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Doctoral thesis
English

Molecular mapping of the activation and desensitization mechanisms of CCR5

Number of pages239
Imprimatur date2024
Defense date2024
Abstract

CCR5, a chemokine receptor within the G protein-coupled receptor (GPCR) superfamily, initially gained attention for its pivotal role as the primary co-receptor for HIV. Subsequently, its significance expanded to its involvement in cancer and inflammatory diseases. The chemokine-receptor binding mechanism consists on a "two-site" model, featuring the chemokine recognition site 1 (CRS1) and chemokine recognition site 2 (CRS2). CRS1 involves the N-terminus of the receptor interacting with the chemokine core via electrostatic interactions, guiding the chemokine N-terminus into the binding pocket of the receptor, known as CRS2, where activation occurs. Through mutagenesis of the N-terminus, potent anti-HIV CCL5 analogs with diverse pharmacological profiles, ranging from antagonists to superagonists, were generated. These analogs, alongside natural ligands CCL3 and CCL5, were employed to elucidate the activation mechanism of CCR5 through mutagenesis and structural studies. Notably, ligands such as CCL3, CCL5, antagonist 5P7-CCL5, and superagonist 6P4-CCL5 revealed distinct binding modes and unveiled four CCR5 activation routes. Despite these insights, several questions persist, including which the key contacts between the chemokine and receptor triggering activation; whether all chemokines use the same activation route, or are there more alternative pathways; and whether it is possible to identify specific amino acid residues CRS2 that could elicit biased signaling?

To address these queries, we constructed a functional map of CCR5 CRS2 by studying point mutants pharmacologically, employing natural ligands and CCL5 analogs. This map has highlighted positions K261.28 and Y371.39 as key for signaling and Y2516.51 for arrestin recruitment. Interestingly, CCL4 exhibited dependence on all four CCR5 activation routes and demonstrated similarities with CCL3 in the hydrophobic floor region.

Upon activation by CCL5 or PSC-CCL5, CCR5 undergoes desensitization via clathrin-mediated endocytosis, following a slow-recycling pathway through the endosome recycling complex (ERC) and retrograde trafficking to the Trans-Golgi Network (TGN) in both primary human T lymphocytes and CHO cells. In contrast, 5P14-CCL5 induces a fast-recycling pathway via the ERC in CHO cells. This prompts further investigation into whether these observed trafficking patterns occur in other physiologically relevant cellular backgrounds.

To address these questions, we developed two cellular models for fluorescence microscopy, including primary human monocyte-derived macrophages expressing CCR5 and the HEK-293T cell line. Additionally, we generated eight vectors expressing markers of ERC, TGN, lysosomal degradation, and retromer complex fused to fluorescent proteins, and initiated their validation, expanding our tools for studying trafficking via fluorescence microscopy.

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Citation (ISO format)
F. DA ROCHA BRANCO SAHIN, Camila Raquel. Molecular mapping of the activation and desensitization mechanisms of CCR5. 2024. doi: 10.13097/archive-ouverte/unige:175585
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Creation03/12/2024 2:49:51 PM
First validation03/13/2024 10:25:53 AM
Update time03/13/2024 10:25:53 AM
Status update03/13/2024 10:25:53 AM
Last indexation03/13/2024 10:26:12 AM
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