PROJECT SUMMARY
Taste family 1 receptors (T1Rs, forming sweet and umami receptors) and taste family 2 receptors
(T2Rs, forming bitter receptors) are important G-protein couple receptors (GPCRs) expressed in taste buds of
the tongue. T1Rs and T2Rs also play important chemosensory roles in tissues all over the body, including the
airway epithelium and in immune cells. We and other have characterized T1Rs and T2Rs as novel players in
innate immunity. They detect bacterial metabolites to stimulate rapid innate immune responses.
While current screening methods have revealed a lot about specific agonists that activate specific T1Rs
and T2Rs, understanding T1R and T2R activation by complex mixtures (e.g., plant extracts or conditioned
media from bacteria) is currently very difficult. Existing screening methods are focused on calcium signaling as
a read-out. Many compounds activate calcium signals independently of taste receptors in cultured cells, and
the HEK293 cells commonly used express endogenous T2Rs. Calcium-focused screening methods have also
likely missed “biased agonists,” that is, agonists which do not activate T2R G protein signaling but activate
arrestin signaling. This has been reported for many GPCRs, but no one has yet screened for biased agonists
of taste receptors. This is a critical gap in the studies of T1R and T2R biology that will be addressed here.
To overcome limitations of calcium-based assays and reveal new insights into T2R-arrestin signaling,
we will adapt a fluorescence-based tripartite GFP-based assay (known as the TRIO assay) that directly
visualizes heterologously-expressed receptor activation through arrestin binding to the activated receptor. This
assay is much less limited by off-target effects of complex mixtures or single compounds compared with
current methods. It is also adaptable to high throughput plate-reader instrumentation. This assay is faster (1-2
hrs) than other GPCR assays which rely on transcription as a readout (12-24 hrs). We have utilized this assay
to study activation and inhibition of protease-activated and adrenergic receptors in prior studies. Preliminary
data suggest this assay works well with T2Rs. We hypothesize that T1R and T2R TRIO assays will reveal
novel ligand-T1R/T2R interactions and may be useful to de-orphanize the remaining orphan T2R isoforms.
In this proposal, Aim 1 will focus on validation of this optical assay using known bitter and sweet
compounds. Aim 2 will use this assay to screen taste receptors against common pathogenic bacteria and
fungi to characterize which receptors are activated by which pathogens. New clinically relevant data will be
revealed, and the expression constructs validated in this study will become useful tools for taste receptor
research. All expression vectors will be made widely available via Addgene after initial publication. This
research has important utility for both immune research as well as molecular sensory nutrition and taste
research.
