G-protein coupled receptors or GPCRs are cell surface proteins, that form the largest class of transmembrane receptors and are targets for nearly 40% FDA-approved drugs. Also known as 7-transmembrane receptors, or 7TMRs, these proteins signal upon stimulation by different types of molecules including neurotransmitters, hormones, odorants, proteins, etc.   

In a recent study, researchers have investigated the characteristics of a unique 7-transmembrane receptor—Duffy antigen receptor for chemokines (or DARC), which is mainly found in red blood cells. The receptor is a glycoprotein that binds chemokines, and the malarial protozoan Plasmodium vivax, helping the parasite to latch onto the red blood cells. It is also a target for toxins released by the bacterium Staphylococcus aureus. The ‘Duffy blood group’ gets its name from this very glycoprotein.  DARC is a potential target for therapeutics against malaria or antimicrobial resistance. 

In their study, researchers have investigated the molecular mechanisms of DARC’s interaction with chemokines and the structural aspects of DARC, particularly its binding with a chemokine—CCL7. Structurally, the Duffy antigen receptor is a 7-transmembrane protein, whose alpha-helical domains span the cell membrane seven times. It also binds to multiple chemokines—proteins produced by the body’s immune system. DARC belongs to the superfamily of  GPCRs. 

Typical 7TMRs signal through two primary transducers, G-proteins and β-arrestin. Some atypical chemokine receptors, also known as ACKRs, signal exclusively through β-arrestin. Despite being classified as a GPCR based on sequence homology, little is known about DARC’s ability to signal through G-protein or β-arrestin . Additionally, DARC is the only 7-transmembrane receptor reported so far, that binds to both, C-C and C-X-C type chemokines, which represent two of the four major chemokine subfamilies.  

Researchers observe DARC to be functionally different from other chemokine-binding GPCRs and atypical chemokine receptors (ACKRs). When stimulated with CCL7, a C-C type chemokine, DARC does not elicit any detectable second messenger response. Here, CCL7 was chosen, as it has the strongest affinity for DARC. Stimulation with CCL7 also failed to induce both G-protein dissociation and β-arrestin recruitment, indicating its distinct nature from other GPCRs and ACKRs. “For the first time, we show that the binding of chemokines to DARC is very different as compared to the binding of chemokines to typical chemokine receptors”, said Dr. Shirsha Saha, a fresh doctorate from IIT Kanpur, and the first author of the paper. 

The researchers further elucidated the unconventional transducer coupling downstream of DARC using phosphoproteomics analysis through a mass spectrometry-based approach.  Phosphoproteomics analysis provides clues in understanding the chain of events in signaling pathways, that follows the binding of chemokine to DARC. The analysis was done using HEK293 cells derived from human embryonic kidneys and those which stably expressed the protein DARC. Researchers identified hundreds of proteins that played a key role in signal transduction, cell proliferation, and chemokine endocytosis. 

Further, STRING analysis was done to identify proteins that interact with DARC. The analysis revealed a protein—CD82, which is a tetraspanin and a membrane glycoprotein mainly involved in suppressing metastasis of tumors.  

It was previously known that interaction of CD82 and DARC stopped the ‘leakage’ or extravasation of tumor cells from blood vessels. Researchers observed a robust interaction between CD82 and DARC in spite of the presence of CCL7. This indicates that CCL7 binds to a different site on DARC, not the one occupied by CD82. 

Structural studies on DARC using cryo-electron microscopy revealed that CCL7-bound DARC showed a ‘kink’ at the cytoplasmic side on the transmembrane helix 3, or TM3. Additionally, researchers observed a dramatic shortening of the transmembrane helices 5 and 6 in the structural studies. “Our structure of DARC represents a previously unanticipated variation encoded within the 7TM fold,” says Dr. Saha.

Typical GPCRs form a cavity on the cytosolic side of the transmembrane receptor, enabling binding of drugs. However, in the case of DARC, the structural changes in the form of the kink and shortening of helices prevent the formation of such a cavity. This further prevents DARC’s interaction with G-proteins, β-arrestin, or GPCR kinases—the typical signal transducers.  

Such structural changes have been reported for the first time in the active-state structures of chemokine receptors. This further emphasizes the distinct nature of DARC. The structural visualisation of DARC would facilitate the design of novel therapeutic molecules that could potentially inhibit the interaction of Plasmodium vivax merozoites and Staphylococcus aureus toxins with DARC. 

Additionally, it is known that DARC is involved in transcytosis of chemokines in endothelial cells, where chemokines bound to DARC at the basolateral end are released at the apical end of the cells. DARC also plays a role in chemokine scavenging. Chemokines produced at the site of infection need to be cleared to prevent chronic inflammation. While clearing, DARC generates a chemokine gradient that guides the movement of white blood cells to the site of inflammation. 

Overall, the research indicates that the 7TM fold in DARC has evolved mainly to scavenge chemokines without showing typical effector binding and activation. The study lays the foundation for deeper molecular and structural insights into signal transduction, facilitating the design of therapeutics against malaria or bacterial infections.