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

Abstract

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of β-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential β-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced β-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

Systematic Assessment of Human CCR7 Signalling Using NanoBRET Biosensors Points towards the Importance of the Cellular Context

“…This is in line with other studies, which report CCL21 to be significantly worse at inducing β-arrestin2 recruitment [ 21 , 22 , 23 , 26 , 27 ]. In a previous study, we obtained similar results regarding G protein activation and β-arrestin recruitment [ 24 ]. Here, we used NanoBRET instead of NanoBiT to monitor β-arrestin recruitment.…”

“…Interestingly, we previously reported that CCL19- and CCL21-induced distinct cellular impedance profiles and correlated this with differences in β-arrestin2 recruitment in HEK293A cells [ 24 ]. It seems, therefore, that even though no strong ligand bias was found in HEK293A cells, subtle differences in pathway activation can still manifest a difference in more physiologically relevant readouts.…”

“…Early studies indeed suggested similarities in G protein activation by CCL19 and CCL21 but differences in GRK and β-arrestin recruitment, as well as receptor internalisation. Later studies challenged this notion, suggesting that CCL21 is a partial agonist with both impaired G protein activation and β-arrestin recruitment [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Many of these studies, however, used different cell lines and/or methodologies and do not quantify biased signalling in a similar way, if at all, hindering direct comparison between studies.…”

“…Error bars were omitted for clarity. Data for Gαi1, Gαi2 and Gαi3 activation in HEK293A cells were previously published in Vanalken et al [ 24 ].…”

“…However, regarding G protein activation, calcium release and chemotaxis reports are equivocal. Hence, whereas some studies consider the CCL19-CCL21-CCR7 axis to be a prototypical example of biased signalling, others challenge this notion and propose that CCL21 is an overall weaker agonist [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Given that a genuine comparison across studies is difficult due to the use of different cellular backgrounds and methodologies, we performed a systematic analysis of the CCR7 signalling at the transducer level in three commonly used cellular backgrounds using a NanoBRET-based methodology and precisely quantified pathway and ligand preferences, as well as ligand bias and the influence of the cellular system thereon.…”