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

[背景]G蛋白偶联受体(GPCRs)作为人类免疫缺陷病毒1型的辅助受体，在N‘端的胞外区(NTR)通常含有几个酪氨酸残基和天冬氨酸或天冬氨酸。这些酪氨酸的硫酸化被证明可以增强辅助受体的活性。在本研究中，为了了解酪氨酸残基在辅受体活性中的作用，我们构建了CCR5和GPR1胞外区的嵌合体。GPR1是一种孤立的GPR，已被证实是HIV-1毒株的辅助受体，具有脑血管周细胞的细胞嗜性。此外，还构建了几个在NTR中存在氨基酸取代的CCR5突变体。[方法]将CCR5、D6和GPR1基因克隆到表达载体pMX-Puro或PCX-BSR中。利用PCR方法，构建了嵌合和氨基酸替代突变体，并将其转导到人脑胶质瘤细胞系NP-2/CD4中。NP-2/CD_4细胞对HIV-1感染具有严格的抵抗力，尽管CD_4基因被转导并高效表达。[结果]转导D6或CCR5突变体的NP-2/CD4细胞对HIV-1毒株的敏感性不受CCR5共受体活性的影响。另一方面，酪氨酸的取代(第15个A.A.)完全取消辅受体活性。其他酪氨酸在活性中的重要性因病毒株的不同而不同。转导D6基因的NP-2/CD4细胞对几种同时具有CCR5和CXCR4功能的HIV-1毒株具有敏感性。[讨论]具有共受体活性的GPCRs的NTRs中可能存在一个保守的结构，这是与HIV-1环境蛋白相互作用的关键。这种结构将成为开发新的抗HIV-1药物的线索。

[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.