Sweet Taste Receptor Research Articles

Exendin-4 inhibits small intestinal glucose sensing and absorption through repression of T1R2/T1R3 sweet taste receptor signalling in streptozotocin diabetic mice

The small intestine, which is the area where sugars are absorbed, should be considered in the approaches developed for the treatment of diabetes. However, studies on small intestine damage in diabetic individuals, and the effects of current treatments on the small intestine are very limited. This is the first study to investigate the effects of exendin-4, a GLP-1 receptor agonist, on small intestine injury in diabetic mice. BALB/c male mice were divided into 4 groups for this study. The first group was given citrate buffer, the second group was given exendin-4, the third group was given streptozotocin (STZ), and the fourth group was given both exendin-4, and STZ. As the results, we determined a decrease in the edema and deterioration in the integrity of the villi, disruption in continuity of the brush border, fibrosis and enterocyte apoptosis, while the TNFα level and crypt cell proliferation were increased in the small intestinal tissue of exendin-4 treated STZ diabetic mice. Furthermore, the levels of duodenal tissue glucose, SGLT1, and GLUT2 were decreased, whereas there was an increase in GIP level in diabetic mice administered with exendin-4. Moreover, we determined that the sweet taste receptors T1R2/T1R3, downstream molecules PLCβ2, α-gustducin and associated secondary messengers IP3, cAMP, which were increased in the duodenal tissue of STZ-diabetic mice, decreased with exendin-4 administration. These findings were evaluated as that exendin-4 reduces glucose absorption by suppressing the T1R2/T1R3 sweet taste signal perception pathway in duodenum of STZ diabetic mice.

Oral Microbiota-Host Interaction Mediated by Taste Receptors.

Taste receptors, originally identified in taste buds, function as the periphery receptors for taste stimuli and play an important role in food choice. Cohort studies have revealed that single nucleotide polymorphisms of taste receptors such as T1R1, T1R2, T2R38 are associated with susceptibility to oral diseases like dental caries. Recent studies have demonstrated the wide expression of taste receptors in various tissues, including intestinal epithelia, respiratory tract, and gingiva, with an emerging role of participating in the interaction between mucosa surface and microorganisms via monitoring a wide range of metabolites. On the one hand, individuals with different oral microbiomes exhibited varied taste sensitivity, suggesting a potential impact of the oral microbiota composition on taste receptor function. On the other hand, animal studies and in vitro studies have uncovered that a variety of oral cells expressing taste receptors such as gingival solitary chemosensory cells, gingival epithelial cells (GECs), and gingival fibroblasts can detect bacterial signals through bitter taste receptors to trigger host innate immune responses, thus regulating oral microbial homeostasis. This review focuses on how taste receptors, particularly bitter and sweet taste receptors, mediate the oral microbiota-host interaction as well as impact the occurrence and development of oral diseases. Further studies delineating the role of taste receptors in mediating oral microbiota-host interaction will advance our knowledge in oral ecological homeostasis establishment, providing a novel paradigm and treatment target for the better management of dental infectious diseases.

Genome assembly of the numbat (Myrmecobius fasciatus), the only termitivorous marsupial.

The numbat (Myrmecobius fasciatus) is an endangered Australian marsupial, and the last surviving member of the Myrmecobiidae family. The numbat regularly undergoes torpor and is unique amongst marsupials as it is the only diurnal and termitivorous species. Here we sequenced the first draft genome of the numbat using 10× Genomics Chromium linked-read technology, resulting in a 3.42 Gbp genome with a scaffold N50 of 223 kbp. A global transcriptome from liver, lung and tongue was also generated to aid genome annotation, identifying 21,465 protein-coding genes. To investigate adaptation to the numbat's termitivorous diet and arid/semi-arid range, we interrogated the most highly expressed transcripts within the tongue and manually annotated taste, vomeronasal and aquaporin gene families. Antimicrobial proteins and proteins involved in digestion were highly expressed in the tongue, alongside umami taste receptors. However, sweet taste receptors were not expressed in this tissue, which combined with the putative contraction of the bitter taste receptor gene repertoire in the numbat genome, may indicate a potential evolutionary adaptation to their specialised termitivorous diet. Vomeronasal and aquaporin gene repertoires were similar to other marsupials. The draft numbat genome is a valuable tool for conservation and can be applied to population genetics/genomics studies and to investigate the unique biology of this interesting species.

Conductor of the Astrocyte-Neuron Metabolic Orchestra

Disorders such as diabetes mellitus, obesity, Parkinson’s, and Alzheimer’s diseases are characterized by central metabolic dysfunctions and pose an enormous economic burden to public health. Annually, several millions of new cases and deaths are reported worldwide, thus substantiating the need to search for new frontiers in combating the growing prevalence and mortality of these diseases. Over the past few years, scientific evidence has consistently shown that the functional sweet taste receptor, T1R2+T1R3 heterodimer, serves to direct (conduct) peripheral glucose metabolism. Recent data have revealed that this heterodimer can also act as a central glucosensor that conducts cerebral glucose metabolism. Emerging reports have confirmed the central role of this receptor as a driver of glucose metabolism in neurons and astrocytes. In this paper, “metabolic orchestra” is used to depict the organizational complexity of the plasma membrane receptor-network involved in coordinating glucose transport and metabolism in the astrocyte-neuron circuitry. In light of recent works, suggesting that the taste receptor is a crucial central glucosensor and master coordinator of glucose metabolism, here, the T1R2+T1R3 heterodimer is referred to as the metabolic conductor of the astrocyte-neuron circuitry, responsible for a highly coordinatedsignaling of glucose molecules and multi-directional cross-talk with other plasma membrane receptors. This concept represents a shift on the astrocyte-neuron metabolic machinery from the GLUT-2 mediated entry of glucose to a more coordinated one, involving multiple players at the plasma membrane. Research focusing on the treatments of brain disorders involving glucose metabolic dysfunctions is also discussed.

A Dynamic Mass Redistribution Assay for the Human Sweet Taste Receptor Uncovers G-Protein Dependent Biased Ligands.

The sweet taste receptor is rather unique, recognizing a diverse repertoire of natural or synthetic ligands, with a surprisingly large structural diversity, and with potencies stretching over more than six orders of magnitude. Yet, it is not clear if different cell-based assays can faithfully report the relative potencies and efficacies of these molecules. Indeed, up to now, sweet taste receptor agonists have been almost exclusively characterized using cell-based assays developed with overexpressed and promiscuous G proteins. This non-physiological coupling has allowed the quantification of receptor activity via phospholipase C activation and calcium mobilization measurements in heterologous cells on a FLIPR system, for example. Here, we developed a novel assay for the human sweet taste receptor where endogenous G proteins and signaling pathways are recruited by the activated receptor. The effects of several sweet taste receptor agonists and other types of modulators were recorded by measuring changes in dynamic mass redistribution (DMR) using an Epic® reader. Potency and efficacy values obtained in the DMR assay were compared to those results obtained with the classical FLIPR assay. Results demonstrate that for some ligands, the two assay systems provide similar information. However, a clear bias for the FLIPR assay was observed for one third of the agonists evaluated, suggesting that the use of non-physiological coupling may influence the potency and efficacy of sweet taste receptor ligands. Replacing the promiscuous G protein with a chimeric G protein containing the C-terminal tail 25 residues of the physiologically relevant G protein subunit Gαgustducin reduced or abrogated bias.

Tuft Cells and Their Role in Intestinal Diseases.

The interests in intestinal epithelial tuft cells, their basic physiology, involvement in immune responses and relevance for gut diseases, have increased dramatically over the last fifteen years. A key discovery in 2016 of their close connection to helminthic and protozoan infection has further spurred the exploration of these rare chemosensory epithelial cells. Although very sparse in number, tuft cells are now known as important sentinels in the gastrointestinal tract as they monitor intestinal content using succinate as well as sweet and bitter taste receptors. Upon stimulation, tuft cells secrete a broad palette of effector molecules, including interleukin-25, prostaglandin E2 and D2, cysteinyl leukotriene C4, acetylcholine, thymic stromal lymphopoietin, and β-endorphins, some of which with immunomodulatory functions. Tuft cells have proven indispensable in anti-helminthic and anti-protozoan immunity. Most studies on tuft cells are based on murine experiments using double cortin-like kinase 1 (DCLK1) as a marker, while human intestinal tuft cells can be identified by their expression of the cyclooxygenase-1 enzyme. So far, only few studies have examined tuft cells in humans and their relation to gut disease. Here, we present an updated view on intestinal epithelial tuft cells, their physiology, immunological hub function, and their involvement in human disease. We close with a discussion on how tuft cells may have potential therapeutic value in a clinical context.

Long term exposure to sucralose alters the sense of sweet tastants in an intestinal enteroendocrine cell model

Artificial sweeteners can bind to the sweet taste receptors T1R2/T1R3 on the cell membrane of taste receptor cells, including intestinal enteroendocrine cells, and induce an increase in intracellular Ca2+ concentration ([Ca2+]i). Acute adaptation or a reduction in receptor signalling was observed after prolonged exposure to a taste stimulus in an animal model. However, at the cell level, it remains unclear whether the expression level of T1R2/T1R3 will be changed by pre-exposure to sweetener molecules, and whether the corresponding change in [Ca2+]i induced by the altered expression of sweet taste receptors varies. In the present work, western blotting and qPCR analysis showed that the expression level of T1R2/T1R3 in NCI-H716 cells was upregulated by sucralose stimulation. In the sucralose-treated cells, the accumulation of intracellular Ca2+ in the sweetener pre-treated cells was significantly enhanced as compared to that in cells without sweetener pre-treatment, although the sweet response differed between acesulfame K and sucralose with the same sweetness. Collectively, the increased expression of T1Rs in sweetener pre-incubated cells enhanced the sweet taste signal response, which implies that long-term administration of artificial sweeteners in beverages or foods could alter the sensitivity of taste receptor cells to sweetener molecules.

Sweet and umami receptors are expressed in the heart and regulate contractility

The tongue can distinguish between five different tastes via the taste receptors, which are G-protein coupled receptors (GPCRs). There are two classes of taste receptors, the TAS1 (T1) and TAS2 (T2) families, and the T1R1-T1R3 dimer senses the umami taste and T1R2-T1R3 senses the sweet taste. Recently, the taste receptors have also been found in the brain, lungs, intestine and pancreas, where they sense changes in the nutrient environment and respond through GPCR signalling. Given the importance of glucose and amino acid metabolism in the heart, we hypothesized that the sweet and umami taste receptors have an important function in the heart.

Role of Proximal Intestinal Glucose Sensing and Metabolism in the Blood Glucose Control in Type 2 Diabetic Rats After Duodenal Jejunal Bypass Surgery.

Although gastric surgery can significantly improve blood glucose homeostasis in type 2 diabetes mellitus (T2DM), its mechanism remains unclear. This study evaluated the role of intestinal glucose sensing, glucose transport, and metabolism in the alimentary limb (A limb) of T2DM rats after duodenal jejunal bypass (DJB) surgery. A T2DM rat model was induced via a high-glucose high-fat diet and low-dose streptozotocin injection. The diabetic rats were divided into two groups: the DJB surgery (T2DM-DJB) group and the sham surgery (T2DM-Sham) group. Wistar rats were used as wild-type control (Control). Small animal PET was used to assess the change in glucose metabolic status in the intestine. The intestinal villi height and the number of EECs after DJB were evaluated. The expressions of sweet taste receptors (T1R2/T1R3), glucose transporters (SGLT1/GLUT2), and key enzymes involved in glucose metabolism (HK2, PFK2, PKM2, G6Pase, and PCK1) in the A limb after DJB was detected by Western blot and qRT-PCR. Small animal PET analysis showed the intestinal glucose metabolism increased significantly 6weeks after DJB surgery. The intestinal villi height and the number of EECs in the A limb 6weeks after surgery increased significantly in T2DM-DJB rats comparing to T2DM-Sham rats. The mRNA and protein expression of T1R1/T1R3 and SGLT1/GLUT2 were downregulated in DJB-T2DM rats, while enzymes involved in glucose metabolism was upregulated in the A limb in T2DM-DJB rats. Proximal intestinal glucose sensing and metabolism play an important role in blood glucose homeostasis by DJB.

The preference for sugar over sweetener depends on a gut sensor cell

Guided by gut sensory cues, humans and animals prefer nutritive sugars over non-caloric sweeteners, but how the gut steers such preferences remains unknown. In the intestine, neuropod cells synapse with vagal neurons to convey sugar stimuli to the brain within seconds. Here, we found that cholecystokinin (CCK)-labeled duodenal neuropod cells differentiate and transduce luminal stimuli from sweeteners and sugars to the vagus nerve using sweet taste receptors and sodium glucose transporters. The two stimulus types elicited distinct neural pathways: while sweetener stimulated purinergic neurotransmission, sugar stimulated glutamatergic neurotransmission. To probe the contribution of these cells to behavior, we developed optogenetics for the gut lumen by engineering a flexible fiberoptic. We showed that preference for sugar over sweetener in mice depends on neuropod cell glutamatergic signaling. By swiftly discerning the precise identity of nutrient stimuli, gut neuropod cells serve as the entry point to guide nutritive choices.

Saccharin Stimulates Insulin Secretion Dependent on Sweet Taste Receptor-Induced Activation of PLC Signaling Axis.

Background: Saccharin is a common artificial sweetener and a bona fide ligand for sweet taste receptors (STR). STR can regulate insulin secretion in beta cells, so we investigated whether saccharin can stimulate insulin secretion dependent on STR and the activation of phospholipase C (PLC) signaling. Methods: We performed in vivo and in vitro approaches in mice and cells with loss-of-function of STR signaling and specifically assessed the involvement of a PLC signaling cascade using real-time biosensors and calcium imaging. Results: We found that the ingestion of a physiological amount of saccharin can potentiate insulin secretion dependent on STR. Similar to natural sweeteners, saccharin triggers the activation of the PLC signaling cascade, leading to calcium influx and the vesicular exocytosis of insulin. The effects of saccharin also partially require transient receptor potential cation channel M5 (TRPM5) activity. Conclusions: Saccharin ingestion may transiently potentiate insulin secretion through the activation of the canonical STR signaling pathway. These physiological effects provide a framework for understanding the potential health impact of saccharin use and the contribution of STR in peripheral tissues.

Ultrasensitive Bioelectronic Tongue Based on the Venus Flytrap Domain of a Human Sweet Taste Receptor.

Sweet taste is an important factor that regulates calorie intake and contributes to food preferences in humans and animals. Therefore, the evaluation of sweet substances is essential for various fields such as healthcare, food, and pharmaceutical industries. Sweet tastants are detected by sweet taste receptors which are class C G-protein-coupled receptors. T1R2 venus flytrap (VFT) of the sweet taste receptor is known as a primary ligand-binding domain for sweet tastants. In this study, we developed an ultrasensitive artificial sweet taste bioelectronic tongue based on the T1R2 VFT of a human sweet taste receptor. Here, the T1R2 VFT of a human sweet taste receptor was successfully overexpressed in a bacterial expression system. A T1R2 VFT-immobilized carbon nanotube field-effect transistor with floating electrodes was exploited as an artificial sweet taste sensory system. Significantly, our T1R2 VFT-functionalized bioelectronic tongue could be used to detect solutions of sweet tastants down to 0.1 fM and selectively discriminate sweet substances from other taste substances. Furthermore, our device could be used to monitor the response of the T1R2 VFT domain of a sweet taste receptor to sweet substances in real food environments such as apple juice and chamomile herb tea. Moreover, our device was used to evaluate the inhibition and enhancement effects on sweet taste receptors by zinc ions and chamomile tea, respectively. In addition, our device demonstrated long-term storability and reusability. In this respect, our sweet taste bioelectronic tongue could be a promising tool for various basic research and industrial applications.

Taste receptors in chronic rhinosinusitus, what is the evidence? A systematic review.

Bitter and sweet taste receptors (T2Rs and T1Rs), respectively, are involved in the innate immune response of the sinonasal cavity and associated with chronic rhinosinusitis (CRS). Growing evidence suggests extraoral TRs as relevant biomarkers, but the current understanding is incomplete. This systematic review synthesizes current evidence of extraoral taste receptors in CRS. PubMed, Embase, Cochrane, Web of Science, and Scopus were reviewed in accordance with Preferred Reporting Items for Systemic Reviews and Meta-Analyses guidelines and included studies of genotypic and phenotypic T2R/T1R status in CRS patients. Twenty-two studies with 3845 patients were included. Seventeen studies evaluated genotype and 10 evaluated taste phenotypes. Four of 6 studies examining the haplotype distribution of the T2R, TAS2R38, demonstrated increased AVI/AVI haplotype ("nontaster") frequency in CRS. Meanwhile, 2 studies demonstrated decreased bitter sensitivity in CRS with nasal polyposis (CRSwNP), whereas 3 other studies reported decreased bitter sensitivity only in CRS without nasal polyposis (CRSsNP). Findings regarding sweet sensitivity were mixed. Three studies with cystic fibrosis patients (n=1393) were included. Studies investigating the association between clinical outcomes and TAS2R38 alleles were limited, but the nonfunctional combination of AVI/AVI was associated with increased utilization of sinus surgery and, in CRSsNP patients, with poorer improvement of symptoms postoperatively. Both genotypic and phenotypic assessments of T2Rs suggest a potential association with CRS, particularly CRSsNP. However, limited evidence and mixed conclusions cloud the role of T2Rs in CRS. Future investigations should aim to increase diverse populations, broaden institutional diversity, examine T1Rs, and utilize uniform assessments.

Sweet taste perception in mice is blunted by PTBP1-regulated skipping of Tas1r2 exon 4.

Taste perception, initiated by activation of taste receptors in taste bud cells, is crucial for regulating nutrient intake. Genetic polymorphisms in taste receptor genes cannot fully explain the wide individual variations of taste sensitivity. Alternative splicing (AS) is a ubiquitous posttranscriptional mode of gene regulation that enriches the functional diversity of proteins. Here, we report the identification of a novel splicing variant of sweet taste receptor gene Tas1r2 (Tas1r2_∆e4) in mouse taste buds and the mechanism by which it diminishes sweet taste responses in vitro and in vivo. Skipping of Tas1r2 exon 4 in Tas1r2_∆e4 led to loss of amino acids in the extracellular Venus flytrap domain, and the truncated isoform reduced the response of sweet taste receptors (STRs) to all sweet compounds tested by generating nonfunctional T1R2/T1R3 STR heterodimers. The splicing factor PTBP1 (polypyrimidine tract-binding protein 1) promoted Tas1r2_∆e4 generation through binding to a polypyrimidine-rich splicing silencer in Tas1r2 exon 4, thus decreasing STR function and sweet taste perception in mice. Taken together, these data reveal the existence of a regulated AS event in Tas1r2 expression and its effect on sweet taste perception, providing a novel mechanism for modulating taste sensitivity at the posttranscriptional level.

T1R3 homomeric sweet taste receptor negatively regulates insulin-induced glucose transport through Gαs-mediated microtubules disassembly in 3T3-L1 adipocytes.

T1R3 is a class C G protein-coupled receptor family member that forms heterodimeric umami and sweet taste receptors with T1R1 and T1R2, respectively, in the taste cells of taste buds. T1R3 is expressed in 3T3-L1 cells in homomeric form and negatively regulates adipogenesis in a Gαs-dependent but cAMP-independent manner. Although T1R3 expression is markedly upregulated during adipogenesis, its physiological role in mature adipocytes remains obscure. Here, we show that stimulation of T1R3 with sucralose or saccharin induces microtubule disassembly in differentiated 3T3-L1 adipocytes. The effect was reproduced by treatment with cholera toxin or isoproterenol but not with forskolin. Treatment with sucralose or saccharin for 3 h inhibited insulin-stimulated glucose uptake by 32% and 45% in differentiated adipocytes, respectively, similar to the inhibitory effect of nocodazole (by 33%). Isoproterenol treatment inhibited insulin-stimulated glucose transport by 26%, whereas sucralose did not affect the intrinsic activity of the glucose transporter, indicating that it inhibited insulin-induced GLUT4 translocation to the plasma membrane. Immunostaining analysis showed that insulin-stimulated GLUT4 accumulation on the plasma membrane was abrogated in sucralose-treated cells, in association with depolymerization of microtubules. Sucralose-mediated inhibition of GLUT4 translocation was reversed by the overexpression of dominant-negative Gαs (Gαs-G226A) or knockdown of Gαs. Additionally, membrane fractionation analysis showed that sucralose treatment reduced GLUT4 levels in the plasma membrane fraction from insulin-stimulated adipocytes. We have identified a novel non-gustatory role for homomeric T1R3 in adipocytes, and activation of the T1R3 receptor negatively regulates insulin action of glucose transport via Gαs-dependent microtubule disassembly.

Taste perception of cyclic oligosaccharides: α, β, and γ cyclodextrins.

Oligosaccharides, a subclass of complex carbohydrates, occur both naturally in foods and as a result of oral starch digestion. We have previously shown that humans can taste maltooligosaccharides (MOS) and that their detection is independent of the canonical sweet taste receptor. While MOSs most commonly occur in a linear form, they can also exist in cyclic structures, referred to as cyclodextrins (CD). The aim of this study was to investigate how the structure of the MOS backbone (i.e. cyclic form) and the size (i.e. degree of polymerization; DP) affect their taste perception. We tested taste detection of cyclodextrins with DP of 6, 7, and 8 (i.e. α-, β-, and γ-CD, respectively) in the presence and absence of lactisole, a sweet receptor antagonist. We found that subjects could detect the taste of cyclodextrins in aqueous solutions at a significant level (P < 0.05), but were not able to detect them in the presence of lactisole (P > 0.05). These findings suggest that the cyclodextrins, unlike their linear analogs, are ligands of the human sweet taste receptor, hT1R2/hT1R3. Study findings are discussed in terms of how chemical structures may contribute to tastes of saccharides.

Multiple interaction modes between saccharin and sweet taste receptors determine a species-dependent response to saccharin.

Saccharin is a commonly used artificial sweetener that exhibits both sweetening and sweet inhibition activities. The species‐dependent response towards saccharin and the interaction between saccharin and the sweet taste receptor T1R2/T1R3 remain elusive. In this study we used mismatched chimeras of T1R2 and T1R3 and calcium mobilization functional analysis to reveal a detailed species‐dependent response towards saccharin of human, squirrel monkey, and mouse sweet taste receptors. Our findings, combined with previous results by others, suggest multiple and complex interaction modes between saccharin and the sweet taste receptor, which are helpful guidelines for effective modulation of the sweet taste by sweetener/sweet inhibitors.

Investigating mechanism of sweetness intensity differences through dynamic analysis of sweetener–T1R2–membrane systems

Knowing the mechanism of action of sweet taste receptors is important for the design of new, healthy sweeteners. However, little is known about the structures and recognition mechanisms of these receptors. 28 sweeteners were assessed by molecular docking, and 8 typical sweeteners were chosen to construct sweetener–T1R2–membrane systems to analyze interactions between receptor and sweeteners. Natural sweeteners with low-intensity sweetness, such as fructose and xylitol, were released from the Venus flytrap domain at ∼30 ns, with displacements greater than 50 Å. In contrast, artificial neotame and advantame bound stably to the receptor, within 5 Å. Van der Waals interactions were significant in high-intensity sweetener systems, imparting an energy difference of over 15 kcal/mol between neotame (artificial sweetener) and fructose (natural). These results provide a deeper understanding of the mechanisms of sweetener function and offer a new direction for the design of sweeteners.

Evolutionary insights into umami, sweet, and bitter taste receptors in amphibians.

Umami and sweet sensations provide animals with important dietary information for detecting and consuming nutrients, whereas bitter sensation helps animals avoid potentially toxic or harmful substances. Enormous progress has been made toward animal sweet/umami taste receptor (Tas1r) and bitter taste receptor (Tas2r). However, information about amphibians is mainly scarce. This study attempted to delineate the repertoire of Tas1r/Tas2r genes by searching for currently available genome sequences in 14 amphibian species. This study identified 16 Tas1r1, 9 Tas1r2, and 9 Tas1r3 genes to be intact and another 17 Tas1r genes to be pseudogenes or absent in the 14 amphibians. According to the functional prediction of Tas1r genes, two species have lost sweet sensation and seven species have lost both umami and sweet sensations. Anurans possessed a large number of intact Tas2rs, ranging from 39 to 178. In contrast, caecilians possessed a contractive bitter taste repertoire, ranging from 4 to 19. Phylogenetic and reconciling analysis revealed that the repertoire of amphibian Tas1rs and Tas2rs was shaped by massive gene duplications and losses. No correlation was found between feeding preferences and the evolution of Tas1rs in amphibians. However, the expansion of Tas2rs may help amphibians adapt to both aquatic and terrestrial habitats. Bitter detection may have played an important role in the evolutionary adaptation of vertebrates in the transition from water to land.

Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line

Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor–ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.