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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.

No MeSH data available.


TAS2R14 co-immunoprecipitation studies with other transfected GPCRs. FLAG-TAS2R14 was transfected into HEK293T cells alone or with one of the following HA-tagged GPCRs: β1AR, NMU2R, or CXCR6. Lysates were immunoprecipitated with HA and immunoblotted with FLAG. The upper section shows co-immunoprecipitation of β2AR and β1AR with TAS2R14. No (or very little) co-immunoprecipitation was observed with HA-NUMU2R or HA-CXCR6 transfection. Results are representative of 4 independent experiments.
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Figure 10: TAS2R14 co-immunoprecipitation studies with other transfected GPCRs. FLAG-TAS2R14 was transfected into HEK293T cells alone or with one of the following HA-tagged GPCRs: β1AR, NMU2R, or CXCR6. Lysates were immunoprecipitated with HA and immunoblotted with FLAG. The upper section shows co-immunoprecipitation of β2AR and β1AR with TAS2R14. No (or very little) co-immunoprecipitation was observed with HA-NUMU2R or HA-CXCR6 transfection. Results are representative of 4 independent experiments.

Mentions: We next tested whether the closely related β1AR subtype formed a complex with TAS2R14, and, whether β1AR activation altered TAS2R14 function. For the co-immunoprecipitation experiments, two phylogenetically distant GPCRs (15), the neuropeptide receptor NMU2R and the chemokine receptor CXCR6, were also studied. HEK-293T cells were transfected with FLAG-tagged TAS2R14 in the absence or presence of HA-tagged β2AR, β1AR, NMU2R, or CXCR6, immunoprecipitated with HA antibody, and immunoblotted with anti-FLAG antibodies (Fig. 10). As shown, β1AR, as well as the previously shown β2AR, formed complexes with TAS2R14 (top panel). In contrast, there was no signal for NMU2R and a minimally detected signal for CXCR6. The other panels are controls for the immunoprecipitation (second panel) and the inputs to the immunoprecipitation reaction (bottom two panels). These results suggested, then, that β1AR activation might also desensitize TAS2R14, as was seen with β2AR. Because human ASM do not express β1AR, we utilized the H1299 cell line that expresses β1AR, β2AR, and TAS2R14 (as determined by quantitative PCR, data not shown). Cells were treated with carrier (control) and ISO with pretreatment with vehicle, the β1AR antagonist betaxolol, or the β2AR antagonist ICI118551. The latter two conditions isolate β2AR and β1AR activation, respectively. After 5 min of agonist exposure, cells were treated with DPD and [Ca2+]i and immediately recorded (Fig. 11). There was a small (17%) but statistically significant desensitization of TAS2R14 function under the conditions of β1AR activation. When β2AR was selectively activated, there was a greater decrease in TAS2R14 function, amounting to ∼50% (see Fig. 11).


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 *
TAS2R14 co-immunoprecipitation studies with other transfected GPCRs. FLAG-TAS2R14 was transfected into HEK293T cells alone or with one of the following HA-tagged GPCRs: β1AR, NMU2R, or CXCR6. Lysates were immunoprecipitated with HA and immunoblotted with FLAG. The upper section shows co-immunoprecipitation of β2AR and β1AR with TAS2R14. No (or very little) co-immunoprecipitation was observed with HA-NUMU2R or HA-CXCR6 transfection. Results are representative of 4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016158&req=5

Figure 10: TAS2R14 co-immunoprecipitation studies with other transfected GPCRs. FLAG-TAS2R14 was transfected into HEK293T cells alone or with one of the following HA-tagged GPCRs: β1AR, NMU2R, or CXCR6. Lysates were immunoprecipitated with HA and immunoblotted with FLAG. The upper section shows co-immunoprecipitation of β2AR and β1AR with TAS2R14. No (or very little) co-immunoprecipitation was observed with HA-NUMU2R or HA-CXCR6 transfection. Results are representative of 4 independent experiments.
Mentions: We next tested whether the closely related β1AR subtype formed a complex with TAS2R14, and, whether β1AR activation altered TAS2R14 function. For the co-immunoprecipitation experiments, two phylogenetically distant GPCRs (15), the neuropeptide receptor NMU2R and the chemokine receptor CXCR6, were also studied. HEK-293T cells were transfected with FLAG-tagged TAS2R14 in the absence or presence of HA-tagged β2AR, β1AR, NMU2R, or CXCR6, immunoprecipitated with HA antibody, and immunoblotted with anti-FLAG antibodies (Fig. 10). As shown, β1AR, as well as the previously shown β2AR, formed complexes with TAS2R14 (top panel). In contrast, there was no signal for NMU2R and a minimally detected signal for CXCR6. The other panels are controls for the immunoprecipitation (second panel) and the inputs to the immunoprecipitation reaction (bottom two panels). These results suggested, then, that β1AR activation might also desensitize TAS2R14, as was seen with β2AR. Because human ASM do not express β1AR, we utilized the H1299 cell line that expresses β1AR, β2AR, and TAS2R14 (as determined by quantitative PCR, data not shown). Cells were treated with carrier (control) and ISO with pretreatment with vehicle, the β1AR antagonist betaxolol, or the β2AR antagonist ICI118551. The latter two conditions isolate β2AR and β1AR activation, respectively. After 5 min of agonist exposure, cells were treated with DPD and [Ca2+]i and immediately recorded (Fig. 11). There was a small (17%) but statistically significant desensitization of TAS2R14 function under the conditions of β1AR activation. When β2AR was selectively activated, there was a greater decrease in TAS2R14 function, amounting to ∼50% (see Fig. 11).

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.