Identification of ligands for orphan G-protein-coupled receptors by using human immunodeficiency virus

(1)我们已经证明了四种g蛋白偶联受体GPR1、RDC1、D6和FML1作为HIV-1感染的共受体。其中，RDC1和GPR1是已知的孤儿受体，即它们的配体尚未确定。GPR1的氨基酸序列与趋化因子受体的氨基酸序列几乎没有相似之处。因此，为了推断其配体，我们利用人类基因组计划确定的核苷酸序列推断出的氨基酸序列的相似性，对被认为由数百种不同类型组成的gpcr进行了系统发育树。趋化因子受体，由CCR或CXCR趋化因子受体组成，形成了树的单个分支。GPR1与另一种孤儿趋化因子受体DEZ最为相似，并且与FML1也明显相似。据报道，FML1是一种来自细菌消化产物的寡肽受体。因此，GPR1也有可能作为外源寡肽受体发挥作用。(2)我们已经证明，来自apelin的肽可以抑制表达APJ（一种apelin受体）的HIV-1 tpNP-2细胞的感染。接下来，我们测试了是否有可能筛选肽作为孤儿gpcr配体的可能性，特别是GPR1和RDC1。合成GPR1、CCR5或CXCR4的n端肽，并检测其对表达GPR1、CCR5或CXCR4共受体的NP-2细胞抑制HIV-1感染的能力。只有GPR1的n端肽，而CCR5或CXR4由27个氨基酸组成，能抑制既表达GPR1又表达CCR5或CXCR4的NP-2细胞感染HIV-1、HIV-2或SIV。被HIV-1或HIV-2感染的NP-2/CD4细胞系统将适合筛选孤儿gpcr的配体。(3)筛选了从大阪蛋白质研究所获得的100多种寡肽。不幸的是，没有一种肽能显著抑制HIV-1或HIV-2感染。

(1)We have shown four G-protein-coupled receptors, GPR1,RDC1,D6 and FML1, function as co-receptors for HIV-1 infection. Among them, RDC1 and GPR1 are known to be orphan receptors, namely, their ligands have not been identified yet. The amino acid sequence of GPR1 shows little similarities to those of chemokine receptors. Therefore, to infer its ligand, we made a phylogenetic tree of GPCRs, which have been thought to be consisting of several hundred different types, using their similarities in their amino acid sequences deduced from their nucleotide sequences determined upon the Human Genome Projects. Chemokine receptors, consisting of receptors for CCR or CXCR chemokines, formed a single branch of the tree. GPR1 was the most similar to another orphan chemokine receptor, DEZ, and was also markedly similar to FML1. FML1 has been reported to be a receptor for oligoneptides derived from digested products of bacterial origins. Therefore, there is a possibility that GPR1 is also functioning as a receptor for exogenous oligoneptides.(2)We have shown that the peptides derived from apelin inhibit infection of HIV-1 tpNP-2 cell expressing APJ, a receptor for apelin. Next we tested whether it is possible to screen peptides for their probabilities as a ligand for orphan GPCRs, especially GPR1 and RDC1. The N-terminal peptides of GPR1, CCR5 or CXCR4 were synthesized and examined for abilities to inhibit HIV-1 infection to NP-2 cells expressing GPR1,CCR5 or CXCR4 as a co-receptor. Only the N-terminal peptide of GPR1 but CCR5 or CXR4, consisting of 27 amino acids, inhibited infection of NP-2 cells expressing not only GPR1 but also CCR5 or CXCR4 with HIV-1,HIV-2 or SIV. The NP-2/CD4 cell system to be infected with HIV-1 or HIV-2 will be suitable for screening of ligands for orphan GPCRs.(3)We have screened more than 100 oligopeptides obtained from the Protein Research Institute, Osaka. Unfortunately, none of the peptides markedly inhibited HIV-1 or HIV-2 infection.