GPR75: a new CCL5 receptor that mediates neuroprotection against HIV

 描述（由申请人提供）：人类免疫缺陷病毒（HIV）研究的一个挑战涉及发现和表征预防或限制HIV感染大脑后神经变性的化合物。趋化因子CCL 5通过影响包膜蛋白gp 120与辅助受体CCR 5的结合来抑制嗜M性HIV毒株进入巨噬细胞/小胶质细胞。有趣的是，CCL 5还防止由嗜T gp 120和病毒蛋白达特介导的神经元细胞死亡，这些蛋白对CCR 5没有亲和力。因此，这种趋化因子的作用机制仍有待充分表征。最近的研究和我们的初步数据表明，CCL 5激活G蛋白偶联受体75（GPR 75），其编码Gq <$家族的540个氨基酸的孤儿受体。这种受体在大脑中比在免疫器官中更丰富。此外，在SH-SY 5 Y细胞中，CCL 5激活各种促存活信号分子，包括三磷酸肌醇、磷脂酰肌醇3-激酶及其下游靶蛋白激酶B和细胞外信号调节激酶。这些细胞不表达CCR 5、CCR 3和CCR 1，这些受体已知与CCL 5结合。此外，与CCR 5、CCR 3和CCR 1结合的其他趋化因子CCL 4、CCL 7和CCL 3未能激活这些细胞中的这些信号分子。因此，GPR 75可能是解释CCL 5神经保护活性的缺失环节。在这个探索性的提议中，我们将测试创新的假设，即通过CCL 5激活GPR 75促进针对病毒蛋白（例如gp 120和达特）的神经保护。为了检验这一假设，我们提出通过Scatchard分析、交联、受体内化、质谱和表面等离子体共振（Biacore）研究（AIM 1）在人类以及啮齿动物神经元中鉴定和表征CCL 5对GPR 75的结合活性和亲和力。这些实验将伴随检查GPR 75在体外神经元中针对嗜T gp 120和达特的CCL 5介导的神经保护中的作用的研究（AIM 2）。我们希望提供有关CCL 5与GPR 75结合的能力及其在预防HIV蛋白神经毒性中的作用的新的重要数据。这些数据将有助于设计小分子GPR 75激动剂作为对抗HIV引起的突触简化的辅助疗法。

 DESCRIPTION (provided by applicant): One challenge in Human Immunodeficiency Virus (HIV) research involves the discovery and characterization of compounds that prevent or limit the neurodegeneration that follows HIV infection of the brain. The chemokine CCL5 inhibits entry of M-tropic HIV strains into macrophages/microglia by affecting the binding of the envelop protein gp120 to the co-receptor CCR5. Interestingly, CCL5 also prevents neuronal cell death mediated by the T-tropic gp120 and the viral protein Tat, which have no affinity for CCR5. Therefore, the mechanism of action of this chemokine remains to be fully characterized. Recent studies and our preliminary data have shown that CCL5 activates a G protein coupled-receptor 75 (GPR75) which encodes for a 540 amino-acid orphan receptor of the Gq¿ family. This receptor is more abundant in the brain than in the immune organs. Moreover, CCL5 activates various pro-survival signaling molecules, including inositol triphosphate, phosphatidylinositol 3-kinase and its downstream targets protein kinase B and extracellular signal-regulated kinases, in SH-SY5Y cells. These cells do not express CCR5, CCR3 and CCR1, receptors known to bind to CCL5. Moreover, CCL4, CCL7 and CCL3, other chemokines that bind to CCR5, CCR3 and CCR1, failed to activate these signaling molecules in these cells. Therefore, GPR75 could be the missing link to explain the neuroprotective activity of CCL5. In this exploratory proposal we will test the innovative hypothesis that GPR75 activation by CCL5 promotes neuroprotection against viral proteins (e.g. gp120 and Tat). To test this hypothesis, we propose to identify and characterize CCL5 binding activity and affinity for GPR75 by Scatchard analysis, cross-linking, receptor internalization, mass spectrometry and surface plasmon resonance (Biacore) studies (AIM 1) in both human as well as rodent neurons. These experiments will be accompanied by studies examining the role of GPR75 in CCL5-mediated neuroprotection against T-tropic gp120 and Tat in neurons in vitro (AIM 2). We expect to provide new significant data on the ability of CCL5 to bind to GPR75 and its role in preventing the neurotoxicity of HIV proteins. These data will help in the design of small molecule GPR75 agonists as an adjunct therapy against synaptic simplification caused by HIV.