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β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *

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ABSTRACT

Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of β2-adrenergic receptors (β2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when β2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected β2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca2+]i with β2AR co-transfection, ∼53% decrease in [Ca2+]i signaling with shRNA knockdown of β2AR in H292 cells, and ∼60% loss of [Ca2+]i responsiveness in βAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with β2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of β2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged β-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with β-agonist co-treatment. Thus the β2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by β-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function.

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β2AR expression dynamically regulates TAS2R14 functional expression. HEK-293T cells were transfected with TAS2R14 + Gα16/G44, without or with β2AR (A and B). The [Ca2+]i response to the TAS2R14 agonists DPD and FFA are increased when β2AR is co-transfected, consistent with the increased expression of TAS2R14 (Fig. 1, A and B). In 4 experiments, the [Ca2+]i stimulation was increased by 2.4 ± 0.11 and 2.1 ± 0.12, respectively, when β2AR was co-expressed (p < 0.01 versus absence of β2AR). In C, H292 cells, which endogenously express TAS2R14 and β2AR were transfected with β2AR shRNA (or sh-control) and treated with vehicle or the TAS2R14 agonist DPD. Knockdown of β2AR by β2AR shRNA resulted in decreased TAS2R14-mediated [Ca2+]i signaling. In D and E, βAR knock-out mouse (10) ASM cells (which express no detectable βAR) were challenged with TAS2R agonists and revealed >50% reduction in TAS2R-stimulated [Ca2+]i. Results are from 4 representative experiments.
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Figure 4: β2AR expression dynamically regulates TAS2R14 functional expression. HEK-293T cells were transfected with TAS2R14 + Gα16/G44, without or with β2AR (A and B). The [Ca2+]i response to the TAS2R14 agonists DPD and FFA are increased when β2AR is co-transfected, consistent with the increased expression of TAS2R14 (Fig. 1, A and B). In 4 experiments, the [Ca2+]i stimulation was increased by 2.4 ± 0.11 and 2.1 ± 0.12, respectively, when β2AR was co-expressed (p < 0.01 versus absence of β2AR). In C, H292 cells, which endogenously express TAS2R14 and β2AR were transfected with β2AR shRNA (or sh-control) and treated with vehicle or the TAS2R14 agonist DPD. Knockdown of β2AR by β2AR shRNA resulted in decreased TAS2R14-mediated [Ca2+]i signaling. In D and E, βAR knock-out mouse (10) ASM cells (which express no detectable βAR) were challenged with TAS2R agonists and revealed >50% reduction in TAS2R-stimulated [Ca2+]i. Results are from 4 representative experiments.

Mentions: Functional signaling of TAS2R14 was ascertained by fluorescent microscopy and by a plate-based fluorescent assay of Fluo-4-loaded cells. Cells were transfected with the chimeric G-protein Gα16/G44 and pcDNA, and TAS2R14 + β2AR. In the imaging studies, β2AR + TAS2R14 co-transfected cells showed no response to vehicle, nor did pcDNA-transfected (control) cells show a [Ca2+]i response (Fig. 3A) to multiple TAS2R agonists. However, increases in [Ca2+]i were observed for quinine, which activates subtypes 10, 14, and 31, and the TAS2R14 agonists diphenhydramine (DPD) and flufenamic acid (FFA). In contrast, bitter taste receptor agonists for TAS2R31 (saccharin) and TAS2R10 (strychnine) caused no increase in [Ca2+]i. Taken together, these data indicate the expected agonist specificity for TAS2R14 (Fig. 3B) (2). In the plate-based studies, co-transfected cells showed the expected dose-responses (2) to DPD (Fig. 3C) and quinine (data not shown). Furthermore, cells transfected with TAS2R14, β2AR, and Gα16/G44 showed a significant increase in [Ca2+]i response to DPD and FFA (Fig. 4, A and B) compared with the TAS2R14 + Gi16/G44 cells, consistent with the increase in cell surface TAS2R14 expression evoked by β2AR as observe by the biotinylation assay (Fig. 1, A and B), and the confocal imaging (Figs. 1D and 2D). In these experiments, expression of β2AR compared with control pcDNA did not decrease TAS2R14 mRNA levels as determined by quantitative PCR (4.1 ± 0.12 versus 3.5 ± 0.08 units, respectively, n = 4, p > 0.05).


β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *
β2AR expression dynamically regulates TAS2R14 functional expression. HEK-293T cells were transfected with TAS2R14 + Gα16/G44, without or with β2AR (A and B). The [Ca2+]i response to the TAS2R14 agonists DPD and FFA are increased when β2AR is co-transfected, consistent with the increased expression of TAS2R14 (Fig. 1, A and B). In 4 experiments, the [Ca2+]i stimulation was increased by 2.4 ± 0.11 and 2.1 ± 0.12, respectively, when β2AR was co-expressed (p < 0.01 versus absence of β2AR). In C, H292 cells, which endogenously express TAS2R14 and β2AR were transfected with β2AR shRNA (or sh-control) and treated with vehicle or the TAS2R14 agonist DPD. Knockdown of β2AR by β2AR shRNA resulted in decreased TAS2R14-mediated [Ca2+]i signaling. In D and E, βAR knock-out mouse (10) ASM cells (which express no detectable βAR) were challenged with TAS2R agonists and revealed >50% reduction in TAS2R-stimulated [Ca2+]i. Results are from 4 representative experiments.
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Figure 4: β2AR expression dynamically regulates TAS2R14 functional expression. HEK-293T cells were transfected with TAS2R14 + Gα16/G44, without or with β2AR (A and B). The [Ca2+]i response to the TAS2R14 agonists DPD and FFA are increased when β2AR is co-transfected, consistent with the increased expression of TAS2R14 (Fig. 1, A and B). In 4 experiments, the [Ca2+]i stimulation was increased by 2.4 ± 0.11 and 2.1 ± 0.12, respectively, when β2AR was co-expressed (p < 0.01 versus absence of β2AR). In C, H292 cells, which endogenously express TAS2R14 and β2AR were transfected with β2AR shRNA (or sh-control) and treated with vehicle or the TAS2R14 agonist DPD. Knockdown of β2AR by β2AR shRNA resulted in decreased TAS2R14-mediated [Ca2+]i signaling. In D and E, βAR knock-out mouse (10) ASM cells (which express no detectable βAR) were challenged with TAS2R agonists and revealed >50% reduction in TAS2R-stimulated [Ca2+]i. Results are from 4 representative experiments.
Mentions: Functional signaling of TAS2R14 was ascertained by fluorescent microscopy and by a plate-based fluorescent assay of Fluo-4-loaded cells. Cells were transfected with the chimeric G-protein Gα16/G44 and pcDNA, and TAS2R14 + β2AR. In the imaging studies, β2AR + TAS2R14 co-transfected cells showed no response to vehicle, nor did pcDNA-transfected (control) cells show a [Ca2+]i response (Fig. 3A) to multiple TAS2R agonists. However, increases in [Ca2+]i were observed for quinine, which activates subtypes 10, 14, and 31, and the TAS2R14 agonists diphenhydramine (DPD) and flufenamic acid (FFA). In contrast, bitter taste receptor agonists for TAS2R31 (saccharin) and TAS2R10 (strychnine) caused no increase in [Ca2+]i. Taken together, these data indicate the expected agonist specificity for TAS2R14 (Fig. 3B) (2). In the plate-based studies, co-transfected cells showed the expected dose-responses (2) to DPD (Fig. 3C) and quinine (data not shown). Furthermore, cells transfected with TAS2R14, β2AR, and Gα16/G44 showed a significant increase in [Ca2+]i response to DPD and FFA (Fig. 4, A and B) compared with the TAS2R14 + Gi16/G44 cells, consistent with the increase in cell surface TAS2R14 expression evoked by β2AR as observe by the biotinylation assay (Fig. 1, A and B), and the confocal imaging (Figs. 1D and 2D). In these experiments, expression of β2AR compared with control pcDNA did not decrease TAS2R14 mRNA levels as determined by quantitative PCR (4.1 ± 0.12 versus 3.5 ± 0.08 units, respectively, n = 4, p > 0.05).

View Article: PubMed Central - PubMed

ABSTRACT

Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of &beta;2-adrenergic receptors (&beta;2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by &sim;5-fold when &beta;2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected &beta;2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca2+]i with &beta;2AR co-transfection, &sim;53% decrease in [Ca2+]i signaling with shRNA knockdown of &beta;2AR in H292 cells, and &sim;60% loss of [Ca2+]i responsiveness in &beta;AR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with &beta;2AR activation rapidly uncoupling TAS2R14 function (&sim;65% desensitization). Cross-talk was independent of &beta;2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged &beta;-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by &sim;50% with &beta;-agonist co-treatment. Thus the &beta;2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by &beta;-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function.

No MeSH data available.


Related in: MedlinePlus