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CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer.

Parenty G, Appelbe S, Milligan G - Biochem. J. (2008)

Bottom Line: This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy.These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor.These results have novel and important implications for the development and use of small-molecule therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein alpha subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.

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FRET and BRET studies confirm hetero-interactions between co-expressed hCXCR2 and hDOP receptors(A) shows tr-FRET. c-Myc–hDOP and FLAG–hDOP or c-Myc–hCXCR2 and FLAG–hDOP were expressed individually in HEK-293 cells that were then mixed (mix) or the two receptors were co-expressed (Co). Following addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, to intact cells tr-FRET was monitored as described in the Experimental section. (B) shows FRET imaging. hDOP–eYFP (eYFP) was transiently expressed in HEK-293 cells with (lower panels) or without (upper panels) hCXCR2–eCFP (eCFP) and fluorescence imaged. Raw FRET (FRET) and calculated normalized FRET (right-hand panels) was then assessed as described in the Experimental section. (C) shows saturation BRET2 studies. hCXCR2–Renilla luciferase and hCXCR2–GFP2 (■), hCXCR2–Renilla luciferase and hDOP–GFP2 (□) or hDOP–Renilla luciferase and hDOP–GFP2 (○) were transiently co-expressed in HEK-293 cells. Following addition of the luciferase substrate/BRET2 energy donor DeepBlueC, BRET measurements were made. Donor and acceptor ratios were assessed as described in the Experimental section. Each experiment is representative of three.
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Figure 2: FRET and BRET studies confirm hetero-interactions between co-expressed hCXCR2 and hDOP receptors(A) shows tr-FRET. c-Myc–hDOP and FLAG–hDOP or c-Myc–hCXCR2 and FLAG–hDOP were expressed individually in HEK-293 cells that were then mixed (mix) or the two receptors were co-expressed (Co). Following addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, to intact cells tr-FRET was monitored as described in the Experimental section. (B) shows FRET imaging. hDOP–eYFP (eYFP) was transiently expressed in HEK-293 cells with (lower panels) or without (upper panels) hCXCR2–eCFP (eCFP) and fluorescence imaged. Raw FRET (FRET) and calculated normalized FRET (right-hand panels) was then assessed as described in the Experimental section. (C) shows saturation BRET2 studies. hCXCR2–Renilla luciferase and hCXCR2–GFP2 (■), hCXCR2–Renilla luciferase and hDOP–GFP2 (□) or hDOP–Renilla luciferase and hDOP–GFP2 (○) were transiently co-expressed in HEK-293 cells. Following addition of the luciferase substrate/BRET2 energy donor DeepBlueC, BRET measurements were made. Donor and acceptor ratios were assessed as described in the Experimental section. Each experiment is representative of three.

Mentions: To examine potential interactions between the chemokine CXCR2 and DOP opioid receptors, the human forms of these receptors were modified to incorporate either the FLAG or c-Myc epitope tag sequences at the N-terminus. We have previously shown the capacity of each of these receptors to form homodimers/oligomers via co-immunoprecipitation studies [21,31]. Expression in HEK-293 cells of FLAG–hCXCR2 resulted in immunological detection in lysates of these cells of a 34 kDa polypeptide with a degree of micro-heterogeneity (Figure 1) representing differential N-glycosylation [21]. Expression of c-Myc–hDOP resulted in the presence of a c-Myc-reactive polypeptide with a molecular mass of 60 kDa (Figure 1). Only with co-expression of FLAG–hCXCR2 and c-Myc–hDOP did immunoprecipitation with an anti-FLAG antibody result in co-immunoprecipitation of c-Myc immunoreactivity (Figure 1) and, even in SDS/PAGE, such immunoreactivity migrated with sizes ranging from 60 kDa to complexes with a substantially higher apparent molecular mass. Because the N-terminal region of GPCRs that are effectively delivered to the cell surface is expected to be extracellular, we also took advantage of the introduced N-terminal tags to perform tr-FRET studies in intact HEK-293 cells [21,31] to detect protein complexes containing both receptors at the cell surface. Co-expression of c-Myc–hCXCR2 and FLAG–hDOP followed by the addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, resulted in strong tr-FRET and output of light at 665 nm when samples were illuminated with 320 nm light (Figure 2A). This did not occur when HEK-293 cell populations individually expressing either c-Myc–hCXCR2 or FLAG–hDOP were combined prior to addition of the combination of Eu3+-labelled and APC-labelled antibodies (Figure 2A). Furthermore, the extent of energy transfer following co-expression of c-Myc–hCXCR2 and FLAG–hDOP was virtually the same as when c-Myc–hDOP and FLAG–hDOP were co-expressed to generate tr-FRET competent, cell-surface hDOP homodimers (Figure 2A). We have also previously shown that in-frame fusion of auto-fluorescent proteins to the C-terminus of each of hCXCR2 [21] and hDOP [31,35] does not significantly alter the function or pharmacology of these receptors. Co-expression of hCXCR2–eCFP and hDOP–eYFP resulted in a capacity to image eCFP to eYFP FRET in individual single cells (Figure 2B), providing further evidence for direct hCXCR2–hDOP interactions. Estimates of the relative affinities of GPCRs to interact can be obtained from ‘saturation’ BRET studies [36,37]. In such experiments, forms of GPCRs C-terminally tagged with Renilla luciferase and with an autofluorescent protein that is able to act as an energy acceptor of light emitted from substrate oxidation by the luciferase are co-expressed in various ratios and the BRET signal is monitored. Co-expression of hCXCR2–Renilla luciferase and hCXCR2–GFP2 in HEK-293 cells resulted in BRET following addition of the luciferase substrate DeepBlueC (Figure 2C). At low energy acceptor (hCXCR2–GFP2) to energy donor (hCXCR2–Renilla luciferase) ratios the BRET signal increased with increasing [acceptor] to [donor] ratios, but this asymptotically approached a maximal value at higher [acceptor] to [donor] ratios (Figure 2C). Half-maximal BRET signal (BRET50) was achieved at an [acceptor] to [donor] ratio of 1.6±0.1. Co-expression of hDOP–Renilla luciferase and hDOP–GFP2 also generated BRET signals that saturated with increasing [acceptor] to [donor] ratios, in this case with BRET50=2.2±0.07. Co-expression of hDOP–Renilla luciferase with hCXCR2–GFP2 generated BRET signals that saturated, and in this case BRET50 was 0.34±0.02 (Figure 2C). These results are consistent with hCXCR2–hDOP hetero-interactions occurring with an even higher affinity than the corresponding hCXCR2–hCXCR2 and hDOP–hDOP homo-interactions.


CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer.

Parenty G, Appelbe S, Milligan G - Biochem. J. (2008)

FRET and BRET studies confirm hetero-interactions between co-expressed hCXCR2 and hDOP receptors(A) shows tr-FRET. c-Myc–hDOP and FLAG–hDOP or c-Myc–hCXCR2 and FLAG–hDOP were expressed individually in HEK-293 cells that were then mixed (mix) or the two receptors were co-expressed (Co). Following addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, to intact cells tr-FRET was monitored as described in the Experimental section. (B) shows FRET imaging. hDOP–eYFP (eYFP) was transiently expressed in HEK-293 cells with (lower panels) or without (upper panels) hCXCR2–eCFP (eCFP) and fluorescence imaged. Raw FRET (FRET) and calculated normalized FRET (right-hand panels) was then assessed as described in the Experimental section. (C) shows saturation BRET2 studies. hCXCR2–Renilla luciferase and hCXCR2–GFP2 (■), hCXCR2–Renilla luciferase and hDOP–GFP2 (□) or hDOP–Renilla luciferase and hDOP–GFP2 (○) were transiently co-expressed in HEK-293 cells. Following addition of the luciferase substrate/BRET2 energy donor DeepBlueC, BRET measurements were made. Donor and acceptor ratios were assessed as described in the Experimental section. Each experiment is representative of three.
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Show All Figures
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Figure 2: FRET and BRET studies confirm hetero-interactions between co-expressed hCXCR2 and hDOP receptors(A) shows tr-FRET. c-Myc–hDOP and FLAG–hDOP or c-Myc–hCXCR2 and FLAG–hDOP were expressed individually in HEK-293 cells that were then mixed (mix) or the two receptors were co-expressed (Co). Following addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, to intact cells tr-FRET was monitored as described in the Experimental section. (B) shows FRET imaging. hDOP–eYFP (eYFP) was transiently expressed in HEK-293 cells with (lower panels) or without (upper panels) hCXCR2–eCFP (eCFP) and fluorescence imaged. Raw FRET (FRET) and calculated normalized FRET (right-hand panels) was then assessed as described in the Experimental section. (C) shows saturation BRET2 studies. hCXCR2–Renilla luciferase and hCXCR2–GFP2 (■), hCXCR2–Renilla luciferase and hDOP–GFP2 (□) or hDOP–Renilla luciferase and hDOP–GFP2 (○) were transiently co-expressed in HEK-293 cells. Following addition of the luciferase substrate/BRET2 energy donor DeepBlueC, BRET measurements were made. Donor and acceptor ratios were assessed as described in the Experimental section. Each experiment is representative of three.
Mentions: To examine potential interactions between the chemokine CXCR2 and DOP opioid receptors, the human forms of these receptors were modified to incorporate either the FLAG or c-Myc epitope tag sequences at the N-terminus. We have previously shown the capacity of each of these receptors to form homodimers/oligomers via co-immunoprecipitation studies [21,31]. Expression in HEK-293 cells of FLAG–hCXCR2 resulted in immunological detection in lysates of these cells of a 34 kDa polypeptide with a degree of micro-heterogeneity (Figure 1) representing differential N-glycosylation [21]. Expression of c-Myc–hDOP resulted in the presence of a c-Myc-reactive polypeptide with a molecular mass of 60 kDa (Figure 1). Only with co-expression of FLAG–hCXCR2 and c-Myc–hDOP did immunoprecipitation with an anti-FLAG antibody result in co-immunoprecipitation of c-Myc immunoreactivity (Figure 1) and, even in SDS/PAGE, such immunoreactivity migrated with sizes ranging from 60 kDa to complexes with a substantially higher apparent molecular mass. Because the N-terminal region of GPCRs that are effectively delivered to the cell surface is expected to be extracellular, we also took advantage of the introduced N-terminal tags to perform tr-FRET studies in intact HEK-293 cells [21,31] to detect protein complexes containing both receptors at the cell surface. Co-expression of c-Myc–hCXCR2 and FLAG–hDOP followed by the addition of a combination of Eu3+-labelled anti-c-Myc, to act as a long-lived energy donor, and APC-labelled anti-FLAG, to act as a potential energy acceptor, resulted in strong tr-FRET and output of light at 665 nm when samples were illuminated with 320 nm light (Figure 2A). This did not occur when HEK-293 cell populations individually expressing either c-Myc–hCXCR2 or FLAG–hDOP were combined prior to addition of the combination of Eu3+-labelled and APC-labelled antibodies (Figure 2A). Furthermore, the extent of energy transfer following co-expression of c-Myc–hCXCR2 and FLAG–hDOP was virtually the same as when c-Myc–hDOP and FLAG–hDOP were co-expressed to generate tr-FRET competent, cell-surface hDOP homodimers (Figure 2A). We have also previously shown that in-frame fusion of auto-fluorescent proteins to the C-terminus of each of hCXCR2 [21] and hDOP [31,35] does not significantly alter the function or pharmacology of these receptors. Co-expression of hCXCR2–eCFP and hDOP–eYFP resulted in a capacity to image eCFP to eYFP FRET in individual single cells (Figure 2B), providing further evidence for direct hCXCR2–hDOP interactions. Estimates of the relative affinities of GPCRs to interact can be obtained from ‘saturation’ BRET studies [36,37]. In such experiments, forms of GPCRs C-terminally tagged with Renilla luciferase and with an autofluorescent protein that is able to act as an energy acceptor of light emitted from substrate oxidation by the luciferase are co-expressed in various ratios and the BRET signal is monitored. Co-expression of hCXCR2–Renilla luciferase and hCXCR2–GFP2 in HEK-293 cells resulted in BRET following addition of the luciferase substrate DeepBlueC (Figure 2C). At low energy acceptor (hCXCR2–GFP2) to energy donor (hCXCR2–Renilla luciferase) ratios the BRET signal increased with increasing [acceptor] to [donor] ratios, but this asymptotically approached a maximal value at higher [acceptor] to [donor] ratios (Figure 2C). Half-maximal BRET signal (BRET50) was achieved at an [acceptor] to [donor] ratio of 1.6±0.1. Co-expression of hDOP–Renilla luciferase and hDOP–GFP2 also generated BRET signals that saturated with increasing [acceptor] to [donor] ratios, in this case with BRET50=2.2±0.07. Co-expression of hDOP–Renilla luciferase with hCXCR2–GFP2 generated BRET signals that saturated, and in this case BRET50 was 0.34±0.02 (Figure 2C). These results are consistent with hCXCR2–hDOP hetero-interactions occurring with an even higher affinity than the corresponding hCXCR2–hCXCR2 and hDOP–hDOP homo-interactions.

Bottom Line: This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy.These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor.These results have novel and important implications for the development and use of small-molecule therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein alpha subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.

Show MeSH
Related in: MedlinePlus