Effect of Ligand Binding on the Diffusion and Distribution of the G Protein-Coupled Receptor CCR5 in the Cell Membrane

Abstract

The human immunodeficiency virus (HIV) almost entirely depends on CCR5, a host-encoded chemokine receptor member of the G protein-coupled receptor (GPCR) superfamily, for infection of target cells and hence for transmission from person to person. Inhibiting CCR5 is a viable strategy to prevent viral infection since individuals expressing a truncated version of this protein are perfectly healthy. While native chemokines can display some weak anti-HIV activity, it has been shown that N-terminally-modified analogues of the native CCR5 ligand RANTES/CCL5 are much more potent inhibitors: analogues such as PSC-RANTES, 6P4-RANTES, 5P12-RANTES, and 5P14-RANTES, have potencies in the picomolar range.PSC-RANTES owes its anti-HIV potency to its capacity to induce long-term sequestration of CCR5 inside target cells. This molecule is a strong agonist of CCR5 and activation of the receptor was believed to be required for internalization. While some recombinant chemokines like 6P4-RANTES exhibit a comparable pharmacological profile to PSC-RANTES, others such as 5P12-RANTES and 5P14-RANTES do not, which led us to conclude that anti-HIV potency can be achieved by different mechanisms: 5P12-RANTES is capable of efficiently blocking CCR5 without removing the receptor from the cell surface and without activating it; 5P14-RANTES displays comparable anti-HIV potency while causing only partial internalization in the absence of detectable receptor activation.Because CCR5 needs to be mobile in the cell membrane for HIV infection, we investigated the effect of RANTES analogues on the diffusion dynamics and spatial distribution of CCR5 in the cell membrane, using single-particle tracking and blink microscopy techniques in combination with quantum-dot or small-fluorophore labeled antibodies. We observe confined diffusion, directed diffusion, as well as an immobile fraction of receptors and show how ligand binding affects the short-range and long-range diffusion coefficients.