Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore novel aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, which share a common chemokine agonist (CXCL12), in terms of their G-protein coupling, β-arrestin (βarr) recruitment, contribution of GRKs, and ERK1/2 MAP kinase activation. We observe that CXCR7 lacks G-protein coupling while maintaining robust βarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected, however, it exhibits significantly reduced βarr-coupling compared to CXCR7 in response to their shared natural agonist, CXCL12. These two receptors induce distinct βarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We further determine the crystal structure of βarr2 in complex with a carboxyl-terminal phosphopeptide derived from CXCR7, which reveals a smaller interdomain rotation than observed previously for activated βarrs. Importantly, structure-guided cellular experiments reveal a key contribution of a single phosphorylation site in CXCR7 on βarr recruitment and endosomal trafficking. Taken together, our study provides molecular insights into intrinsic bias encoded in the CXCR4-CXCR7 system, and it has broad implications for therapeutically important framework of biased agonism.

The Duffy antigen receptor, also known as FY glycoprotein or CD234, is a seven transmembrane protein expressed primarily at the surface of red blood cells, which displays promiscuous binding to multiple chemokines. Not only does it serve as the basis of the Duffy blood group system but it also acts as the primary attachment site for malarial parasite Plasmodium vivax on erythrocytes and as one of the nucleating receptors for the pore forming toxins secreted by Staphylococcus aureus. Despite a predicted 7TM architecture and efficient binding to a spectrum of chemokines, it fails to exhibit canonical second messenger response such as calcium release, likely due to a lack of G protein coupling. Unlike prototypical GPCRs and β-arrestin-biased atypical chemokine receptors, the Duffy antigen receptor also appears to lack β-arrestin binding, making it an enigmatic 7TM chemokine receptor. In order to decipher the molecular mechanism of this intriguing functional divergence exhibited by the Duffy antigen receptor, we have determined its cryo-EM structure in complex with a C-C type chemokine, CCL7. The structure reveals a relatively superficial binding mode of CCL7, with the N-terminus of the receptor serving as the key interaction interface, and a partially formed orthosteric binding pocket lacking the second site for chemokine recognition compared to prototypical chemokine receptors. The structural framework allows us to employ HDX-MS approach to uncover ligand-induced structural changes in the receptor and draw important insights into the promiscuous nature of chemokine binding. Interestingly, we also observe a dramatic shortening of TM5 and 6 on the intracellular side, compared to prototypical GPCRs, which precludes the coupling of canonical signal-transducers namely G proteins, GRKs and β-arrestins, as demonstrated through extensive cellular assays. Taken together, our study uncovers a previously unknown structural mechanism that imparts unique functional divergence on the 7TM fold encoded in the Duffy antigen receptor while maintaining its scavenging function and should facilitate the designing of novel therapeutics targeting this receptor.