The human family of TAS2Rs is comprised of at least 25 GPCRs that are highly divergent in sequence, sharing about 30–70% amino acid homology 
, which is reflected in the ability of TAS2Rs to recognize a diverse variety of chemical moieties. Despite this diversity, only a single inhibitor of these GPCRs has been described to date 
. Here we present evidence that an FDA-approved therapeutic is an allosteric inhibitor of a subset of human TAS2R receptors. The inhibitory properties of probenecid were unexpected since probenecid is commonly used to improve the cellular uptake of fluorescent dyes into cells to increase
the sensitivity of GPCR calcium flux assays 
. Our results show that probenecid can selectively inhibit the function of the bitter taste receptors hTAS2R16, hTAS2R38, and hTAS2R43 in vitro
, while leaving intact the function of other bitter taste receptors and GPCRs, including hTAS2R31, CXCR4, CCR5, and βAR. Interestingly, the inhibition of multiple bitter taste receptors was also observed for GIV3727, a recently described hTAS2R antagonist 
. Both probenecid and GIV3727 inhibit hTAS2R43 but only weakly inhibit hTAS2R31 (if at all), while each inhibitor has additional activity on a non-overlapping subset of receptors. The ability of both compounds to inhibit subsets of hTAS2Rs suggests that at least two different structural motifs may exist within each of these subsets of hTAS2R receptors. A better understanding of the structures of the TAS2Rs may reveal some of these common structural motifs.
Although the interaction of probenecid with a TAS2R receptor cannot be directly measured by binding or competition assays because bitter taste ligands bind too weakly (high µM to mM EC50s), our work provides several lines of evidence that the mechanism of action for probenecid inhibition occurs by direct binding to the hTAS2R16 receptor. First, analysis of hTAS2R16 point mutations define amino acid residues involved in probenecid binding or signaling that result in decreased sensitivity to probenecid while maintaining normal responses to the ligand salicin. Second, mechanism of action studies for probenecid against the hTAS2R16 receptor demonstrate rapid kinetics for complete inhibition (within 5 minutes of probenecid treatment) and near-instantaneous action for partial (>50%) inhibition, consistent with an effect on an upstream signaling component, such as the receptor itself. The effect of probenecid is also observed in the presence of inhibitors against MRP transporters (reported IC50, 100–150 µM) 
, which are responsible for probenecid's ability to increase fluorescent dye uptake. Probenecid is also known to inhibit other proteins such as the pannexin1 hemichannels in taste bud cells (IC50, 150 µM) 
, but it is unlikely that inhibition of such proteins would effect GPCR signaling or explain the structural (point mutation) and mechanism of action (rapid inhibition) studies here.
The identification of point mutations at residues 44 (P44T) and 96 (N96T) of hTAS2R16 that significantly suppress the ability of probenecid to inhibit salicin activity help to define probenecid's mechanism of action on the receptor. Both mutations affect probenecid activity without affecting salicin activity, suggesting an allosteric mechanism with distinct sites on the receptor for salicin and probenecid. This is in contrast to GIV3727, where mutations in hTAS2R43 and hTAS2R46 that confer resistance to inhibition alter both the specificity and activity of agonist compounds, suggesting an overlapping binding site 
. Nevertheless, P44 and N96 are likely to constitute only part of the probenecid interaction site, as these residues are not completely conserved between the hTAS2Rs that are sensitive to probenecid (i.e. hTAS2R16, hTAS2R38, and hTAS2R43 do not all contain P44 and N96 equivalents).
As suggested by structural studies of class A GPCRs 
and computational studies of TAS2Rs 
, the binding site for bitter receptor ligands would be expected to be in the transmembrane region of the receptor, with the site open to the extracellular portion of the receptor. Based on structure predictions, P44 and N96 are located in the first intracellular loop and the C-terminal half of the third transmembrane domain, respectively 
. Previous studies have implicated N172, located in the second extracellular loop of hTAS2R16, in the activity of diverse agonists 
. More recently, salicin ligand docking studies and mutational analysis of hTAS2R16 demonstrate the presence of at least 7 residues in TM3, TM5, and TM6 (distinct from P44 and N96) involved in ligand recognition for hTAS2R16, with all residues located towards the extracellular face of the receptor 
. The disparate locations of these residues and P44/N96, as well as the equivalence of the salicin response between WT and P44T/N96T, is suggestive of distinct binding sites for salicin and probenecid and point to an allosteric mechanism of action for probenecid.
The intracellular location of the probenecid binding site suggests that probenecid may potentially act by uncoupling G proteins from the receptor. If so, probenecid may be a useful reagent for understanding signal transduction for TAS2R receptors. Additional mutational analysis to further define the binding sites for probenecid and salicin on hTAS2R16 will be important for a complete understanding of the molecular mechanism of probenecid inhibition and may provide insight for the rational design of effective bitter blockers.
It is interesting to note that well-known polymorphisms in several TAS2Rs are found in the first intracellular loop, near the location of the P44 mutation in hTAS2R16 that confers probenecid resistance. For example, P49 of hTAS2R38 is located in the first intracellular loop and is part of the well-known PAV (taster) haplotype that confers sensitivity of individuals to PTC 
. Polymorphisms of a comparable residue, W35, in the receptors hTAS2R43 and hTAS2R31 significantly modulate the activity of the receptors with their respective ligands 
. The effect of mutations in the first intracellular loop of hTAS2R38, hTAS2R43, and hTAS2R31 highlight the role of this domain as a conserved modulator of TAS2R function.
Our studies using probenecid analogs suggest that inhibitor hydrophobicity is important for the pharmacological activity of probenecid. In particular, the propyl groups of probenecid may provide a better fit for the size and/or hydrophobicity of the putative probenecid-binding site on hTAS2R16. The ability of probenecid to cross the plasma membrane 
due to its negative charge at physiological pH and the hydrophobic character of probenecid's di-n
-propyl groups, is consistent with the binding of probenecid to a site on the intracellular face of the receptor, such as one that blocks binding of a G protein to the receptor.
Finally, the ability of probenecid to completely inhibit the cellular response to salicin in vitro provides a mechanistic explanation for its ability to inhibit human bitter taste perception of salicin in vivo. Future perceptual testing with a variety of diverse bitter compounds will help to determine whether the inhibitory effect of probenecid in vivo includes additional TAS2Rs.