Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

Rummel, Pia Cwarzko; Thiele, Stefanie; Hansen, Laerke Smidt; Petersen, Trine P.; Sparre-Ulrich, Alexander Hovard; Ulven, Trond; Rosenkilde, Mette M.

doi:10.1124/mol.113.086702

Cited by 17 publications

(19 citation statements)

References 62 publications

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“…This domain is generally important for receptor function by contributing to protein folding and conformational constraining in addition to forming the ligand-binding sites (76)(77)(78). ECL-2 is, on average, the longest of the 3 ECLs and encompasses the most divergent loop with sequence variations even within otherwise conserved class A 7TM receptor families (52,79). The length and sequence of ECL-2 are surprisingly conserved for the BILF1 receptors, indicating an essential role of this receptor region.…”

Section: Discussionmentioning

confidence: 99%

Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

Spieß

Fares

Sparre-Ulrich

et al. 2015

J Virol

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Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-proteincoupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein G␣i and induced activation of the NF-B transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function. IMPORTANCE Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

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Section: Discussionmentioning

confidence: 99%

Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

Spieß

Fares

Sparre-Ulrich

et al. 2015

J Virol

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“…However, in these studies, small molecule-induced activation (and thereby the ability of the mutant receptors to be activated independently of chemokine binding) was not tested. We recently reported that the disulfide bridges play different roles in the receptors CCR1 and CCR5 (44). In CCR1, the 7TM bridge was found to be essential for activation with both chemokine and small molecule ligands, whereas the CKR bridge was not required for small moleculemediated activation.…”

Section: Discussionmentioning

confidence: 99%

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

Barington

Rummel

Lückmann

et al. 2016

Journal of Biological Chemistry

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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...

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“…Chemokines mainly interact with extracellular receptor regions (68), and therefore the two conformationally constraining disulfide bridges have a major impact on chemokine binding (113, 115, 139). Yet, the roles of these bridges vary – even between homologous chemokine receptors like CCR1 and CCR5 (139) and thereby contribute to the explanation for why small drug-like substances act differently on (1) different chemokines for the same receptor (136) or (2) different chemokine receptors (137, 138).…”

Section: Resultsmentioning

confidence: 99%

“…Yet, the roles of these bridges vary – even between homologous chemokine receptors like CCR1 and CCR5 (139) and thereby contribute to the explanation for why small drug-like substances act differently on (1) different chemokines for the same receptor (136) or (2) different chemokine receptors (137, 138). These pharmacodynamic challenges combined with the redundancy of the chemokine system may be central for the lack of success when it comes to the development of anti-inflammatory compounds targeting chemokine receptors.…”

Section: Resultsmentioning

confidence: 99%

Biased and G Protein-Independent Signaling of Chemokine Receptors

2014

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Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand–receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of “classic” redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors – in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution.

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Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

Cited by 17 publications

References 62 publications

Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

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

Biased and G Protein-Independent Signaling of Chemokine Receptors

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