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Identification of novel coreceptors in GPCRs harboring several tyrosines in the N'-terminal region

鉴定 N 端区域含有多个酪氨酸的 GPCR 中的新型辅助受体

基本信息

批准号：
14370099
负责人：
SHIMIZU Nobuaki
金额：
$ 3.97万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370099/
关键词：
HIV GPCR Coreceptor Cell tropism Infection AIDS Diversity Mutation Envタンパク質

项目摘要

[Background]G protein-coupled receptors(GPCRs), which have capacity to act as coreceptors for human immunodeficiency virus type 1, commonly contain several tyrosine residues with asparagines or aspartic acids in the N'-terminal extracellular region(NTR). Sulfation of these tyrosines were demonstrated to enhance coreceptor activity. In this study, in order to know roles of the tyrosine residues in coreceptor activity, we constructed chimeras of extracellular domains among CCR5 and GPR1. GPR1 is an orphan GPCR, which had been identified to act as a coreceptor for HIV-1 strains with the cell tropism for pericytes in brain blood vessels. Moreover, several CCR5 mutants with amino acid substitutions in the NTR were also constructed. Coreceptor activity of a chemokine receptor, D6, which also contains several tyrosine residues in the NTR, was examined.[Method] CCR5, D6, and GPR1 genes were cloned in the expression plasmids, pMX-puro or pCX-bsr. Using the PCR method, chimeric and amino acid substitution mutants were constructed and transduced into a human glioma-derived cell line, NP-2/CD4. NP-2/CD4 cells were strictly resistant to HIV-1 infection, although the CD4 gene was transduced and highly expressed. Susceptibilities of NP-2/CD4 cells transduced with D6 or CCR5 mutants to HIV-1 strains were determined.[Results] A substitution of the tyrosine (the 3rd amino acid position) into alanine had no effect on the coreceptor activity of CCR5. On the other hand, the substitution of the tyrosine (the 15th a.a.) completely abolish the coreceptor activity. Importance of the other tyrosines in the activity was varied depending on viral strains. NP-2/CD4 cells transduced with D6 gene showed susceptibility to several HIV-1 strains with both CCR5 and CXCR4 uses.[Discussion] There may be a conserved structure in NTRs of the GPCRs with coreceptor activities, which is critical for the interaction with the Env protein of HIV-1. This structure will be a clue to develop new anti-HIV-1 drugs.

【背景】G蛋白偶联受体（GPCR）能够作为人类免疫缺陷病毒1型的辅助受体，其N'端胞外区（NTR）通常含有多个带有天冬酰胺或天冬氨酸的酪氨酸残基。这些酪氨酸的硫酸化被证明可以增强辅助受体活性。在本研究中，为了了解酪氨酸残基在辅助受体活性中的作用，我们构建了CCR5和GPR1的胞外结构域嵌合体。 GPR1 是一种孤儿 GPCR，已被鉴定为 HIV-1 毒株的辅助受体，具有脑血管周细胞的细胞趋向性。此外，还构建了几个在NTR中具有氨基酸取代的CCR5突变体。研究了NTR中也含有多个酪氨酸残基的趋化因子受体D6的辅助受体活性。[方法]将CCR5、D6和GPR1基因克隆到表达质粒pMX-puro或pCX-bsr中。使用PCR方法，构建嵌合和氨基酸取代突变体并转导至人神经胶质瘤来源的细胞系NP-2/CD4中。尽管CD4基因被转导并高度表达，但NP-2/CD4细胞对HIV-1感染具有严格的抵抗力。测定转染D6或CCR5突变体的NP-2/CD4细胞对HIV-1株的敏感性。【结果】将酪氨酸（第3位氨基酸）替换为丙氨酸对CCR5的辅助受体活性没有影响。另一方面，酪氨酸（第 15 个氨基酸）的取代完全消除了辅助受体活性。其他酪氨酸在活性中的重要性根据病毒株的不同而不同。转染D6基因的NP-2/CD4细胞对多种同时使用CCR5和CXCR4的HIV-1毒株表现出易感性。[讨论]具有辅助受体活性的GPCR的NTR中可能存在保守结构，这对于与HIV-1的Env蛋白相互作用至关重要。这一结构将成为开发新的抗HIV-1药物的线索。