Functional Interaction between T2R Taste Receptors and G-Protein α Subunits Expressed in Taste Receptor Cells

Abstract

Bitter taste perception is a conserved chemical sense against the ingestion of poisonous substances in mammals. A multigene family of G-protein-coupled receptors, T2R (so-called TAS2R or TRB) receptors and a G-protein alpha subunit (Galpha), gustducin, are believed to be key molecules for its perception, but little is known about the molecular basis for its interaction. Here, we use a heterologous expression system to determine a specific domain of gustducin necessary for T2R coupling. Two chimeric Galpha16 proteins harboring 37 and 44 gustducin-specific sequences at their C termini (G16/gust37 and G16/gust44) responded to different T2R receptors with known ligands, but G16/gust 23, G16/gust11, and G16/gust5 did not. The former two chimeras contained a predicted beta6 sheet, an alpha5 helix, and an extreme C terminus of gustducin, and all the domains were indispensable to the expression of T2R activity. We also expressed G16 protein chimeras with the corresponding domain from other Galpha(i) proteins, cone-transducin (Galpha(t2)), Galpha(i2), and Galpha(z) (G16/t2, G16/i2, and G16/z). As a result, G16/t2 and G16/i2 produced specific responses of T2Rs, but G16/z did not. Because Galpha(t2) and Galpha(i2) are expressed in the taste receptor cells, these G-protein alpha(i) subunits may also be involved in bitter taste perception via T2R receptors. The present Galpha16-based chimeras could be useful tools to analyze the functions of many orphan G-protein-coupled taste receptors.

Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans

“…3, 17, 29, 32, 33, and 115). Briefly, receptor cDNA constructs were transiently transfected in human embryonic kidney HEK293T cells stably expressing the chimeric G protein G␣16gust44 using Lipofectamine 2000 (Invitrogen) (33,115,116). Twenty-three to 26 h later the cells were loaded with Fluo-4 AM (Molecular Probes, Karlsruhe, Germany) in the presence of probenecid (2.5 mM, Sigma-Aldrich) in serum-free medium.…”

The Human Bitter Taste Receptor TAS2R10 Is Tailored to Accommodate Numerous Diverse Ligands

“…HEK 293T-G␣16gust44 (Ueda et al, 2003) cells were transiently transfected with receptor constructs as described previously (Brockhoff et al, 2007). After incubation for ϳ24 h, cells were loaded with Fluo4-AM (Invitrogen).…”

Molecular mechanism for the umami taste synergism

“…Cells were selected with G418 (Invitrogen, 0.4 mg/ml) and zeocin (Invitrogen, 0.1 mg/ml) in low glucose DMEM. hT1R1-2/rT1R3 stable cell lines were generated by transfecting linearized pEAK10-derived T1R1-T1R2 and pCDNA3.1/Zeo-derived (Invitrogen) T1R3 vectors into G16gust44 cell, a HEK293 line stably expressing the chimeric G16gust44 protein (24). Cells were selected with puromycin (Calbiochem, 0.5 g/ml) and zeocin (Invitrogen, 50 g/ml) in glutamine-free DMEM supplemented with GlutaMAX.…”