The third intracellular loop plays a critical role in bitter taste receptor activation.

Bitter taste receptors (T2Rs) belong to the superfamily of G protein-coupled receptors (GPCRs). T2Rs are chemosensory receptors with important therapeutic potential. In humans, bitter taste is perceived by 25 T2Rs, which are distinct from the well-studied Class A GPCRs. The activation mechanism of T2Rs is poorly understood and none of the structure-function studies are focused on the role of the important third intracellular loop (ICL3). T2Rs have a unique signature sequence at the cytoplasmic end of fifth transmembrane helix (TM5), a highly conserved LxxSL motif. Here, we pursue an alanine scan mutagenesis of the ICL3 of T2R4 and characterize the functionality of 23 alanine mutants. We identify four mutants, H214A, Q216A, V234A and M237A, that exhibit constitutive activity. To our surprise, the H214A mutant showed very high constitutive activity over wild type T2R4. Interestingly, His214 is highly conserved (96%) in T2Rs and is present two amino acids below the LxxSL motif in TM5. Molecular modeling shows a dynamic network of interactions involving residues in TM5-ICL3-TM6 that restrain the movement of the helices. Changes in this network, as in the case of H214A, Q216A, V234A and M237A mutants, cause the receptor to adopt an active conformation. The conserved LxxSL motif in TM5 performs both structural and functional roles in this process. These results provide insight into the activation mechanism of T2Rs, and emphasize the unique functional role of ICL3 even within the GPCR subfamilies.