TYROSINE SULFATION OF CHEMOKINE RECEPTORS

趋化因子受体的酪氨酸硫酸化

• 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
• 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

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.

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