Systematic Assessment of Chemokine Signaling at Chemokine Receptors CCR4, CCR7 and CCR10

Abstract

Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and β-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced β-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and β-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and β-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism

“…The human CCR7 receptor was previously shown to be differentially activated by its natural ligands, CCL19 and CCL21. Although some conflicting data exist concerning the level of G protein activation induced by both chemokines, it is well established that CCL19-induced CCR7 activation results in more potent β-arrestin recruitment and receptor internalisation compared to CCL21 [ 19 , 20 , 21 , 22 , 23 ]. In this study, we analysed CCR7 signalling using CEI and demonstrate that this label-free technology holds promise as a methodology to investigate and decipher GPCR signalling.…”

“…Early studies reported similar findings [ 19 , 20 ]. Two more recent studies, however, showed that there was a difference in potency, but not efficacy, between CCL19 and CCL21 concerning cAMP modulation [ 22 , 23 ]. In the latter studies, CCR7 signalling was studied in Chinese hamster ovary cells.…”

“…CCR7 signalling bias has been extensively studied in vitro. While it seems to be the consensus that CCL19 is more potent in recruiting β-arrestins and inducing internalisation than CCL21, reports regarding G protein activation are ambiguous as it is still under debate whether they activate G proteins with similar potency and efficacy [ 17 , 18 , 19 , 20 , 21 , 22 , 23 ].…”

Engineering Broad-spectrum Inhibitors of Inflammatory Chemokines from a New Family of Tick Evasins

“…Detached cells were seeded (50,000 cells/well) in a white 96-well plate (culture plates; PerkinElmer) and incubated overnight at 37 °C, 5% CO2. Cells were washed twice and equilibrated with Hank's Balanced Salt Solution (HBSS) for 30 minute at 37 °C followed by the addition of the Rluc substrate coelenterazine-h (Nanolight Technology) at a final concentration of 5 µM for 10 minute 39 . For initial experiments, concentration-response curves of chemokines were measured by activating the chemokine receptors with different concentrations of chemokines.…”

Swapping N-terminal regions among tick evasins reveals cooperative interactions influencing chemokine binding and selectivity

“…For transient transfection, 5 μg of plasmid containing CAMYEL biosensor was mixed with 500 μl of PBS and vortexed thoroughly. PEI (30 μg) was added, and the mixture was vortexed and incubated for 20 min at 25 °C, then added dropwise into a 10-cm culture dish containing four million adherent cells ( 32 ). After 24 h incubation, the transfected cells were detached using PBS-EDTA solution and transferred to a 96-well culture plate (50,000 cells/well), then further incubated for 18 h. Cells were then washed twice and equilibrated with Hank’s Balanced Salt Solution (Gibco) for 10 min at 37 °C, incubated with 5 μM Coelenterazine-H (Nanolight Technology) for 10 min.…”