Cell surface localization of receptors considered nonfunctional and orphan bitter taste receptors

Despite considerable efforts to identify bitter agonists for the ∼25 putatively functional human bitter taste receptors, four receptors remained orphan until now. Whether the problem of identifying agonists for those receptors is due to technical issues related to functional heterologous expression or to the lack of suitable activators is unknown. A similar problem occurs with some variants of bitter taste receptors considered nonfunctional. While some researchers believe that these variants are nonfunctional, others speculate that agonist specificity may have shifted toward yet unknown bitter compounds. Efficient cell-surface localization is generally required for canonical TAS2R signaling in heterologous functional assays and is an important determinant of receptor responsiveness. To address the lack of responsiveness in heterologous expression assays, we quantitatively assessed cell-surface localization of these receptors in living mammalian cells using the HiBiT protein tagging system established in our laboratory. HiBiT-tagged putatively nonfunctional and orphan receptors were expressed in HEK 293T-Gα16gust44 cells and subjected to luminescence measurements using a membrane-impermeable detection reagent. It was observed that all four orphan receptors, TAS2R19, -R42, -R45, and -R60, as well as the putatively nonfunctional receptor variants of TAS2R9, and -R38, were present at the cell surface, albeit at grossly different levels. Immunofluorescence experiments confirmed these results. We conclude that some orphan bitter taste receptors do not show obvious signs of routing or misfolding problems in heterologous cells, suggesting the existence of yet-to-be-discovered bitter activators, particularly for the two receptors, TAS2R19 and TAS2R60, which exhibit pronounced cell-surface localization.