Traffic control: Mechanisms of ligand-specific internalization and intracellular distribution of CCR5.

CC chemokine receptor (CCR) 5 promotes inflammatory responses by driving cell migration and scavenging chemokine. A CCR5 inhibitor Maraviroc has been approved for blocking HIV entry; however, inhibitors for the treatment of other diseases have had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor intracellular retention versus presence on the cell membrane are poorly understood. In this study, we systematically compared the time-dependent trafficking behavior of CCR5 following stimulation with its endogenous agonist, CCL5, and 2 CCL5 variants that promote CCR5 intracellular retention. Using a broad panel of pharmacologic assays, fluorescence microscopy, and live cell ascorbic acid peroxidase proximity labeling proteomics, we identified distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All 3 chemokines internalize CCR5 via β-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on G protein-coupled receptor 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 cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. SIGNIFICANCE STATEMENT: CC chemokine receptor (CCR) 5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer, and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably, some ligands induce retention of the receptor inside the cell. This study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.