TYROSINE SULFATION OF CHEMOKINE RECEPTORS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 CCR5趋化因子受体是已知的HIV1进入的辅助受体。CCR5胞外N末端片段上酪氨酸的硫酸化已被证明介导了病毒外壳蛋白gp120的结合。我们最近阐明了CCR5 N-末端的酪氨酸硫化模式和时间序列(Seibert C，Cadene M，Sanfiz A，Chait BT，Sakmar TP，酪氨酸转硫酶1和2对CCR5 N-末端的酪氨酸硫化遵循离散模式和时间序列)。《学报》S A.2002.08.20；99(17)：11031-6.到目前为止，人们对酪氨酸硫化在趋化因子受体功能中的作用或这种修饰的一般性知之甚少。特别是，需要更多的工作来了解酪氨酸硫化如何影响受体结合其天然趋化因子配体的能力，并确定它在哪些受体上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，我们进行了全长CCR5和其他趋化因子受体(见下文)中酪氨酸硫化的表征。 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信号对胚胎发育是必不可少的，对免疫细胞归巢至关重要，并与多种类型的癌症的转移有关。CXCR4也是嗜T细胞系(X4)HIV-1毒株进入细胞的主要辅助受体。CXCR4 N端的酪氨酸残基在翻译后被硫酸盐化，参与了SDF-1与CXCR4的高亲和力结合。然而，三个潜在的酪氨酸硫化位点的具体作用还不是很清楚。我们用重组人酪氨酸蛋白硫化转移酶TPST-1和TPST-2修饰CXCR4受体的1-38位氨基酸(CXCR41-38)，研究了CXCR4的硫化模式和序列。我们结合反相高效液相色谱、蛋白质裂解和质谱学对反应产物进行了分析。我们发现CXCR4 1-38被两种TPST酶有效地硫化，导致最终产物含有三个硫代酪氨酸残基。将硫酸盐逐步添加到多肽中，产生含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盐化模式表明，在这两种酶中，Tyr-21首先被硫酸盐化，然后是Tyr-12或Tyr-7。利用异核核磁共振光谱，我们证明了CXCR4 1-38的SDF-1结合亲和力随着硫代酪氨酸数量的增加而增加，这表明CXCR4 N-末端的所有三个位点都具有潜在的硫化生理作用。这些结果为理解翻译后酪氨酸硫化在SDF-1诱导的CXCR4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： 酪氨酸蛋白磺基转移酶对CXCR4的酪氨酸顺序硫化反应。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。

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.