CC 趋化因子受体 5 (CCR5) 通过驱动细胞迁移和清除趋化因子形成定向趋化因子梯度来促进炎症反应。一种针对 CCR5 的药物已被批准用于阻止 HIV 进入细胞。然而，由于 CCR5 受体复杂的生物学和药理学，靶向 CCR5 治疗炎症性疾病和癌症的成功有限。 CCR5 被许多天然和工程趋化因子激活，这些趋化因子引发不同的受体信号传导和运输反应，包括一些将受体隔离在细胞内的趋化因子。隔离现象可能在治疗上可利用，但不同配体将 CCR5 运输到不同细胞位置的机制尚不清楚。在这里，我们采用活细胞抗坏血酸过氧化物酶邻近标记和定量质谱蛋白质组学，在用其内源激动剂 CCL5 和两种促进受体细胞内保留的 CCL5 变体刺激后，公正地发现 CCR5 的时间分辨蛋白质邻域。连同靶向药理学测定，这些数据揭示了具有时间分辨率的不同配体依赖性 CCR5 运输模式。所有三种趋化因子均通过 β-抑制蛋白依赖性、网格蛋白介导的内吞作用内化 CCR5，但程度不同、动力学不同且对 GPCR 激酶亚型的依赖性不同。激动剂的不同之处在于它们将受体靶向溶酶体以及高尔基体区室和跨高尔基体网络进行降解的能力，并且这些运输模式转化为不同水平的配体清除。这些结果提供了对 CCR5 细胞内隔离背后的分子机制的深入了解，并为基于趋化因子的 CCR5 靶向分子的开发提出了可行的模式。CCR5 在免疫系统中发挥着至关重要的作用，并且在炎症、癌症等许多生理和病理过程中发挥着重要作用和艾滋病毒传播。除了其功能多样性之外，不同的 CCR5 配体可以诱导不同的受体信号反应和运输行为；后者包括细胞内受体隔离，这为抑制 CCR5 功能提供了潜在的治疗策略。这项研究利用时间分辨邻近标记蛋白质组学和靶向药理学实验，揭示了受体隔离的分子基础，包括可用于开发促进受体保留在细胞内的 CCR5 靶向分子的信息。

CC chemokine receptor 5 (CCR5) contributes to inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A drug against CCR5 has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of inflammatory diseases and cancer has had limited success because of the complex biology and pharmacology of this receptor. CCR5 is activated by many natural and engineered chemokines that elicit distinct receptor signaling and trafficking responses, including some that sequester the receptor inside the cell. The sequestration phenomenon may be therapeutically exploitable, but the mechanisms by which different ligands traffic CCR5 to different cellular locations are poorly understood. Here we employed live cell ascorbic acid peroxidase proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveals distinct ligand-dependent CCR5 trafficking patterns with temporal resolution. All three chemokines internalize CCR5 via β-arrestin- dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and with varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the molecular mechanisms behind CCR5 intracellular sequestration and suggest actionable patterns for the development of chemokine-based CCR5 targeting molecules.CCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and HIV transmission. Along with its functional diversity, different CCR5 ligands can induce distinct receptor signaling responses and trafficking behaviors; the latter includes intracellular receptor sequestration which offers a potential therapeutic strategy for inhibiting CCR5 function. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the molecular basis for receptor sequestration including information that can be exploited for the development of CCR5 targeting molecules that promote retention of the receptor inside the cell.