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

Jan 19, 2021The Journal of biological chemistry

Specific parts of bitter taste receptors contain important sites for regulation by GRK enzymes in TAS2R14

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Abstract

Agonist-promoted functional desensitization of TAS2R14 occurs through GRK phosphorylation of carboxy-terminal residues.

The carboxy-terminal tail is essential for desensitization of TAS2R14, as mutations in this region disrupt the process.
Phosphorylation of the third intracellular loop is involved in β-arrestin binding, which is necessary for receptor function.
Inhibition of GRK2 prevents desensitization of TAS2R14 in human airway smooth muscle cells.
Mutant forms of TAS2R14 that lack phosphorylation sites show impaired internalization and trafficking.
The study indicates a critical role for specific residues in both the carboxy-terminal tail and third intracellular loop for receptor activity.

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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.

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

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