Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

• Published in: The Journal of biological chemistry Vol. 291; no. 31; pp. 16208 - 16220
• Main Authors: Barington, Line, Rummel, Pia C., Lückmann, Michael, Pihl, Heidi, Larsen, Olav, Daugvilaite, Viktorija, Johnsen, Anders H., Frimurer, Thomas M., Karlshøj, Stefanie, Rosenkilde, Mette M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.07.2016; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; CCR8; Chemokine CCL1 - chemistry; Chemokine CCL1 - metabolism; chemokine receptor; Chemokines, CC - chemistry; Chemokines, CC - metabolism; Chlorocebus aethiops; COS Cells; Disulfides - chemistry; Disulfides - metabolism; G protein-coupled receptor (GPCR); Humans; Inositol 1,4,5-Trisphosphate - chemistry; Inositol 1,4,5-Trisphosphate - metabolism; metal ion-protein interaction; molecular pharmacology; mutagenesis in vitro; Protein Binding; Protein Domains; Protein Structure, Secondary; protein-protein interaction; receptor structure-function; Receptors, CCR8 - chemistry; Receptors, CCR8 - genetics; Receptors, CCR8 - metabolism; Signal Transduction; Viral Proteins - chemistry; Viral Proteins - metabolism; molecular pharmacology; G protein-coupled receptor (GPCR); mutagenesis in vitro; CCR8; protein-protein interaction; receptor structure-function; chemokine receptor; metal ion-protein interaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.706747

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr187 in TMIV (Phe171) and TMV (Trp194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.