Regulation of the chemokine receptors CXCR4 and ACKR3 by receptor activity-modifying proteins

The chemokine CXCL12 and its two cognate receptors-CXCR4 and ACKR3-are key players in various homeostatic and pathophysiological processes, including embryonic development, autoimmune diseases, tissue repair, and cancer. Recent reports identified an interaction of CXCR4 and ACKR3 with receptor activity-modifying proteins (RAMPs), and RAMP3 has been shown to facilitate ACKR3's recycling properties. Yet, the functional effects of RAMPs on the CXCL12 signaling axis remain largely elusive. Here, we characterize the effects of RAMPs on CXCR4 and ACKR3 function. We show that, in the absence of a ligand, RAMPs do not affect the cell membrane localization or constitutive internalization of the two receptors. RAMP3 inhibits ligand-stimulated internalization of ACKR3, which retains the receptor at the membrane and inhibits its ability to scavenge CXCL12. In addition, while cAMP inhibition by CXCR4 is unaffected by RAMPs, basal and ligand-stimulated β-arrestin recruitment to both CXCR4 and ACKR3 is reduced in the presence of RAMP3 due to complex formation at the cell surface. The effects on ACKR3 are observed for chemokine, small molecule, and peptide agonists as well as for a N-terminal truncated receptor variant, suggesting that RAMP regulation involves contacts with the transmembrane domain of the receptor. Taken together, our results show that RAMPs regulate the CXCL12 signaling axis by directly interfering with receptor function. These findings could have direct implications for the interplay between receptors in vivo as well as future drug design in the therapeutic targeting of the CXCL12 signaling axis.