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

Area of Science:

• Immunology
• Cell Biology
• Pharmacology

Background:

• Human C-C chemokine receptor type 7 (CCR7) binds endogenous ligands CCL19 and CCL21.
• These ligands exhibit biased agonism, notably in differential β-arrestin recruitment.
• Traditional methods for detecting biased agonism are label-based and pathway-specific.

Purpose of the Study:

• To evaluate Cellular Electrical Impedance (CEI) as an alternative, label-free method for studying CCR7 signaling.
• To investigate the biased agonism of CCR7 ligands using CEI.
• To deconvolute the signaling pathways involved in CCR7 activation.

Main Methods:

• Utilized CCR7-expressing HEK293 cells stimulated with CCL19 or CCL21.
• Measured cellular responses using the label-free CEI technology.
• Performed label-based, pathway-specific assays to corroborate CEI findings.

Main Results:

• CEI detected distinct impedance profiles for CCL19 and CCL21 stimulation, indicating biased agonism during the desensitization phase.
• Differential β-arrestin recruitment modulated the impedance profile but was not the sole contributor.
• Pathway deconvolution showed Gαi-mediated signaling as the primary contributor to the impedance profile, with Gαq and Gα12/13 also involved.
• Label-based assays confirmed CCL19's higher potency for β-arrestin2 recruitment and receptor internalization, while Gαi activation was similar for both ligands.

Conclusions:

• CEI is a powerful, label-free technology for analyzing complex receptor signaling, including biased agonism.
• CEI provides integrated cellular responses reflecting multiple signaling pathways.
• This method offers a valuable alternative to traditional label-based assays for studying GPCRs like CCR7.