The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.

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. Lactisole's 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 lactisole's 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 T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.Taste is a primal sense that enables diverse organisms to identify and ingest sweet-tasting nutritious foods and to reject bitter-tasting environmental poisons (1). Taste perception can be categorized into five distinct qualities: salty, sour, bitter, umami (amino acid taste), and sweet (1). Salty and sour depend on the actions of ion channels. Bitter, umami, and sweet depend on G-protein-coupled receptors (GPCRs) 1 and coupled signaling pathways. Sweet taste in large part depends on a heterodimeric receptor comprised of T1R2ϩT1R3 (2-5).The T1R taste receptors (T1R1, T1R2, and T1R3) are most closely related to metabotropic glutamate receptors (mGluRs), Ca 2ϩ -sensing receptors (CaSRs), and some pheromone receptors (6 -10). All of these receptors are class-C GPCRs, with the large clam shell-shaped extracellular amino-terminal domain (ATD) characteristic of this family. Following the ATD is a cysteine-rich region that connects the ATD to the heptahelical transmembrane domain (TMD); following the TMD is a short intracellular carboxyl-terminal tail. The solved crystal structure of the ATD of mGluR1 identifies a "Venus flytrap module" (VFTM) involved in ligand binding (11). The canonical agonist glutamate binds within the VFTM in a cleft formed by the two lobes of this module to stabilize a closed active conformation of the mGluR1 ATD. In contrast, several positive and negative allosteric modulators of class-C GPCRs have been identified and shown to act via binding not within the VFTM but instead within the TMD (12-16).Over the past few decades, multiple models of the sweet receptor's hypothetical ligand binding site have been generated based on the structures of existing sweeteners but without direct knowledge of the nature of the sweet receptor itself. A consensus feature of these models is the presence of A-H-B groups, in which the AH group is a hydrogen donor and the B group is an electronegative center. These models have explanatory and predictive value for some, but not all sweeteners, suggesting th...

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.Obesity and diabetes have reached epidemic proportions in developed societies. Although in part this is because of a more sedentary lifestyle, our strong preference for sweet tasting foods and their abundance is a major factor. Replacing sugar with low-or non-caloric sweeteners may be of benefit. To design more effective sweeteners it is important to understand at the molecular level how the sweet taste receptor functions. It has been demonstrated that the combination of T1R2 ϩ T1R3 recognizes and responds to many sweet ligands, including sugars, small molecule artificial sweeteners, and protein sweeteners (1, 2).T1R2 and T1R3 are subclass 3 G-protein-coupled receptors (1-7). Other members of this subclass are metabotropic glutamate receptors (mGluRs), 1 calcium-sensing receptors, pheromone receptors, and other taste/olfactory receptors (T1R1, 5.24 odor receptor) (8). Each member of this family has a large extracellular amino-terminal domain (ATD) followed by a cysteine-rich linker domain and a seven-transmembrane-spanning helical region.The solved crystal structures of the ATD of homodimeric metabotropic glutamate type 1 receptor (mGluR1) show that the mGluR1 ligand-binding region consists of two amino-terminal protomers (9). Each protomer comprises LB1 and LB2 domains that form a clamshell-like structure with the ligandbinding domain lying between LB1 and LB2. The free-form I (open-open_R) is thought to be in the resting state, whereas the free-form II (closed-open_A) is thought to be the active state. Agonist binding stabilizes the active closed-open_A conformer and promotes a shift of equilibrium toward the active state. The role of the cysteine-rich region, which links the ATD to the transmembrane domain, is presently unknown.Based on sequence homology and predicted secondary structural similarity to mGluR1, it seems likely that the T1R2 ϩ T1R3 sweet receptor will also have open-open and open-closed forms and that small sweet compounds may stabilize the ac...

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...

Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues within these regions, that contribute to their ligand binding sites. Differences in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine accessibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of approximately 80 membrane-spanning residues that differ between the D2 and D4 receptors, only 20 were found to be accessible, and 6 of these 20 are conservative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mutations in a D4 receptor background. The combined substitution of four to six of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The mutated residues are in the second, third, and seventh membrane-spanning segments (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affinity for clozapine to D4-like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.