A novel human receptor involved in bitter tastant detection identified using the model organism Dictyostelium discoideum

Abstract

SummaryDetection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ 2 , which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) in wild-type but not grlJ 2 cells. Finally, we have identified an uncharacterized human protein c-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ 2 sensitivity to phenylthiourea in cell movement and PIP 3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.

Bitter tastant responses in the amoeba Dictyostelium correlate with rat and human taste assays

“…There are many potential mechanisms behind this effect. Dictyostelium is a well-studied model for cell movement (Dang et al, 2013;Artemenko et al, 2014) and has been explored in a range of pharmacological studies for identifying chemical targets (Robery et al, 2013;Waheed et al, 2014;Lockley et al, 2015). Indeed, a large number of studies have identified changes in cell behavior (particularly in movement) caused by deletion of individual proteins (Chattwood et al, 2014;Fets et al, 2014;Wessels et al, 2014).…”

“…This suggests that, although TAS2 receptors are involved in bitter taste perception in mammals, perhaps other molecular targets may also be involved in Dictyostelium and mammalian systems. An example of this is provided by a recent study investigating novel targets of a standard strong bitter tastant, phenylthiourea (PTU), in Dictyostelium, where the bitter tastant inhibited cell movement (Robery et al, 2013), and a genetic screen identified a PTU-sensitive receptor with homology to a poorly characterized human GABAB protein, where the human protein restored the sensitivity to PTU in Dictyostelium (Robery et al, 2013). Another study, again in Dictyostelium, identified an ion channel (PDK2) to be targeted by a bitter taste-related compound, naringenin (Glendinning, 1994), a flavonoid found in high levels in citrus fruit (Waheed et al, 2014).…”

“…In all these cases, discoveries in Dictyostelium have been successfully translated to humans or other mammals (Xu et al, 2007;Chang et al, 2013Chang et al, , 2014. Finally, Dictyostelium was able to identify pungent (e.g., capsaicin) and bitter (quinine, denatonium, phenylthiourea) tastants (Robery et al, 2013;Otto et al, 2015) and led to the discovery of a novel human receptor implicated in detection of the bitter tastant phenylthiourea (Robery et al, 2013). These wide-ranging studies demonstrated the potential utility for Dictyostelium in the identification of novel pharmaceutical compounds with a bitter taste liability.…”

“…Since Dictyostelium responded to chlorhexidine and earlier studies showed a response to the bitter tastants phenylthiourea and denatonium (Robery et al, 2013), we then sought to develop an approach to quantify cell behavior changes using another bitter substance, azelastine (Clapham et al, 2012) (250 µM; Fig. 2A).…”

“…Stability study of the antihistamine drug azelastine HCl along with a kinetic investigation and the identification of new degradation products. Anal Sci 30, 691-697. http://dx.doi.org/10.2116/ analsci.30.691 G proteins (Robery et al, 2013), may identify the molecular mechanism underlying the responses evoked by the structurally diverse bitter tastants in this study.…”