Seven structures of atypical chemokine receptor 3 reveal the molecular bases for its promiscuity and signaling bias.

Atypical chemokine receptor 3 (ACKR3) is a seven transmembrane (TM) spanning heptahelical receptor that is involved in different stages of cancer progression. Unlike canonical G protein-coupled receptors (GPCRs), ACKR3 does not couple to heterotrimeric G proteins when activated by its endogenous ligand, CXCL12. It is therefore a naturally occurring fully arrestin biased receptor. ACKR3 however desensitizes normally and contributes to cancer progression through arrestin-mediated signaling pathways and/or by active sequestration of chemokine ligands, which blocks CXCL12-mediated downregulation of CXCR4. Here, we report seven cryo-electron microscopic structures of ACKR3 bound to three different agonistic ligands, including CXCL12, a potent CXCL12 variant (CXCL12 ), and a small molecule agonist (CCX662). Although the chemokines bind to ACKR3 in a truly unique way, all three ligands still extend deep in the orthosteric pocket. In all the structures, the TM spans of ACKR3 assume a configuration like those of active GPCRs, although the cytoplasmic pocket is more compact in ACKR3. In addition, compared to other chemokine receptors, the structure of the intracellular loop 2 (ICL2) of ACKR3 is quite different and lacks a kink at the cytoplasmic end of TM4. This is predicted to prevent ACKR3 from binding heterotrimeric G proteins in a canonical way, thereby blocking nucleotide exchange. In summary, our structures provide insight into the promiscuous recognition of ACKR3 relative to other chemokine receptors, and into its distinct functions relative to other canonical chemokine receptors. The structure models may also guide future drug discovery efforts targeting ACKR3 for cancer therapy.