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.Chemokines (chemotactic cytokines) regulate the differentiation, activation, and recruitment of leukocytes. They also play important roles in several physiological mechanisms outside the immune system such as organogenesis and angiogenesis (1, 2). With ϳ50 members, these cytokines exert their effects through chemokine receptors (23 members), which belong to class A of the family of seven-transmembrane (7TM) 2 G protein-coupled receptors (3). The implications of the chemokine system in a vast number of human diseases (3) have increased the interest in developing potent, selective, and clinically useful chemokine receptor antagonists.The binding of a chemokine to its cognate receptor is initially driven by electrostatic interactions between the overall positively charged chemokine and the negatively charged extracellular surface of the receptor. Then interactions between the chemokine N terminus and residues in the main binding pocket of the receptor trigger receptor activation (4 -6). In contrast, small molecule ligands bind deeper in the main binding pocket and constrain the receptors in either active or inactive conformations (7,8). Whereas most mapping studies of small molecules have focused on the transmembrane areas, newer studies as well as crystal structures of class A receptors suggest that extracellular receptor regions, in particular ex...

Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization

“…Extracellular loops are known to play important functional roles in ligand binding and receptor activation (Avlani et al, 2007;Barington et al, 2016;Conner et al, 2007;Kclo et al, 2005;Nguyen et al, 2016a, 2016b, Palczewski et al, 2000Peeters et al, 2011;Ragnarsson et al, 2015;Scarselli et al, 2007;Wheatley et al, 2012Wheatley et al, , 2007. Recently reported crystal structures show that the conserved disulfide bridge forces ECL-2 to form a lid over the binding pocket, and that ECL-2 contains well defined but rather different secondary structures within the different receptor families (Palczewski et al, 2000;Rasmussen et al, 2007;Shi and Javitch, 2004;Shimamura et al, 2011;Warne et al, 2008).…”

“…There were several studies disrupting the conserved disulfide bond in different GPCRs by mutagenesis studies, but it has been described that mutant receptors were restricted to endoplasmic reticulum showing lower membrane expression (Barington et al, 2016;Conner et al, 2007;Rummel et al, 2013;Scholl and Wells, 2000), thus the effect of disulfide bridges on ligand binding or function can be masked by the effect due to the mutation itself.…”

Understanding the common themes and diverse roles of the second extracellular loop (ECL2) of the GPCR super-family

“…In some cases it has been shown to be essential for receptor function; With the C5aR, the double alanine substitution did not produce a signalling response upon ligand stimulation (however constitutively active substitutions remained active) (Klco et al, 2005). In the chemokine CCR8 receptor, the conserved disulphide bond between TM3 and ECL2 was essential for receptor function with the CCL peptide agonist but also small molecular antagonists that bind deeper in the TM bundle (Barington et al, 2016). However in other receptors the disulphide bond is functionally redundant; Double alanine mutations (to remove the bond and any free sulphydryl groups) on the family A adenosine A2A receptor (Naranjo et al, 2015) and the family B CGRP and GLP1 receptors (Woolley et al, 2013, Mann et al, 2010 retained receptor function.…”

“…In the V1AR vasopressin receptor, aromatic residues that are highly conserved in the neurohypophysial GPCR subfamily were important for agonist binding and receptor activation (Conner et al, 2007). Tyrosine residues in the chemokine CCR8 receptor were important for agonist and antagonist binding (Barington et al, 2016). Aromatic and hydrophobic residues of ECL2 line the orthosteric binding pocket of the ET B receptor (Shihoya et al, 2016).…”

Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation

“…On the basis of amino acid residue conservation, we created and characterized 25 EBV-BILF1 extracellular domain mutants in terms of MHC class I downregulation and signaling. We also engineered the receptor to contain a binding site for small ‘‘model’’ compounds, the metal ion chelators (32–34). We tested the aromatic chelators bipyridine and phenanthroline, in a complex with Zn +2 , for their ability to modulate receptor signaling and MHC class I downregulation.…”