The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception

Abstract

Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis . A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types . We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) . Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds-for example, pharmaceuticals and selected phytochemicals.

Figure 1

Dose-Response Curves of the Calcium Concentrations in Cells Cotransfected with hTAS2R38 Variants after Stimulation with Increasing PTC and PROP Concentrations (A) Effects of a PTC concentration series on cells expressing the TAS2R38 variants (descending order): PAV (dashed line), PAI (solid line), PVI (solid line), AAI (dashed line), AAV (solid line), PVV (dashed line), AVV (solid line), or AVI (dashed line). See (C) for symbols key. The amplitudes of PTC (A) and PROP (B) responses have been normalized to those of the peptide hormone somatostatin-14 SST (1 μM), which activates an endogenous receptor. All receptor variants were challenged with PROP and PTC up to 1 mM, at which point PAV responses saturated. Each point represents the mean ± the standard error of the mean of at least three independent experiments carried out in triplicate. See Supplemental Experimental Procedures for methods, and for additional detail see Bufe et al., 2002 [9]. (B) Effects of a PROP concentration series on cells expressing the eight TAS2R38 variants. Symbols are as above. (C) Half maximal response (EC₅₀) and threshold values of all receptor variants for PROP and PTC. Amino acids identical to those of the AVI variant are listed in bold in the three-letter haplotype name to the left. Responses were normalized to somatostatin responses. EC₅₀ and threshold numerical values for the three haplotypes tested from subjects PAV, AAI, AVI are in bold.

Figure 2

Presence of hTAS2R38 mRNA in Human Taste Papillae (A) Agarose gel electrophoresis of 765 bp fragments amplified by RT-PCR with primers specific for hTAS2R38 mRNA. (1) 6:100 bp ladder used as size standard; (2) hTAS2R38 plasmid DNA, positive control; (3) negative control, no template present; (4) circumvallate papillae; and (5) reverse transcriptase has been omitted from the reaction to assess the presence of contaminating genomic DNA. (B) Allele-specific gene expression of hTAS2R38 in human fungiform taste tissue. mRNA levels have been measured by quantitative RT-PCR in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels in four subjects of the indicated diplotype: A/P, AVI/PAV; A/A, AVI/AVI; and P/P, PAV/PAV. Grey bars represent mRNA levels for PAV variant, and black bars represent mRNA for the AVI variant. (C) In situ hybridization of human circumvallate papilla with hTAS2R38 sense (left panel) or antisense cRNA probe (middle and right panels), processed by alkaline phosphatase. Scale bars represent 100 μm (left and middle panels) and 10 μm (right panel) (3 of 11 cells stained in this section). Star denotes the taste bud shown in higher magnification in the right panel.

Figure 3

Suprathreshold Concentration-Intensity Functions for PTC and PROP Tasting in 32 Subjects Genotyped for hTAS2R38 The y axis represents standardized perceived intensity as rated on the general labeled magnitude scale. The x axis represents the concentration of the respective compounds tested. Each line represents the average responses over two trials for an individual subject. The top row of panels represents PTC responses, and the bottom row of panels represents PROP responses. The left column represents AVI/AVI subjects, the middle column represents AVI/PAV subjects, and the right column represents PAV/PAV subjects. Symbols identify haplotype groups: black circles, AVI/AVI (left column); red down-triangles, AVI/PAV (middle column); and green squares, PAV/PAV (right column). Insets (left column) represent geometric mean taste responses with geometric standard errors for PTC and PROP for the various haplotype groups; symbols and colors are the same as with individual curves. The single AAI/AAI subject’s concentration-intensity functions are added to the insets (blue diamonds).

Figure 4

Psychometric Functions for PTC and PROP Tasting in One PAV/PAV Subject, One AVI/AVI Subject, and One AAI/AAI Subject Genotyped for TAS2R38 Variants The y axis represents percent of trials correct in a two-alternative forced-choice task. The x axis represents the concentrations of the respective compounds that were tested. The dashed, dotted, and solid fitted functions represent the PAV (green), AAI (blue), and AVI (black) subjects’ sensitivities. Asterisks represent the concentrations of the three individuals’ modified Harris-Kalmus (mH-K) bitterness recognition thresholds and are placed on their respective psychometric functions. Large filled squares that lie on the x axis represent the mean mH-K bitterness recognition thresholds for the 32 subjects (from Figure 3), who were tested separately and genotyped as PAV/PAV (green), PAV/AVI (red), or AVI/AVI (black). Vertical dashed lines indicate detection thresholds (~IC₅₀). Numerical threshold values for the three individuals’ and the group means are presented by haplotype in the table beneath the figure.