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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.


Label-free dose response curves on CHO-C5aR cells and HMDM. (a) Label-free response of all ligands on the EPIC® optical system on CHO-C5aR cells (b) Label free response of all ligands on the xCELLigence impedance system on CHO-C5aR cells (c) Ligand response on Alphascreen secondary messenger ERK assay on CHO-C5aR cells and (d) I125 binding assay of all ligands on CHO-C5aR membranes (e) Label-free response of all ligands on the EPIC® optical system on HMDM (f) Label-free response of all ligands on the xCELLigence impedance system on HMDM (g) Ligand response on Alphascreen secondary messenger ERK assay on HMDM and (h) I125 binding assay of all ligands on HMDM. Error bars represent standard error of the mean (n = 3 − 15).
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biosensors-02-00273-f003: Label-free dose response curves on CHO-C5aR cells and HMDM. (a) Label-free response of all ligands on the EPIC® optical system on CHO-C5aR cells (b) Label free response of all ligands on the xCELLigence impedance system on CHO-C5aR cells (c) Ligand response on Alphascreen secondary messenger ERK assay on CHO-C5aR cells and (d) I125 binding assay of all ligands on CHO-C5aR membranes (e) Label-free response of all ligands on the EPIC® optical system on HMDM (f) Label-free response of all ligands on the xCELLigence impedance system on HMDM (g) Ligand response on Alphascreen secondary messenger ERK assay on HMDM and (h) I125 binding assay of all ligands on HMDM. Error bars represent standard error of the mean (n = 3 − 15).

Mentions: There has been some inconsistency in the literature with regards to the activity of C5a to its reported “inactive” form, C5a des-Arg, in different cell types and across different assay platforms [30,33,40,41,42,43,44,45,46,47]. Most suggest a 10–1,000 fold reduction in potency for C5a des-Arg compared to C5a. It has also been shown that the de-glycosylated form of C5a des-Arg is 10-fold more potent than the native glycosylated form [41]. In HMDM the IC50 of C5a was 1.2 nM compared to 2.5 nM (~2-fold difference) for C5a des-Arg (Table 1). However, for CHO-C5aR cells, an IC50 of 0.2 nM and 1.2 nM (Table 2) (~6-fold difference) was observed for C5a and C5a des-Arg, respectively (Figure 3). The observed difference in binding affinity between the two cell types, may be due to the presence of other interacting partners/modulators in the HMDM that are absent in the CHO-C5aR cell line. Thus the binding affinities are dependent on the cellular background and can vary from one cell type to the other. The peptide partial agonist bound more tightly in HMDM relative to CHO-C5aR cells; however, this was not observed for any other ligand. The binding observed on the CHO-C5aR membrane was specific, since there was no specific binding to CHO-K1 membrane by 125I-C5a (data not shown).


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)

Label-free dose response curves on CHO-C5aR cells and HMDM. (a) Label-free response of all ligands on the EPIC® optical system on CHO-C5aR cells (b) Label free response of all ligands on the xCELLigence impedance system on CHO-C5aR cells (c) Ligand response on Alphascreen secondary messenger ERK assay on CHO-C5aR cells and (d) I125 binding assay of all ligands on CHO-C5aR membranes (e) Label-free response of all ligands on the EPIC® optical system on HMDM (f) Label-free response of all ligands on the xCELLigence impedance system on HMDM (g) Ligand response on Alphascreen secondary messenger ERK assay on HMDM and (h) I125 binding assay of all ligands on HMDM. Error bars represent standard error of the mean (n = 3 − 15).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00273-f003: Label-free dose response curves on CHO-C5aR cells and HMDM. (a) Label-free response of all ligands on the EPIC® optical system on CHO-C5aR cells (b) Label free response of all ligands on the xCELLigence impedance system on CHO-C5aR cells (c) Ligand response on Alphascreen secondary messenger ERK assay on CHO-C5aR cells and (d) I125 binding assay of all ligands on CHO-C5aR membranes (e) Label-free response of all ligands on the EPIC® optical system on HMDM (f) Label-free response of all ligands on the xCELLigence impedance system on HMDM (g) Ligand response on Alphascreen secondary messenger ERK assay on HMDM and (h) I125 binding assay of all ligands on HMDM. Error bars represent standard error of the mean (n = 3 − 15).
Mentions: There has been some inconsistency in the literature with regards to the activity of C5a to its reported “inactive” form, C5a des-Arg, in different cell types and across different assay platforms [30,33,40,41,42,43,44,45,46,47]. Most suggest a 10–1,000 fold reduction in potency for C5a des-Arg compared to C5a. It has also been shown that the de-glycosylated form of C5a des-Arg is 10-fold more potent than the native glycosylated form [41]. In HMDM the IC50 of C5a was 1.2 nM compared to 2.5 nM (~2-fold difference) for C5a des-Arg (Table 1). However, for CHO-C5aR cells, an IC50 of 0.2 nM and 1.2 nM (Table 2) (~6-fold difference) was observed for C5a and C5a des-Arg, respectively (Figure 3). The observed difference in binding affinity between the two cell types, may be due to the presence of other interacting partners/modulators in the HMDM that are absent in the CHO-C5aR cell line. Thus the binding affinities are dependent on the cellular background and can vary from one cell type to the other. The peptide partial agonist bound more tightly in HMDM relative to CHO-C5aR cells; however, this was not observed for any other ligand. The binding observed on the CHO-C5aR membrane was specific, since there was no specific binding to CHO-K1 membrane by 125I-C5a (data not shown).

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.