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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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Distribution and functional contributions of TAS2R14 internalization by β-agonist. HEK-293T cells were transfected with HA-β2AR and FLAG-TAS2R14. Cells were treated with ISO for 5 min or 1 h, and the change in intracellular expression of each receptor determined by fluorescence microscopy (A) and the change in cell surface expression by the biotinylation assay (see “Experimental Procedures”) (B). As expected, intracellular β2AR increased in a time-dependent manner (A), with concomitant decrease in cell surface expression (B) after 5 min exposure to ISO. In contrast, there was no statistically significant redistribution of TAS2R14 from the cell surface to cytosol at 5 min. However, by 1 h intracellular accumulation and loss of cell surface TAS2R14 was detected. Results are representative of 5 independent experiments, where β2AR cell surface expression loss was 52 ± 2.1 and 86.75 ± 1.1%, and TAS2R14 loss was 11 ± 1.3 and 64 ± 2.6%, at the 5-min and 1-h time points, respectively. In C, the response to TAS2R14 agonist DPD is quantitated after 1 h of ISO in the absence or presence of the internalization inhibitor dynasore. The loss-of-function of TAS2R14 from 1 h ISO was only partially rescued by blocking internalization, consistent with the rapid receptor:receptor uncoupling process still in effect. Results are from a single representative experiment performed in triplicate. See text for statistical analysis from multiple experiments.
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Figure 7: Distribution and functional contributions of TAS2R14 internalization by β-agonist. HEK-293T cells were transfected with HA-β2AR and FLAG-TAS2R14. Cells were treated with ISO for 5 min or 1 h, and the change in intracellular expression of each receptor determined by fluorescence microscopy (A) and the change in cell surface expression by the biotinylation assay (see “Experimental Procedures”) (B). As expected, intracellular β2AR increased in a time-dependent manner (A), with concomitant decrease in cell surface expression (B) after 5 min exposure to ISO. In contrast, there was no statistically significant redistribution of TAS2R14 from the cell surface to cytosol at 5 min. However, by 1 h intracellular accumulation and loss of cell surface TAS2R14 was detected. Results are representative of 5 independent experiments, where β2AR cell surface expression loss was 52 ± 2.1 and 86.75 ± 1.1%, and TAS2R14 loss was 11 ± 1.3 and 64 ± 2.6%, at the 5-min and 1-h time points, respectively. In C, the response to TAS2R14 agonist DPD is quantitated after 1 h of ISO in the absence or presence of the internalization inhibitor dynasore. The loss-of-function of TAS2R14 from 1 h ISO was only partially rescued by blocking internalization, consistent with the rapid receptor:receptor uncoupling process still in effect. Results are from a single representative experiment performed in triplicate. See text for statistical analysis from multiple experiments.

Mentions: Interestingly, β2AR internalization from the cell surface to the interior is also underway after 5 min of agonist exposure (11, 12). We considered, then, that within the context of the heterodimer, TAS2R14 may co-internalize with β2AR, thereby resulting in an overall decreased cellular response to TAS2R14 agonist. However, whereas loss of β2AR cell surface expression was clearly apparent after 5 min of ISO exposure, TAS2AR14 cell surface expression was not changed (Fig. 6). In quantitative imaging studies, there was a readily detectable increase in intracellular β2AR after 5 min of treatment with agonist, and no statistically significant parallel increase in intracellular TAS2R14 at this time point (Fig. 7A). After a 1-h exposure to ISO, both β2AR and TAS2R14 intracellular expressions were increased, with β2AR > TAS2R14 (Figs. 6 and 7A). These imaging studies were confirmed using the cell surface biotinylation assay. In these assays we found β2AR cell surface loss was rapid and amounted to >25%, whereas TAS2R14 cell surface loss was minimally detected at 5 min. However, by 1-h ISO exposure, TAS2R14 cell surface expression was clearly decreased and approached >50% loss (Fig. 7B). So, at least with brief β-agonist exposure, the evidence does not support co-internalization. However, by 1-h ISO exposure, there was detectable loss of cell surface expression of TAS2R14, measured as a gain of intracellular expression (Figs. 6 and 7A), or loss of cell surface immunoreactivity in the biotinylation assays (Fig. 7B). This suggested that internalization of TAS2R14 during prolonged β-agonist exposure could be the basis for the further loss of cellular responsiveness to TAS2R14 agonist at this more prolonged time point. To further explore this, we blocked receptor internalization with the dynamin inhibitor dynasore. As shown in Fig. 7C, dynamin inhibition only partially rescued TAS2R14 desensitization by β-agonist after a 1-h exposure, indicating two processes at play: an early event that may be uncoupling of TAS2R14 to its G-protein due to interactions within the heterodimer, and a later internalization of TAS2R14 such that cell surface expression is reduced.


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 *
Distribution and functional contributions of TAS2R14 internalization by β-agonist. HEK-293T cells were transfected with HA-β2AR and FLAG-TAS2R14. Cells were treated with ISO for 5 min or 1 h, and the change in intracellular expression of each receptor determined by fluorescence microscopy (A) and the change in cell surface expression by the biotinylation assay (see “Experimental Procedures”) (B). As expected, intracellular β2AR increased in a time-dependent manner (A), with concomitant decrease in cell surface expression (B) after 5 min exposure to ISO. In contrast, there was no statistically significant redistribution of TAS2R14 from the cell surface to cytosol at 5 min. However, by 1 h intracellular accumulation and loss of cell surface TAS2R14 was detected. Results are representative of 5 independent experiments, where β2AR cell surface expression loss was 52 ± 2.1 and 86.75 ± 1.1%, and TAS2R14 loss was 11 ± 1.3 and 64 ± 2.6%, at the 5-min and 1-h time points, respectively. In C, the response to TAS2R14 agonist DPD is quantitated after 1 h of ISO in the absence or presence of the internalization inhibitor dynasore. The loss-of-function of TAS2R14 from 1 h ISO was only partially rescued by blocking internalization, consistent with the rapid receptor:receptor uncoupling process still in effect. Results are from a single representative experiment performed in triplicate. See text for statistical analysis from multiple experiments.
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Figure 7: Distribution and functional contributions of TAS2R14 internalization by β-agonist. HEK-293T cells were transfected with HA-β2AR and FLAG-TAS2R14. Cells were treated with ISO for 5 min or 1 h, and the change in intracellular expression of each receptor determined by fluorescence microscopy (A) and the change in cell surface expression by the biotinylation assay (see “Experimental Procedures”) (B). As expected, intracellular β2AR increased in a time-dependent manner (A), with concomitant decrease in cell surface expression (B) after 5 min exposure to ISO. In contrast, there was no statistically significant redistribution of TAS2R14 from the cell surface to cytosol at 5 min. However, by 1 h intracellular accumulation and loss of cell surface TAS2R14 was detected. Results are representative of 5 independent experiments, where β2AR cell surface expression loss was 52 ± 2.1 and 86.75 ± 1.1%, and TAS2R14 loss was 11 ± 1.3 and 64 ± 2.6%, at the 5-min and 1-h time points, respectively. In C, the response to TAS2R14 agonist DPD is quantitated after 1 h of ISO in the absence or presence of the internalization inhibitor dynasore. The loss-of-function of TAS2R14 from 1 h ISO was only partially rescued by blocking internalization, consistent with the rapid receptor:receptor uncoupling process still in effect. Results are from a single representative experiment performed in triplicate. See text for statistical analysis from multiple experiments.
Mentions: Interestingly, β2AR internalization from the cell surface to the interior is also underway after 5 min of agonist exposure (11, 12). We considered, then, that within the context of the heterodimer, TAS2R14 may co-internalize with β2AR, thereby resulting in an overall decreased cellular response to TAS2R14 agonist. However, whereas loss of β2AR cell surface expression was clearly apparent after 5 min of ISO exposure, TAS2AR14 cell surface expression was not changed (Fig. 6). In quantitative imaging studies, there was a readily detectable increase in intracellular β2AR after 5 min of treatment with agonist, and no statistically significant parallel increase in intracellular TAS2R14 at this time point (Fig. 7A). After a 1-h exposure to ISO, both β2AR and TAS2R14 intracellular expressions were increased, with β2AR > TAS2R14 (Figs. 6 and 7A). These imaging studies were confirmed using the cell surface biotinylation assay. In these assays we found β2AR cell surface loss was rapid and amounted to >25%, whereas TAS2R14 cell surface loss was minimally detected at 5 min. However, by 1-h ISO exposure, TAS2R14 cell surface expression was clearly decreased and approached >50% loss (Fig. 7B). So, at least with brief β-agonist exposure, the evidence does not support co-internalization. However, by 1-h ISO exposure, there was detectable loss of cell surface expression of TAS2R14, measured as a gain of intracellular expression (Figs. 6 and 7A), or loss of cell surface immunoreactivity in the biotinylation assays (Fig. 7B). This suggested that internalization of TAS2R14 during prolonged β-agonist exposure could be the basis for the further loss of cellular responsiveness to TAS2R14 agonist at this more prolonged time point. To further explore this, we blocked receptor internalization with the dynamin inhibitor dynasore. As shown in Fig. 7C, dynamin inhibition only partially rescued TAS2R14 desensitization by β-agonist after a 1-h exposure, indicating two processes at play: an early event that may be uncoupling of TAS2R14 to its G-protein due to interactions within the heterodimer, and a later internalization of TAS2R14 such that cell surface expression is reduced.

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 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.

No MeSH data available.


Related in: MedlinePlus