Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

Fanny Momboisse (1) , Giacomo Nardi (2) , Philippe Colin (3) , Melany Hery (1) , Nelia Cordeiro (1) , Olivier Schwartz (1) , Nathalie Sauvonnet (4) , Fernando Arenzana-Seisdedos (5) , Thibault Lagache (2) , Bernard Lagane (3) , Jean-Christophe Olivo-Marin (2) , Anne Brelot (1)

1 CNRS-UMR3569 - Virus et Immunité - Virus and immunity
2 BIA - Analyse d'images biologiques - Biological Image Analysis
3 Infinity - Institut Toulousain des Maladies Infectieuses et Inflammatoires
4 BCI - Département de Biologie cellulaire et infection - Department of Cell Biology and infection
5 Populations virales et Pathogenèse - Viral Populations and Pathogenesis

Abstract G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on β-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

Domaines: Sciences du Vivant [q-bio]

Fanny Momboisse, Giacomo Nardi, Philippe Colin, Melany Hery, Nelia Cordeiro, et al.. Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status. 2022. ⟨pasteur-03821243v1⟩