Abstract
Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-α or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T-cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at ˜ 45 min after Gro-α stimulation. This multiplexing demonstrated that Gro-α-induced CXCR2 internalization was tightly correlated with Gro-α-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.
Keywords: High content screening, CXCR2, ERK, NFAT, high throughput screening
Combinatorial Chemistry & High Throughput Screening
Title: High Content Screening of CXCR2-Dependent Signalling Pathways
Volume: 13 Issue: 1
Author(s): Michael Wolff, Simone Krede, Dorothea Haasen, Jorg Wiedenmann, G. Ulrich Nienhaus, Barbara Kistler, Franz Oswald and Ralf Heilker
Affiliation:
Keywords: High content screening, CXCR2, ERK, NFAT, high throughput screening
Abstract: Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-α or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T-cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at ˜ 45 min after Gro-α stimulation. This multiplexing demonstrated that Gro-α-induced CXCR2 internalization was tightly correlated with Gro-α-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.
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Cite this article as:
Wolff Michael, Krede Simone, Haasen Dorothea, Wiedenmann Jorg, Nienhaus Ulrich G., Kistler Barbara, Oswald Franz and Heilker Ralf, High Content Screening of CXCR2-Dependent Signalling Pathways, Combinatorial Chemistry & High Throughput Screening 2010; 13 (1) . https://dx.doi.org/10.2174/138620710790218249
DOI https://dx.doi.org/10.2174/138620710790218249 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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