TYROSINE SULFATION OF CHEMOKINE RECEPTORS

趋化因子受体的酪氨酸硫酸化

• 批准号: 7954067
• 负责人: THOMAS P SAKMAR
• 金额: $ 0.4万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954067
• 关键词: Affect; Affinity; Amino Acids; Binding; Biochemistry; CCR5 gene; CXC Chemokines; Capsid Proteins; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; Embryonic Development; Enzymes; Funding; G-Protein-Coupled Receptors; Grant; HIV Envelope Protein gp120; HIV-1; Heteronuclear NMR; High Pressure Liquid Chromatography; Homing; Human; Immune; Inorganic Sulfates; Institution; Length; Ligands; Manuscripts; Mass Spectrum Analysis; Mediating; Modification; N-terminal; NMR Spectroscopy; Neoplasm Metastasis; Pattern; Peptides; Phase; Physiological; Publishing; Reaction; Recombinants; Research; Research Personnel; Resources; Role; Signal Transduction; Site; Source; Stromal Cell-Derived Factor 1; T-Lymphocyte; Tail; Tyrosine; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Virus Diseases; Work; base; cancer type; chemokine; chemokine receptor; extracellular; macromolecule; protein-tyrosine sulfotransferase; receptor; receptor function; sulfation; tyrosine O-sulfate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CCR5 chemokine receptor is a known coreceptor of HIV1 entry. Sulfation of tyrosines at the extracellular N-terminal segment of CCR5 has been shown to mediate binding of the viral coat protein gp120. We recently elucidated the pattern and temporal sequence of tyrosine sulfation of a peptide corresponding to the N-terminus of CCR5 (Seibert C, Cadene M, Sanfiz A, Chait BT, Sakmar TP, Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence. Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11031-6.) So far little is known about the role of tyrosine sulfation in chemokine receptors function, or the generality of this modification. In particular, more work is needed to understand how tyrosine sulfation affects the receptor's ability to bind its natural chemokine ligands and to determine in which receptors it is critical to viral infection. To better understand these aspects of chemokine receptors function, we have undertaken the characterization of tyrosine sulfation in full-length CCR5 as well as other chemokine receptors (see below). 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. A manuscript describing this work has been published: Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases. Seibert C, Veldkamp CT, Peterson FC, Chait BT, Volkman BF, Sakmar TP. Biochemistry. 2008 Oct 28;47(43):11251-62.

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构为 中心，但不一定是研究者所在的机构。 CCR 5趋化因子受体是已知的HIV 1进入的辅助受体。在CCR 5的细胞外N-末端区段处的酪氨酸的硫酸化已显示介导病毒外壳蛋白gp 120的结合。我们最近阐明了对应于CCR 5的N-末端的肽的酪氨酸硫酸化的模式和时间序列（Seibert C，Cadene M，Sanfiz A，Chait BT，Sakmar TP，Tyrosine sulfation of CCR 5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence.美国国家科学院2002年8月20日;99（17）：11031-6.）到目前为止，对酪氨酸硫酸化在趋化因子受体功能中的作用或这种修饰的普遍性知之甚少。特别是，需要更多的工作来了解酪氨酸硫酸化如何影响受体结合其天然趋化因子配体的能力，并确定哪些受体对病毒感染至关重要。为了更好地理解趋化因子受体功能的这些方面，我们已经进行了全长CCR 5以及其他趋化因子受体中酪氨酸硫酸化的表征（见下文）。 CXC趋化因子受体4（CXCR 4）是基质细胞衍生因子-1（SDF-1/CXCL 12）的G蛋白偶联受体。SDF-1诱导的CXCR 4信号传导对于胚胎发育是必不可少的，对于免疫细胞归巢至关重要，并且与许多类型癌症的转移有关。CXCR 4也是T细胞系嗜性（X4）HIV-1毒株进入细胞的主要辅助受体。CXCR 4的N-末端尾部中的酪氨酸残基，其在透析后硫酸化，涉及SDF-1与CXCR 4的高亲和力结合。然而，三个潜在的酪氨酸硫酸化位点的具体作用还没有很好地理解。我们通过使用重组人酪蛋白磺基转移酶TPST-1和TPST-2修饰对应于受体（CXCR 4 1-38）的氨基酸1-38的肽来研究CXCR 4硫酸化的模式和序列。我们用反相HPLC、蛋白水解裂解和质谱法的组合分析了反应产物。我们发现CXCR 4 1-38被两种TPST酶有效地硫酸化，导致具有三个磺基酪氨酸残基的最终产物。逐步向肽中加入硫酸盐，产生具有一个或两个磺基酪氨酸的特定中间体。这些中间体中的硫酸化模式表明，对于两种酶，Tyr-21首先被硫酸化，然后是Tyr-12或Tyr-7。使用杂波NMR光谱，我们证明了CXCR 4 1-38的SDF-1结合亲和力随着磺基酪氨酸的存在而增加，这表明CXCR 4 N-末端的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CXCR 4信号转导中的作用提供了结构基础。描述这项工作的手稿已经出版： 酪氨酰蛋白磺基转移酶对CXCR 4的连续酪氨酸硫酸化。Seibert C，Veldkamp CT，Peterson FC，Chait BT，Bristman BF，Sakmar TP.生物化学。2008年10月28日;47（43）：11251-62。