Arrestin recruitment to c-c chemokine receptor 5: potent c-c chemokine ligand 5 analogs reveal differences in dependence on receptor phosphorylation and isoform-specific recruitment bias

Article Article scientifique Article Arrestin recruitment to c-c chemokine receptor 5: potent c-c chemokine ligand 5 analogs reveal differences in dependence on receptor phosphorylation and isoform-specific recruitment bias eng 2020 author Martins Elsa 847920 author Brodier Hellena 751309 author Rossitto-Borlat Irène 23207 author Cadosch Ilke 813481 author Villard Mélanie 732748 author Hartley Oliver 0000-0002-2283-8379 507767 599-611 10.1124/molpharm.120.000036 32943494 0026-895X 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. Animals Arrestin/metabolism Arrestins/metabolism CHO Cells Cell Line Chemokine CCL5/metabolism Chemokines/metabolism Chemokines CC/metabolism Cricetulus HEK293 Cells Humans Ligands Phosphorylation/physiology Protein Isoforms/metabolism Receptors Chemokine/metabolism Signal Transduction/physiology Beta-Arrestin 1/metabolism Dewey 616.07 Swiss National Science Foundation Studying the modulation of CCR5 trafficking by chemokine analogues with potent anti-HIV activity 163085 CH Project funding Swiss National Science Foundation Studying the modulation of CCR5 trafficking by chemokine analogues with potent anti-HIV activity 184828 CH Project funding 137 Département de pathologie et immunologie 21F false 497 Protein and peptide engineering for medicine 835 Molecular Pharmacology 98 5 d2af0299-9ef4-4695-86a1-0b1c7fa1cf51 Article (Published version) Public 599.full-1.pdf 2122741 application/pdf CC BY-NC openAccess Faculté de médecine / Section de médecine fondamentale / Département de pathologie et immunologie

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Arrestin recruitment to c-c chemokine receptor 5: potent c-c chemokine ligand 5 analogs reveal differences in dependence on receptor phosphorylation and isoform-specific recruitment bias	Molecular Pharmacology			2020	417	201

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