that the SDF-1 binding affinity of CXCR4 1-38 increases with the number of sulfotyrosines present, which suggests a potential physiological role for sulfation of all three sites in the N-terminus of CXCR4. These results provide a structural basis for understanding the role of post-translational tyrosine sulfation in SDF-1-induced CXCR4 signaling.
Seibert
Christoph
C
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065, USA. seiberc@mail.rockefeller.edu
Veldkamp
Christopher T
CT
Peterson
Francis C
FC
Chait
Brian T
BT
Volkman
Brian F
BF
Sakmar
Thomas P
TP
D000595:Amino Acid Sequence; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives*
D019718:Receptors, CXCR4; D011994:Recombinant Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase