Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

There is a reliable and valid variation in human umami taste of l-glutamate. Variations in perception of umami taste correlated with variations in the human TAS1R3 gene. The putative human taste receptor TAS1R1-TAS1R3 responds specifically to l-glutamate mixed with the ribonucleotide IMP. Thus, this receptor likely contributes to human umami taste perception.

T cell activation requires two signals: specific recognition of antigen through the T cell receptor (TCR) and a costimulatory signal provided primarily by CD28 in naïve T cells. We cloned a novel gene with considerable homology to RIBP/TSAd/Lad, an adaptor involved in T cell activation and interleukin-2 (IL-2) promoter activation. Expression of this gene is limited to the spleen and thymus. We have named this gene ALX, adaptor in lymphocytes of unknown function X. Because the related adaptor RIBP is involved in IL-2 regulation, we investigated whether ALX had a similar function. ALX overexpression in Jurkat T cells results in inhibition of IL-2 promoter activation after stimulation with superantigen. The IL-2 promoter contains several binding sites for transcription factors including the composite element RE/AP, which is the primary site of CD28 transcriptional activation. ALX overexpression had the greatest effect on the activation of a RE/AP reporter as opposed to an AP-1 reporter. Interestingly, ALX overexpression strongly inhibited RE/AP activation in response to anti-CD28/phorbol 12-myristate 13-acetate (PMA) stimulation but had minimal effect when anti-TCR/PMA was used. Therefore, it appears that ALX may function downstream of CD28 costimulation during T cell activation. In addition, the mobility of ALX shifts upon TCR/CD28 costimulation to a greater extent than what is observed with either stimulus alone demonstrating that ALX is a target of both TCR and CD28 costimulatory signaling pathways.The activation of T cells is critical to the generation of an immune response. Minimally, two signals are required to activate resting T cells into effector T cells: an antigen-specific signal through the T cell receptor (TCR) 1 and a second antigenindependent "costimulatory" signal, which is primarily pro- 2). The biochemical events downstream of CD28 and how they synergize with TCR signals to result in T cell activation are much less understood. A critical step in T cell activation is the induction of the IL-2 gene, which occurs through both a transcriptional up-regulation of its promoter and increased stability of its mRNA (3, 4).One molecule involved in TCR signaling is the adaptor molecule TSAd (also designated Lad, and for the mouse ortholog RIBP) (5). This adaptor was identified in separate two-hybrid screens for proteins that associated with the Tec family kinases Rlk and Itk (6), the Src family kinase Lck (7), and the mitogenactivated protein kinase kinase kinase MEKK2 (8). Although the mechanism of TSAd action is not well characterized, a role for TSAd in the regulation of IL-2 was demonstrated in the RIBP knock-out mouse (6). These mice have no gross abnormalities in T cell development, but mature T cells show a moderate defect (ϳ70% decrease) in proliferation and IL-2 production upon TCR or TCR/CD28 stimulation. TSAd has been reported to localize to the cytoplasm in T cells and to translocate to the immunological synapse during T cell activation (8), although it also has been reported to be primarily nucle...

Identifying the structures that contribute to monoclonal antibody (mAb) binding sites (epitopes) within native G protein-coupled receptors (GPCRs) can be useful for developing topological models of the accessible receptor surface, for selecting the most relevant mAbs for therapeutic, diagnostic, and research applications and for distinguishing the intellectual property positions of otherwise similar mAbs. While conventional site-directed mutagenesis studies can identify individual amino acid residues that are critical to mAb binding, defining comprehensive epitopes is difficult and time-consuming for these structurally complex proteins. For example, in studies over the past decade, 13 residues (cumulatively) in the GPCR CCR5 have been reported to contribute to the interactions of five well-studied mAbs. 1-5 However, crystallographically defined epitopes contain an average of 20 contact residues each, 6 so these 13 residues likely represent only a portion of all the amino acids that constitute these five epitopes. Because of the importance of CCR5 in HIV infection and inflammation,7 more mAbs have been raised against the native form of this receptor than most other GPCRs, providing a useful set of tools to map its immunodominant structural regions. Here, we have used a high-throughput structure-function analysis strategy, which we refer to as "shotgun mutagenesis", to comprehensively map the critical residues, and in some cases the critical atoms, for these five epitopes of CCR5.To map mAb epitopes, we used an arrayed library of mutations covering nearly all the amino acids in the protein to identify amino acid changes that resulted in loss of mAb reactivity. This approach enabled each epitope to be rapidly mapped within a period of weeks. To create the mutant library, a parental CCR5 plasmid was first created, containing the full length (1059 bp) cDNA for wild type CCR5, flanked by a N-terminal HA epitope tag and a C-terminal V5 epitope tag. Cellular expression of the wild type tagged construct was confirmed by Western blot, immunofluorescence, and flow cytometry. Random mutations were next introduced into the parental CCR5 cDNA using a PCR-based method (Diversify PCR Random Mutagenesis kit, Clontech). Sequenced clones, most exhibiting one to two substitutions, were then selected from these random mutants to create a library with substitutions spanning the entire protein.The final library comprised 734 mutant CCR5 plasmids with substitutions in 346 of the 352 residues of CCR5 (>98% coverage). The average mutation rate per clone was 1.86 amino acids, and each amino acid position was substituted multiple times (an average of 3.95) across the entire library.We used this selective library of CCR5 mutants to map the epitopes of the anti-CCR5 mAbs CTC8, 45523, 45529, 45533 (R&D Systems), and 2D7 (Becton Dickinson). All five mAbs were originally derived, in three independent immunizations, by injecting mice with cells transiently overexpressing human CCR5. 4, 8 These mAbs are therefore representative of the murine immu...

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC 50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC 50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). (Journal of Biomolecular Screening 2008:424-429)