Ligand-regulated ACKR3 nanoclustering shapes CXCR4 signaling at the plasma membrane

Recent advances in imaging have revealed that chemokine receptors integrate extracellular cues via dynamic nanoscale organization at the plasma membrane. While the atypical chemokine receptor ACKR3 is a known regulator of chemokine availability, its spatial organization and specific signaling contributions remain poorly defined. In this study, we employed single‑particle tracking and quantitative total internal reflection fluorescence microscopy to characterize the spatio-temporal distribution and molecular dynamics of ACKR3 on T cells. We further investigated its interplay with the canonical chemokine receptor CXCR4. Our results demonstrate that ACKR3 exists as a mixture of monomers, dimers, and higher‑order nanoclusters, the formation of which is driven by ligand binding. Stimulation with CXCL12 or the ACKR3-selective small molecule agonist VUF15485 induces robust ACKR3 oligomerization independent of CXCR4 expression, G‑protein coupling, β‑arrestin recruitment, or actin cytoskeleton remodeling. Dual‑color single‑molecule imaging further reveals dynamic CXCR4/ACKR3 heterocomplexes that are modulated by CXCL12. Functionally, ACKR3 co‑expression attenuates CXCL12‑induced CXCR4–Gαi signaling and subsequent cAMP inhibition. Together, these findings identify ACKR3 as an active organizer of chemokine receptor nanoscale architecture, providing a mechanistic framework for how ACKR3 fine‑tunes CXCR4 signaling beyond its established role as a chemokine scavenger.