Intracellular and Dual-Site Inhibition of a bitter taste GPCR

Bitter taste receptors (TAS2Rs) are G protein-coupled receptors expressed in both gustatory and extraoral tissues and activated by a broad range of compounds. TAS2R14 is among the most promiscuous members of this family, responding to many structurally diverse ligands. Cryo-electron microscopy structures of TAS2R14 have revealed agonists binding in an intracellular pocket, raising the question of the main sites of interaction for known TAS2R14 antagonists. To address this, we examined the effects of mutations at residues located in the extracellular and intracellular regions on receptor inhibition by three antagonist compounds. Mutations in the extracellular region reduced the inhibitory effect of LF22, whereas all three compounds showed reduced inhibition in the intracellular mutants. Computational co-folding of these ligands with TAS2R14 supported these observations, indicating that LF22 interacts with both top and bottom binding sites, whereas LF1 and probenecid engage predominantly the intracellular site adjacent to the G protein interface. Interestingly, LF1 is much more potent for TAS2R16 than its known inhibitor probenecid. These findings reveal distinct inhibitory mechanisms among TAS2R antagonists and provide new insights towards designing inhibitors of bitter taste.