Identification of the functional domains in an orphan G protein-coupled receptor, GPR1, which acts as a coreceptor for the brain-derived cell tropism of HIV-1, HIV-2, and SIV

[目的]周细胞位于脑血管周围，与内皮细胞共同构成血脑屏障。细胞感染人类免疫缺陷病毒1型(HIV-1)被认为是发生获得性免疫缺陷综合征(AIDS)相关性脑炎的原因之一。孤儿G蛋白偶联受体GPR1作为HIV-1毒株的辅助受体，感染脑血管衍生的周细胞。本研究的目的是确定GPR1的辅受体活性的重要结构域。[方法](1)与HIV-1相互作用的氨基末端结构域(NTR)和三个胞外环(ECL)。因此，通过聚合酶链式反应(PCR)获得了编码4个胞外区的GPR1的DNA片段，并将其与作为嗜巨噬细胞HIV-1毒株的辅助受体的CCR5重组。(2)通过聚合酶链式反应在GPR1基因中引入核苷酸突变，使NTR的氨基酸发生缺失或替换突变。(3)…之间的嵌合体更多的EN GPR1和CCR5以及NTR的GPR1突变体被导入到胶质瘤细胞系NP-2/CD4中，即使检测到CD4的表达，该细胞对HIV-1、HIV-2(HIV-2)和猴免疫缺陷病毒(SIV)的感染也是严格耐受的。检测建立的细胞对不同的HIV-1、HIV-2和SIV毒株的敏感性。[结果](1)所有ELC嵌合体均丧失其协同受体活性。(2)NTR嵌合体作为辅助受体。(3)NtR中第一个酪氨酸被丙氨酸取代的GPR1氨基酸突变体丧失了共受体功能。另一方面，第二、第三和第四个酪氨酸被丙氨酸取代对功能没有影响。(4)NtR氨基末端11个氨基酸的缺失对GPR1的辅受体活性没有影响。[结论]NTR对GPR1的辅受体功能具有重要作用。NTR中含有四个酪氨酸残基的区域对于其辅助受体功能是重要的。这些结果可用于了解HIV-1感染脑血管周细胞的机制，并可用于抗HIV-1药物的开发。较少

【Aim】 Pericytes located around the brain blood vessels constitute the blood-brain barrier with endothelial cells. The infection of the cells with human immunodeficiency virus type-1 (HIV-1) is thought to be a causative factor to develop acquired immune deficiency syndrome (AIDS)-related encephalitis. An orphan G protein-coupled receptor, GPR1, acts as a coreceptor for HIV-1 strains which infect the brain vessels-derived pericytes. The aim of this study was to identify the important domain of GPR1 for its coreceptor activity. 【Methods】 (1) The amino-terminal domain (NTR) and three extracellular loops (ECLs) interact with HIV-1. Therefore, the DNA fragments of GPR1 coding four extracellular domains were prepared by polymerase chain reaction (PCR) and recombined with CCR5 which acts as a coreceptor for macrophage tropic HIV-1 strains. (2) Some nucleotide changes were introduced into GPR1 gene by PCR to make the amino acid deletion or substitution mutants of the NTR. (3) The chimeras betwe … More en GPR1 and CCR5 and the GPR1 mutants of the NTR were introduced into a glioma-derived cell line NP-2/CD4 which is strictly resistant to the infection with HIV-1, HIV type-2 (HIV-2), and simian immunodeficiency virus (SIV) even though the expression of CD4 is detected. Susceptibility of the established cells to various HIV-1, HIV-2, and SIV strains were detected. 【Results】 (1) All of ELC chimeras lost its coreceptor activities. (2) The NTR chimeras acted as the coreceptor. (3) The amino acid mutant of GPR1 in which the first tyrosine in the NTR was substituted with alanine lost the coreceptor function. On the other hand, the substitutions of the second, the third, and the fourth tyrosine with alanine had no effects on the function. (4) The deletion of the amino-terminal 11 amino acids of the NTR had no effects on the coreceptor activity of GPR1. 【Conclusion】 The NTR is important for the coreceptor function of GPR1. The region containing four tyrosine residues in the NTR is important for its coreceptor functions. These results are available to know the mechanisms of the HIV-1 infection to the brain vessels pericytes and applicable to the development of anti-HIV-1 drugs. Less