TYROSINE SULFATION OF CHEMOKINE RECEPTORS

趋化因子受体的酪氨酸硫酸化

基本信息

批准号：
8169113
负责人：
THOMAS P SAKMAR
金额：
$ 0.41万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8169113
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 protein-tyrosine sulfotransferase receptor receptor function sulfation tyrosine O-sulfate

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。 CCR5 趋化因子受体是 HIV1 进入的已知辅助受体。 CCR5 胞外 N 末端片段酪氨酸的硫酸化已被证明可介导病毒外壳蛋白 gp120 的结合。我们最近阐明了与 CCR5 N 末端相对应的肽酪氨酸硫酸化的模式和时间序列（Seibert C、Cadene M、Sanfiz A、Chait BT、Sakmar TP、酪氨酰蛋白磺基转移酶对 CCR5 N 末端肽的酪氨酸硫酸化 1 2 遵循离散模式和时间顺序。Proc Natl Acad Sci U S A. 2002。 Aug 20;99(17):11031-6.) 迄今为止，人们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，需要做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，并确定在哪些受体中它对病毒感染至关重要。为了更好地了解趋化因子受体功能的这些方面，我们对全长 CCR5 以及其他趋化因子受体中的酪氨酸硫酸化进行了表征（见下文）。 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 信号传导对于胚胎发育不可或缺，对于免疫细胞归巢至关重要，并且与多种癌症的转移有关。 CXCR4 还充当 T 细胞系嗜性 (X4) HIV-1 毒株进入细胞的主要辅助受体。 CXCR4 N 末端尾部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磺基转移酶 TPST-1 和 TPST-2 修饰对应于受体氨基酸 1-38 的肽 (CXCR4 1-38)，研究了 CXCR4 硫酸化的模式和序列。我们结合反相 HPLC、蛋白水解和质谱分析了反应产物。我们发现 CXCR4 1-38 可以被两种 TPST 酶有效硫酸化，从而产生具有三个磺基酪氨酸残基的最终产品。将硫酸盐逐步添加到肽中，产生具有一种或两种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，使用这两种酶时，Tyr-21 首先被硫酸化，然后是 Tyr-12 或 Tyr-7。使用异核核磁共振波谱，我们证明了 CXCR4 1-38 的 SDF-1 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。描述这项工作的手稿已经出版：酪氨酰蛋白磺基转移酶对 CXCR4 进行连续酪氨酸硫酸化。 Seibert C、Veldkamp CT、Peterson FC、Chait BT、Volkman BF、Sakmar TP。生物化学。 2008 年 10 月 28 日；47(43)：11251-62。