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Post-translational allosteric activation of the P2X 7 receptor through glycosaminoglycan chains of CD44 proteoglycans

View Article: PubMed Central - PubMed

ABSTRACT

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation.

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ATP-gated P2X7 receptor is expressed on the CHO cells surface. The P2X7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. (a) The amount of P2X7 receptor expressed in whole CHO cells. (b) P2X7 receptor expressed at the surface of CHO cells. (c) Immunofluorescence labeling of P2X7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μm.
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fig3: ATP-gated P2X7 receptor is expressed on the CHO cells surface. The P2X7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. (a) The amount of P2X7 receptor expressed in whole CHO cells. (b) P2X7 receptor expressed at the surface of CHO cells. (c) Immunofluorescence labeling of P2X7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μm.

Mentions: Alterations of the P2X7 response to the agonists ATP and BzATP observed in CHO-K1 and CHO-745 cells likely reflect either differences in P2X7 expression patterns or modulation mechanisms. Antibodies against the P2X7 receptor were used to investigate the cellular expression of this receptor using flow cytometry and confocal microscopy. The quantification of the P2X7 receptor expression through flow cytometry revealed that CHO-K1 and CHO-745 cells expressed similar amounts in the whole cell (permeabilized cells; Figure 3a). The quantification of only the P2X7 receptor external label (without permeabilization) also showed similar amounts in both cell lines (Figure 3b). These results suggest that the secretion of the P2X7 receptor was not altered in CHO-745 cells. To corroborate this hypothesis, confocal microscopy was performed after labeling the P2X7 receptor, cellular membrane, endoplasmic reticulum and Golgi (Figure 3c). We did not observe differences in P2X7 receptor pattern expression and cellular localization between CHO-K1 and CHO-745 cells (Figure 3c). These results indicate that the differences of Ca2+cyt concentration curves in response to ATP and BzATP are not associated with the cellular expression or cellular localization of the receptor. Altogether, these data strongly suggest that the presence of GAGs on the surface of CHO cells modulates P2X7 receptor activity in response to agonists.


Post-translational allosteric activation of the P2X 7 receptor through glycosaminoglycan chains of CD44 proteoglycans
ATP-gated P2X7 receptor is expressed on the CHO cells surface. The P2X7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. (a) The amount of P2X7 receptor expressed in whole CHO cells. (b) P2X7 receptor expressed at the surface of CHO cells. (c) Immunofluorescence labeling of P2X7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μm.
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fig3: ATP-gated P2X7 receptor is expressed on the CHO cells surface. The P2X7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. (a) The amount of P2X7 receptor expressed in whole CHO cells. (b) P2X7 receptor expressed at the surface of CHO cells. (c) Immunofluorescence labeling of P2X7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μm.
Mentions: Alterations of the P2X7 response to the agonists ATP and BzATP observed in CHO-K1 and CHO-745 cells likely reflect either differences in P2X7 expression patterns or modulation mechanisms. Antibodies against the P2X7 receptor were used to investigate the cellular expression of this receptor using flow cytometry and confocal microscopy. The quantification of the P2X7 receptor expression through flow cytometry revealed that CHO-K1 and CHO-745 cells expressed similar amounts in the whole cell (permeabilized cells; Figure 3a). The quantification of only the P2X7 receptor external label (without permeabilization) also showed similar amounts in both cell lines (Figure 3b). These results suggest that the secretion of the P2X7 receptor was not altered in CHO-745 cells. To corroborate this hypothesis, confocal microscopy was performed after labeling the P2X7 receptor, cellular membrane, endoplasmic reticulum and Golgi (Figure 3c). We did not observe differences in P2X7 receptor pattern expression and cellular localization between CHO-K1 and CHO-745 cells (Figure 3c). These results indicate that the differences of Ca2+cyt concentration curves in response to ATP and BzATP are not associated with the cellular expression or cellular localization of the receptor. Altogether, these data strongly suggest that the presence of GAGs on the surface of CHO cells modulates P2X7 receptor activity in response to agonists.

View Article: PubMed Central - PubMed

ABSTRACT

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation.

No MeSH data available.


Related in: MedlinePlus