Marianna Max

Tas1r3 , encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac

The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7–9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.

Trp8, a transient receptor potential channel expressed in taste receptor cells

We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as α-gustducin, Gγ13, phospholipase C-β2 (PLC-β2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.

Gγ13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The α subunit of gustducin (α-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein γ subunit (Gγ13) that colocalized with α-gustducin in taste receptor cells. Of 19 α-gustducin/Gγ13-positive taste receptor cells profiled, all expressed the G protein β3 subunit (Gβ3); ~80% also expressed Gβ1. Gustducin heterotrimers (α-gustducin/Gβ1/Gγ13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Gγ13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via α-gustducins regulation of phosphodiesterase (PDE) and Gβγs activation of phospholipase C (PLC).

Lactisole Interacts with the Transmembrane Domains of Human T1R3 to Inhibit Sweet Taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisoles inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisoles action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3s seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.

Identification of the Cyclamate Interaction Site within the Transmembrane Domain of the Human Sweet Taste Receptor Subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.

The Heterodimeric Sweet Taste Receptor has Multiple Potential Ligand Binding Sites

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

G protein subunit Gγ13 is coexpressed with Gαo, Gβ3, and Gβ4 in retinal ON bipolar cells

We investigated the expression of Gγ13, a recently discovered G protein subunit, and a selection of Gβ subunits in retinal bipolar cells, by using a transgenic mouse strain in which green fluorescent protein is strongly expressed in a single type of cone bipolar cell. The cells have ON morphology, and patch‐clamp recordings in slices confirmed that they are of the physiological ON type. Immunohistochemistry showed that Gγ13 is expressed in rod bipolar cells and ON cone bipolar cells, where it is colocalized in the dendrites with Gαo. ON and OFF cone bipolar cells and rod bipolar cells were identified among dissociated cells by their green fluorescence and/or distinct morphology. Hybridization of single‐cell polymerase chain reaction products with cDNA probes for G protein subunits Gβ1 to 5 showed that Gβ3, Gβ4, and Gγ13 are coexpressed in ON bipolar cells but not present in OFF bipolar cells. Gβ1, 2, and 5 are expressed in partially overlapping subpopulations of cone bipolar cells. Gγ13 and Gβ3 and/or Gβ4, thus, seem selectively to participate in signal transduction by ON bipolar cells. J. Comp. Neurol. 455:1–10, 2003.

Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

G protein subunit G gamma 13 is coexpressed with G alpha o, G beta 3, and G beta 4 in retinal ON bipolar cells.

We investigated the expression of Gγ13, a recently discovered G protein subunit, and a selection of Gβ subunits in retinal bipolar cells, by using a transgenic mouse strain in which green fluorescent protein is strongly expressed in a single type of cone bipolar cell. The cells have ON morphology, and patch‐clamp recordings in slices confirmed that they are of the physiological ON type. Immunohistochemistry showed that Gγ13 is expressed in rod bipolar cells and ON cone bipolar cells, where it is colocalized in the dendrites with Gαo. ON and OFF cone bipolar cells and rod bipolar cells were identified among dissociated cells by their green fluorescence and/or distinct morphology. Hybridization of single‐cell polymerase chain reaction products with cDNA probes for G protein subunits Gβ1 to 5 showed that Gβ3, Gβ4, and Gγ13 are coexpressed in ON bipolar cells but not present in OFF bipolar cells. Gβ1, 2, and 5 are expressed in partially overlapping subpopulations of cone bipolar cells. Gγ13 and Gβ3 and/or Gβ4, thus, seem selectively to participate in signal transduction by ON bipolar cells. J. Comp. Neurol. 455:1–10, 2003.

Direct NMR Detection of the Binding of Functional Ligands to the Human Sweet Receptor, a Heterodimeric Family 3 GPCR

We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.