Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.
"Afterwards, the same authors proposed that modulation of ORF74 activity by means of chemokines represented an example of chemokine-driven neoplastic condition . Moreover, it has also been reported that the CC chemo- kine/US28 receptor, a G-protein-coupled receptor (GPCR) encoded by human cytomegalovirus (HCMV) and homologous to the human CCR1, binds in vivo with high affinity a few chemokines of the CC family as well as the CX3C chemokine fractalkine by means of hydrophobic interaction between aromatic residues . We found in CXCR3(1–48) four aromatic residues that may enhance and give specificity to the interaction with the CXCL11 that is the only one among the three natural ligands of CXCR3 to contain aromatic residues (2) in the interacting region between receptor N-terminal region and chemokine N-loop. "
[Show abstract][Hide abstract] ABSTRACT: Our study on the highly charged N-terminal peptide of the human chemokine receptor CXCR3 by spectroscopic methods in solution and by means of molecular dynamics simulations showed that the charge content modulates the intrinsic structural preference of its flexible backbone. Collectively, our findings suggest that the structural organization of a protein should be seen as a part of a continuum in which the ratio between electrostatic and hydrophobic interactions and the intrinsic flexibility are important properties used to optimize the folding. When this ratio changes and the structure is intrinsically flexible, the structural organization of the system moves along the continuum of the possible conformational states. By all this combined information, one can describe the structure of CXCR3(1–48) as an ensemble of conformations. In fact, the peptide shows stretches of negative charges embedded in a flexible sequence which can be used to maximize promiscuous interactions relevant to molecular recognition but globally the peptide appears as a poly-structured globule-like ensemble that is dynamically stabilized by H-bonds. We have approached the study of the most populated ensembles with subset selection to explain our experimental data also by evidencing that the changes into the fraction of charged residues discriminate between dynamically poly-structured states, conceivably because of small free energy barriers existing between the different conformations of CXCR3(1–48). Therefore, the overlap of a highly flexible backbone, negatively charged residues and sites which can be modified by post-translational modifications represent the structural organization that controls the molecular mechanisms underlying the biological functions carried out by CXCR3(1–48).
"US28 encoded by human cytomegalovirus binds several human CC-chemokines (CCL1–CCL5), but has particular high affinity for the soluble form of CX3CL1 (42). CCL2–5 act as agonists in some pathways and neutral ligands in others, whereas CX3CL1 is a partial inverse agonist in most pathways (43–49). Similar phenomena have been observed for the viral CXC-chemokine receptors ECRF3 (from herpesvirus Saimiri) and ORF74 (from human herpesvirus 8) (34, 50–55). "
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Immunology 06/2014; 5:277. DOI:10.3389/fimmu.2014.00277
"For example, CCL5/RANTES has been shown to behave as both a neutral agonist, having no measurable effect on constitutive PLC-β signaling or transcription factor activation, and as a positive agonist leading to the release of intracellular calcium and activation of FAK , , . CX3CL1/Fractalkine has been suggested to behave as an inverse agonist, as it significantly blocks the ability of US28 to promote PLC-β activity, and as a positive agonist leading to the release of intracellular calcium and activation of FAK , , , , . Moreover, CCL5/RANTES and CX3CL1/Fractalkine have been reported to exhibit cell-specific effects as CCL5/RANTES promotes smooth muscle cell migration and inhibits macrophage migration while CX3CL1/Fractalkine promotes macrophage migration and inhibits smooth muscle cell migration , . "
[Show abstract][Hide abstract] ABSTRACT: Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca(++) release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca(++) release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.
PLoS ONE 11/2012; 7(11):e50524. DOI:10.1371/journal.pone.0050524 · 3.23 Impact Factor
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