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A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.


3D bar graph comparison of data across all platforms for the different ligands on CHO-C5aR cells and HMDM. Note break in x-axis scale at 500 and 3,000 nM.
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biosensors-02-00273-f004: 3D bar graph comparison of data across all platforms for the different ligands on CHO-C5aR cells and HMDM. Note break in x-axis scale at 500 and 3,000 nM.

Mentions: It is understandable that being a relatively new technology that excludes the use of “labels”, in this field of science arouses questions regarding the exact nature of the signal. The signalling profiles generated on the two label-free platforms assessed in this study, are shown in Figure 2 and the summary of all the ligand potencies via different pathways, and the extent to which they are up- or down-regulated in our assays, is summarised in Figure 4. Since these technologies were first introduced, considerable effort has been invested into deciphering the nature of the signal and what it means in the biological context. Major cell events, such as cell growth and proliferation lead to large signals. However, GPCR receptor activation events lead to smaller changes in cell morphology and mass redistribution in the cell that can be monitored, as shown in this study, with the xCELLigence and EPIC® systems, respectively. Orthogonal studies, along with pharmacological and chemical studies, have been used to propose that GPCRs that couple to different G proteins have unique DMR signalling profiles [59]. This was supported in a study by Schroder et al., where they used the EPIC® label-free system and found the signalling fingerprints to be in agreement with each of the major Gα protein classes proposed previously [60]. In our study, we observed a similar EPIC® signalling profile for C5a, in both cell types (Figure 2), to that attributed to the Gαi protein in Schroder’s study. However, the signalling profile for C5a on the xCELLigence system differed not only to the EPIC® profile, but between cell types. It is apparent from these profiles that the xCELLigence system yields more complex data and the profile for the same receptor varies significantly from cell type to cell type. The kinetics of the xCELLigence responses observed on the HMDM differ to the CHO-C5aR cells, specifically the sharp decline in the cell index after the peak is reached, a pattern not so apparent in the transfected cells. The decline in the HMDM signal observed after the peak on the EPIC®, is also slightly faster than that observed for the CHO-C5aR, however; it is not as prominent as that observed on the xCELLigence profile. As mentioned above, the C5L2 “scavenger” receptor is present in HMDM, and this may play a role in removing excess C5a, so that the signalling events are down regulated more rapidly and hence a more rapid decline is observed. Although we know that the signal generated on the label-free platforms examined in this study are due to the activation of C5aR (control cells showed no response), the exact nature (ERK, cAMP, calcium etc.) of the signal is unknown. It is beyond the scope of this study to decipher what causes the individual peaks shown in Figure 2. However, studies have been undertaken to understand what signalling components generate the DMR signal for receptors such as the EGF receptor. By utilising inhibitors for a range of known intracellular signalling components, Fang et al., were able to show that the DMR profile for EGFR was attributable to Ras/MAPK signalling [58]. However, how these results translate across platforms is unknown. It would not be entirely surprising that signature profiles are consistent within a detection system, but differ between systems because the detection modes are different. For example, the Corning EPIC® system used in this study, exhibits a sample penetration depth of ~150 nm [17], but the xCELLigence system has no such limit. However, the results summarised in Table 1 and Table 2 show that, despite this difference, the ligand potencies ascertained from both the label-free platforms were comparable. Indeed, for both of these platforms, the potencies were consistently lower than the potencies calculated using the secondary messenger reporter assays. This is not in agreement with the findings of Schroder et al. [60], where no significant difference between EPIC® and secondary messenger assay potencies were reported. Interestingly, Dodgson et al. [61] found that the potencies of some compounds were similar between the Corning EPIC® system and the FLIPR system, but with other compounds there was a 10-fold decrease in potency on the label-free platform compared to the label-based platform. The latter observation is similar to what has been found in this study. The shifts in potency on label-free platforms might be attributed to the signal representing an integrated response. All events ranging from cell stimulation to receptor recycling are measured and integrated, therefore a less potent response may be observed. By analogy, if one was to take individual potencies for all possible pathways activated upon receptor stimulation, including those that down-regulate the signalling response, and average them, the potency is more likely to reflect what is detected with label-free biosensors. In secondary messenger reporter assays, only downstream amplified secondary messengers are measured, which may present more potent responses [61] as no accountability is taken for signal regulation. It is not currently known whether the discrepancy between label-free and label-based assays is related to the type of GPCR assayed or characteristics of the ligand or both.


A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

3D bar graph comparison of data across all platforms for the different ligands on CHO-C5aR cells and HMDM. Note break in x-axis scale at 500 and 3,000 nM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263554&req=5

biosensors-02-00273-f004: 3D bar graph comparison of data across all platforms for the different ligands on CHO-C5aR cells and HMDM. Note break in x-axis scale at 500 and 3,000 nM.
Mentions: It is understandable that being a relatively new technology that excludes the use of “labels”, in this field of science arouses questions regarding the exact nature of the signal. The signalling profiles generated on the two label-free platforms assessed in this study, are shown in Figure 2 and the summary of all the ligand potencies via different pathways, and the extent to which they are up- or down-regulated in our assays, is summarised in Figure 4. Since these technologies were first introduced, considerable effort has been invested into deciphering the nature of the signal and what it means in the biological context. Major cell events, such as cell growth and proliferation lead to large signals. However, GPCR receptor activation events lead to smaller changes in cell morphology and mass redistribution in the cell that can be monitored, as shown in this study, with the xCELLigence and EPIC® systems, respectively. Orthogonal studies, along with pharmacological and chemical studies, have been used to propose that GPCRs that couple to different G proteins have unique DMR signalling profiles [59]. This was supported in a study by Schroder et al., where they used the EPIC® label-free system and found the signalling fingerprints to be in agreement with each of the major Gα protein classes proposed previously [60]. In our study, we observed a similar EPIC® signalling profile for C5a, in both cell types (Figure 2), to that attributed to the Gαi protein in Schroder’s study. However, the signalling profile for C5a on the xCELLigence system differed not only to the EPIC® profile, but between cell types. It is apparent from these profiles that the xCELLigence system yields more complex data and the profile for the same receptor varies significantly from cell type to cell type. The kinetics of the xCELLigence responses observed on the HMDM differ to the CHO-C5aR cells, specifically the sharp decline in the cell index after the peak is reached, a pattern not so apparent in the transfected cells. The decline in the HMDM signal observed after the peak on the EPIC®, is also slightly faster than that observed for the CHO-C5aR, however; it is not as prominent as that observed on the xCELLigence profile. As mentioned above, the C5L2 “scavenger” receptor is present in HMDM, and this may play a role in removing excess C5a, so that the signalling events are down regulated more rapidly and hence a more rapid decline is observed. Although we know that the signal generated on the label-free platforms examined in this study are due to the activation of C5aR (control cells showed no response), the exact nature (ERK, cAMP, calcium etc.) of the signal is unknown. It is beyond the scope of this study to decipher what causes the individual peaks shown in Figure 2. However, studies have been undertaken to understand what signalling components generate the DMR signal for receptors such as the EGF receptor. By utilising inhibitors for a range of known intracellular signalling components, Fang et al., were able to show that the DMR profile for EGFR was attributable to Ras/MAPK signalling [58]. However, how these results translate across platforms is unknown. It would not be entirely surprising that signature profiles are consistent within a detection system, but differ between systems because the detection modes are different. For example, the Corning EPIC® system used in this study, exhibits a sample penetration depth of ~150 nm [17], but the xCELLigence system has no such limit. However, the results summarised in Table 1 and Table 2 show that, despite this difference, the ligand potencies ascertained from both the label-free platforms were comparable. Indeed, for both of these platforms, the potencies were consistently lower than the potencies calculated using the secondary messenger reporter assays. This is not in agreement with the findings of Schroder et al. [60], where no significant difference between EPIC® and secondary messenger assay potencies were reported. Interestingly, Dodgson et al. [61] found that the potencies of some compounds were similar between the Corning EPIC® system and the FLIPR system, but with other compounds there was a 10-fold decrease in potency on the label-free platform compared to the label-based platform. The latter observation is similar to what has been found in this study. The shifts in potency on label-free platforms might be attributed to the signal representing an integrated response. All events ranging from cell stimulation to receptor recycling are measured and integrated, therefore a less potent response may be observed. By analogy, if one was to take individual potencies for all possible pathways activated upon receptor stimulation, including those that down-regulate the signalling response, and average them, the potency is more likely to reflect what is detected with label-free biosensors. In secondary messenger reporter assays, only downstream amplified secondary messengers are measured, which may present more potent responses [61] as no accountability is taken for signal regulation. It is not currently known whether the discrepancy between label-free and label-based assays is related to the type of GPCR assayed or characteristics of the ligand or both.

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.