The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14

Abstract

For most G protein-coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein-coupled receptor kinase (GRK)-mediated phosphorylation, leading to β-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:β-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the β-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and β-arrestin binding. However, β-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.

Keywords: G protein–coupled receptor kinases; G protein–coupled receptors; TAS2R; desensitization; β-arrestin.

Figure 1

TAS2R14-based peptides are phosphorylated at Ser/Thr by GRK2.A, localization of the Ser/Thr in the third intracellular loop (IL3) and C-terminal tail (CT) of TAS2R14. B–D, peptides based on WT IL3 and CT, but not Ala substituted for Ser/Thr peptides, are phosphorylated by activated GRK2 (representative experiments). E, GST-IL-WT and GSTCT-WT are phosphorylated by activated GRK2 to the same extent. The phosphorylation levels were normalized to the amount of loaded protein as determined by Coomassie blue staining. The small amount of signal observed in the mutant peptide lanes was not different than “0” as determined by the one-sample t test. ∗p < 0.001 versus analogous WT peptide. Data are shown as mean ± SD, with individual results, from five independent experiments. GRK2, G protein–coupled receptor kinase 2; GST glutathione-S-transferase; TAS2R14, bitter taste 2 receptor member 14.

Figure 2

Functional agonist-promoted desensitization profiles of the [Ca²⁺]_iresponse of WT and mutant TAS2R14 receptors.A, results (mean ± SE) from five experiments. ∗Slope from 10 to 50 s as determined by linear regression differs from WT, p < 0.01 (see also Table 1). B, representative Western blot showing that expression of the WT and mutant receptors was comparable for experiments shown in panel A. [Ca²⁺]_i, intracellular calcium; CT, carboxy-terminal tail; IL, intracellular loop; TAS2R14, bitter taste 2 receptor member 14.

Figure 3

Agonist-promoted desensitization of the TAS2R14 [Ca²⁺]_iresponse is attenuated by GRK2 knockdown.A, representative Western blot showing the extent of GRK2 knockdown in cells used for experiments in panel B. B, results (mean ± SE) from five experiments. ∗Slope from 10 to 50 s as determined by linear regression is different from control (scrambled) siRNA, p < 0.01 (see also Table 1). [Ca²⁺]_i, intracellular calcium; GRK, G protein–coupled receptor kinase; TAS2R14, bitter taste 2 receptor member 14.

Figure 4

Qualitative assessment of agonist-promoted β-arrestin2 recruitment by WT and mutant TAS2R14 receptors. WT and IL-5A receptors exposed to agonist (10 min) displayed a redistribution of β-arrestin2–GFP from the cytosol to the cell membrane and a punctate accumulation pattern at the cell membrane. This appearance was not observed with CT-5A and IL/CT-10A receptors. Shown is a representative of five experiments performed. CT, carboxy-terminal tail; DPD, diphenhydramine; IL, intracellular loop; TAS2R14, bitter taste 2 receptor member 14.

Figure 5

Proximity ligation assay (PLA) reveals differential β-arrestin2 binding to WT and mutant receptors.A, representative experiment showing agonist-promoted β-arrestin2 binding (red signal) with the indicated receptors. B, mean ± SD, and individual results, from five PLA experiments. ∗p < 0.01 versus WT; #not significant versus 0% change as determined by the one-sample t test. DAPI, 4′,6-diamidino-2-phenylindole; DPD, diphenhydramine; IL, intracellular loop.

Figure 6

Agonist-promoted TAS2R14 internalization and sorting to early endosomes is altered by Ser/Thr mutations.A, representative experiment showing agonist (DPD)-promoted loss of cell surface receptors as determined by Western blotting of the membrane fraction. B, mean ± SD, and individual results, from four cell surface expression experiments. ∗p < 0.05 versus WT. C, agonist-promoted colocalization (yellow signal) of WT receptor with the early endosome marker EEA1, which was not observed with any of the indicated mutant TAS2R14 receptors. Shown is a representative experiment. The scale bars are shown in the bottom right-hand panel for each cell line. B, mean ± SD, and individual results, from four colocalization experiments. ∗p Value not significantly different compared with vehicle control. CT, carboxy-terminal tail; DAPI, 4′,6-diamidino-2-phenylindole; DPD, diphenhydramine; EEA1, early endosome antigen 1; IL, intracellular loop; TAS2R14, bitter taste 2 receptor member 14.