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

Abstract

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.Taste is a primal sense that is essential for humans and other organisms to detect the nutritive quality of a potential food source while avoiding environmental toxins (1-3). Taste perception can be categorized into five distinct qualities: sweet, bitter, salty, sour, and umami (amino acid taste) (1-3). Sweet, bitter, and umami tastes are mediated in large part by G-protein-coupled receptors (GPCRs) 2 and their linked signaling pathways. Sour and salty tastes are thought to be mediated by direct effects on specialized ion channels (1-3).The detection of sweet taste is mediated by two GPCR subunits, T1R2 and T1R3, which are specifically expressed in taste receptor cells (4 -13). When expressed in vitro, T1R2 ϩ T1R3 heterodimer responds to a broad spectrum of chemically diverse sweeteners, ranging from natural sugars (sucrose, fructose, glucose, and maltose), sweet amino acids (D-tryptophan, D-phenylalanine, and D-serine), and artificial sweeteners (acesulfame-K, aspartame, cyclamate, saccharin, and sucralose) to sweet tasting proteins (monellin, thaumatin, and brazzein) (10, 11, 14 -16). To date, all sweeteners tested in vitro activate the T1R2 ϩ T1R3 heterodimer. In vivo, genetic ablation in mice of T1R2, T1R3, or both either reduces or eliminates responses to sweet compounds (12, 13). Thus, the T1R2 ϩ T1R3 heterodimer is broadly tuned and functions as the principal or sole sweet taste receptor in vivo.T1R2 and T1R3 are class C GPCR subunits (4 -9), members of a group that also includes T1R1 (a component of the umami taste receptor), metabotropic glutamate receptors, the calcium-sensing receptor, ␥-aminobutyric acid type B receptors, and vomeronasal receptors. Like most other GPCRs, each class C receptor has a heptahelic...

T1R3 and gustducin in gut sense sugars to regulate expression of Na + -glucose cotransporter 1

“…The concentrations of GLP-1 and GIP (total) (active form) in the culture medium were determined by using GLP-1 and GIP ELISA kits (Linco Research, St. Charles, MO). For the Ca 2ϩ imaging experiments GLUTag cells were transiently transfected by using Lipofectamine 2000 (Invitrogen) with the following plasmids: G␣16/gust44 [a plasmid encoding a G␣16-G␣ gust chimeric G protein ␣-subunit that couples to sweet taste receptors (29)], YC3.60 (a ratiometric fluorescent indicator of free Ca 2ϩ ) (30), and REEP-EI (a plasmid encoding a receptor-enhancing protein that promotes function of olfactory (30) and gustatory receptors (E.I. and R.F.M., unpublished data).…”

Signal transduction and information processing in mammalian taste buds

“…This contrasts with the N-terminal, extracellular binding domains for sugars and sweet proteins. Moreover, the cyclamate-binding pocket also appears to be where certain antagonists such as lactisole interact with the sweet receptor [68,69]. These multiple binding pockets for different sweettasting compounds are illustrated in Fig.…”

“…They constructed chimeras of the mouse and human T1R1+T1R3 and located a cysteine-rich region in T1R3 near the base of the N terminus that appears to be a binding pocket for brazzein and other sweet-tasting proteins. Using chimeric constructs of human and rat T1R1+T1R3 [68], or human and mouse T1R2+T1R3 followed by site-directed mutagenesis [69], researchers have narrowed down the cyclamate-binding pocket to portions of transmembrane segments 3, 5, and 6 of T1R3 and extracellular loop 2, suggesting that this pocket is mainly burrowed into the membrane. This contrasts with the N-terminal, extracellular binding domains for sugars and sweet proteins.…”

Binding of glutamate to the umami receptor

“…Homology modeling of the closely related sweet taste receptors (T1R2 + T1R3) has facilitated an understanding of the interactions of sweeteners with their receptor [7][8][9][10][11][12].…”