Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Mol Pharmacol. 2020 Nov;98(5):599-611. doi: 10.1124/molpharm.120.000036. Epub 2020 Sep 17.
C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.
C-C 趋化因子受体 5（CCR5）是趋化因子受体家族中的一种 G 蛋白偶联受体（GPCR）。作为一种已确立的抗人类免疫缺陷病毒药物靶点，CCR5 在癌症和神经炎症方面引起了极大的关注。几种 C-C 趋化因子配体 5（CCL5）的 N 端工程化类似物，即 CCR5 的天然配体，是高度有效的 CCR5 抑制剂。某些类似物的抑制机制与受体脱敏的调节有关，但细胞和分子机制尚未完全阐明。在这里，我们利用一组 CCR5 磷酸化突变体和 arrestin 变体来研究 CCL5 类似物在引发 CCR5 磷酸化和 arrestin 募集方面与 CCL5 的差异，参考当前的“核心”和“尾部”arrestin-GPCR 相互作用模型。我们表明，CCL5 以募集的方式招募 arrestin 2 和 arrestin 3 到 CCR5，特别是 arrestin 2 的募集强烈依赖于 arrestin 尾部的相互作用。5P12-RANTES 不会引发受体磷酸化或 arrestin 募集。相比之下，PSC-RANTES 诱导 CCR5 过度磷酸化，导致 arrestin 募集增强，对 arrestin 尾部相互作用的依赖性降低。5P14-RANTES 诱导的受体磷酸化水平与 CCL5 相当，但 arrestin 募集绝对依赖于 arrestin 尾部的相互作用，并且在使用的一种细胞背景下，募集显示出对 arrestin 3 而非 arrestin 2 的同工型偏向。在四个涉及的丝氨酸残基上，没有观察到配体特异性的受体磷酸化模式差异的证据。我们的结果提高了对 CCR5 分子药理学的理解，并有助于进一步阐明一组强效抑制剂的抑制机制。意义声明：C-C 趋化因子受体 5（CCR5）是人类免疫缺陷病毒、癌症和炎症的关键药物靶点。高度有效的趋化因子类似物抑制剂通过调节受体脱敏发挥作用，该过程由 arrestin 蛋白的募集引发。这项研究表明，强效的 C-C 趋化因子配体 5 类似物在它们引发的 CCR5-arrestin 关联的程度和质量上彼此不同，与母体趋化因子也不同，这为 CCR5 药理学和细胞生物学提供了有价值的见解，将有助于开发针对这一重要受体的新药。
