title: Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.
abstract: 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, Tyr(184) (Cys + 1) and Tyr(187) (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 Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). 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 Tyr(187) 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.
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Line
authors.initials: L
authors.lastname: Barington
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Pia C
authors.initials: PC
authors.lastname: Rummel
authors.affiliation: From the Department of Neuroscience and Pharmacology, the Novo Nordisk Foundation Center for Basic Metabolic Research, and.
authors.forename: Michael
authors.initials: M
authors.lastname: Lückmann
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Heidi
authors.initials: H
authors.lastname: Pihl
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Olav
authors.initials: O
authors.lastname: Larsen
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Viktorija
authors.initials: V
authors.lastname: Daugvilaite
authors.affiliation: the Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark.
authors.forename: Anders H
authors.initials: AH
authors.lastname: Johnsen
authors.affiliation: the Novo Nordisk Foundation Center for Basic Metabolic Research, and the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark and.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Frimurer
authors.affiliation: From the Department of Neuroscience and Pharmacology.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Karlshøj
authors.affiliation: From the Department of Neuroscience and Pharmacology, rosenkilde@sund.ku.dk.
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
mesh_terms: D000818:Animals; D019556:COS Cells; D054402:Chemokine CCL1 / Q000737:chemistry* / Q000378:metabolism; D019742:Chemokines, CC / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D054401:Receptors, CCR8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C065971:CCL1 protein, human; C103330:CCR8 protein, human; D054402:Chemokine CCL1; D019742:Chemokines, CC; D004220:Disulfides; C435144:MC148 protein, poxvirus molluscum contagiosum; D054401:Receptors, CCR8; D014764:Viral Proteins; D015544:Inositol 1,4,5-Trisphosphate
keywords: CCR8; G protein-coupled receptor (GPCR); chemokine receptor; metal ion-protein interaction; molecular pharmacology; mutagenesis in vitro; protein-protein interaction; receptor structure-function