Effects of Small Molecule Ligands on ACKR3 ReceptorsS

Chemokines such as stromal derived factor 1 and their G pro-tein coupled receptors are well-known regulators of the devel-opment and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C che-mokine motif receptor 4 (CXCR4) and atypical chemokine re-ceptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the fl-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mamma-lian Ga isoforms and have generated a comprehensive map of the Ga activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective ago-nists for ACKR3 as assessed by their ability to recruit fl-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the trans-membrane domains of ACKR3. The development of more se-lective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signal-ing axis and better understand the structural determinants for ACKR3 activation.SIGNIFICANCE STATEMENT We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions be-tween these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.