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

Donghwa Kim,Maria Castaño,Lauren K Lujan,Jung A Woo,Stephen B Liggett

doi:10.1074/jbc.ra120.016056

Donghwa Kim, Maria Castaño + Show 3 more

Open Access

https://doi.org/10.1074/jbc.ra120.016056

Copy DOI

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.

Highlights

G protein–coupled receptors (GPCRs) are recognized as multifunctional signaling units, with the capacity to evoke intracellular events mediated by G protein and non-G protein mechanisms [1]
The taste 2 receptor member 14 (TAS2R14) has five Ser/Thr in the third intracellular loop (IL3) and five Ser in the cytoplasmic carboxy-terminal tail (CT). These sequences were screened for potential G protein– coupled receptor kinase (GRK) phosphorylation sites by expressing the glutathione-Stransferase (GST)–tagged fusion proteins representing WT TAS2R14 sequence or the analogous sequence with Ser/Thr substituted with Ala
In experiments where the proteins were electrophoresed on the same gel with equal protein loading, we found that the third loop peptide and the C-tail peptide phosphorylated to the same extent by active GRK2 (Fig. 1, D and E)

Summary

Introduction

G protein–coupled receptors (GPCRs) are recognized as multifunctional signaling units, with the capacity to evoke intracellular events mediated by G protein and non-G protein mechanisms [1]. To ascertain the functional effects of potential GRK phosphorylation of TAS2R14, three mutant receptor complementary DNAs were constructed substituting Ala in the encoded receptor for all Ser/Thr in the IL3 (IL-5A), the cytoplasmic tail (CT-5A), or both (IL/CT-10A). The [Ca2+]i response to agonist (Fig. 3B) was markedly altered by the GRK2 knockdown, revealing a pattern consistent with less desensitization, with mean slope of −0.03 ± 0.35 versus −1.74 ± 0.03 for siCtrl-transfected cells (p < 0.001).

Results

Conclusion