Seibert Christoph, Veldkamp Christopher T, Peterson Francis C, Chait Brian T, Volkman Brian F, Sakmar Thomas P
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065, USA.
Biochemistry. 2008 Oct 28;47(43):11251-62. doi: 10.1021/bi800965m. Epub 2008 Oct 4.
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.
CXC趋化因子受体4（CXCR4）是基质细胞衍生因子-1（SDF-1/CXCL12）的G蛋白偶联受体。SDF-1诱导的CXCR4信号传导对胚胎发育不可或缺，对免疫细胞归巢至关重要，并且与多种癌症的转移有关。CXCR4还是T细胞系嗜性（X4）HIV-1毒株细胞进入的主要共受体。CXCR4 N末端尾巴中的酪氨酸残基在翻译后被硫酸化，与SDF-1与CXCR4的高亲和力结合有关。然而，三个潜在酪氨酸硫酸化位点的具体作用尚不清楚。我们通过使用重组人酪氨酰蛋白硫酸转移酶TPST-1和TPST-2修饰与受体氨基酸1-38相对应的肽（CXCR4 1-38），研究了CXCR4硫酸化的模式和序列。我们结合反相高效液相色谱、蛋白水解切割和质谱分析反应产物。我们发现CXCR4 1-38能被两种TPST酶有效硫酸化，产生具有三个磺基酪氨酸残基的最终产物。硫酸盐逐步添加到肽上，产生具有一个或两个磺基酪氨酸的特定中间体。这些中间体的硫酸化模式表明，两种酶都首先硫酸化Tyr-21，然后是Tyr-12或Tyr-7。使用异核核磁共振光谱，我们证明CXCR4 1-38与SDF-1的结合亲和力随着磺基酪氨酸数量的增加而增加，这表明CXCR4 N末端所有三个位点的硫酸化具有潜在的生理作用。这些结果为理解翻译后酪氨酸硫酸化在SDF-1诱导的CXCR4信号传导中的作用提供了结构基础。
