Dr Andrea Krumm explains how a fluorescent assay detects ligand bias at GPCRs
G-protein-coupled receptors (GPCRs) transmit extracellular stimuli into intracellular reactions. They are the target of approximately 35% of all approved drugs and are being intensively examined. GPCRs can signal via both G-protein and arrestin routes. Some ligands tend to prefer one way over the other, which can affect the downstream cellular effects. For example, arrestin biased agonists at the angiotensin II type 1 (AT1R) receptor lower arterial pressure and increase cardiac performance, while unbiased or G-protein-biased ligands do not improve cardiac performance. In a recent project, the goal was to develop better tools for the reliable identification and quantification of agonist distortion. Therefore methods that capture β-arrestin and G-protein second messengers in real time were developed. With a BMG Labtech ClarioStar plate reader, signals from any signal path can be collected simultaneously and with high accuracy.
Temporal patterns of GPCR signalling processes affect the detection and quantification of biased agonism and these patterns vary with different receptors and agonists. However, this “kinetic context” is missing in standard endpoint measurements. To record the signal responses of the second messenger over time, the scientists developed red and green fluorescent biosensors for β-arrestin, cAMP, DAG, Ca2+ and PIP2. Each biosensor consists of a single fluorescent protein, the fluorescence intensity of which changes when a signal is transmitted via the corresponding molecule. Red and green biosensors can be combined in a single experiment to simultaneously measure the response functions of arrestin and G protein-mediated signals in the same cell population. A green biosensor for β-arrestin was combined with a red DAG sensor or R-GECO, a red biosensor for Ca2+, in living cells that express the angiotensin receptor AT1R. The receptor was activated with known ligands and monitored the fluorescence intensity over time with the ClarioStar microplate reader.
HEK293T cells were transduced in suspension with BacMam vectors carrying the indicated sensors (Montana Molecular) and plated in 96-well plates. After 24 hours, cell culture media was exchanged for DPBS and cells were placed at room temperature for 30 minutes prior to drug addition.
Drug addition and dose response
For all experiments, baseline ﬂuorescence measurements were acquired, after which the plate was removed, and drug was added using an electronic multichannel pipette. Changes in ﬂuorescence intensity were measured immediately after drug addition and over several minutes to capture response functions for each of ﬁve ATR1 ligands.
Five AT1R receptor agonists (30 µM) produce different β-arrestin, DAG, and Ca2+ response functions over time (Fig 2). The agonists were Angiotensin II regarded as a balanced agonist, the SII agonist known to favor arrestin signalling and intermediate agonists (TRV). Kinetic analysis of the response function from each ligand can be used to reliably assess agonist bias. Fig. 2 shows the fluorescent signal of the β-arrestin biosensor in green. The downward sensor decreases in intensity when activated. The red curves indicate Ca2+ (A) or DAG (B). All agonists trigger β-arrestin responses, though they do to a different extent. The indicators of Gq signalling (Ca2+ and DAG) differ among the five agonists with the known balanced agonist Angiotensin II exhibiting the highest Gq-related response for DAG (Fig. 2B).
Biosensor detection results
Fluorescent biosensor assays for β-arrestin and G-protein signalling can be combined in living cells and monitored on a Clariostar with the highest precision.
Dr Andrea Krumm is with BMG Labtech