The Cysteine-rich Region of T1R3 Determines Responses to Intensely Sweet Proteins

Abstract

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...

Glucose transporters and ATP-gated K + (K ATP ) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells

“…KO mice deficient in either Tas1r2 or Tas1r3 display profound deficits in their responses to sweeteners (11,12). Heterologous expression of the human form of T1r2 + T1r3 generates receptor activity responsive to a broad array of sweet compounds, including monosaccharide and disaccharide sugars (e.g., glucose, fructose, sucrose), sugar alcohols, small-molecule noncaloric sweeteners (e.g., saccharin, cyclamate, sucralose, aspartame, neotame), and large-molecule noncaloric sweeteners (e.g., monellin, thaumatin, brazzein) (7,(13)(14)(15)(16)(17). Heterologous expression of the rodent form of T1r2 + T1r3 generates a similarly broad-acting receptor but without activity toward the "human-specific sweeteners" (cyclamate, aspartame, neotame, monellin, and thaumatin) recognized as sweet by humans and old world primates but not by rodents.…”

Positive allosteric modulators of the human sweet taste receptor enhance sweet taste

“…Every one of these three domains contains agonist binding sites, explaining sweet receptor activation by a vast repertoire of chemically distinct molecules. Sucrose and noncaloric sweeteners such as aspartame and neotame interact within the VFT of T1R2 (9,10), other noncaloric sweeteners such as cyclamate and neohesperidin dihydrochalcone (NHDC) interact within the TMD of T1R3 (10-12), S819, a synthetic sweet agonist, interacts with the TMD of T1R2 (10), and the sweet-tasting protein brazzein requires the cysteine-rich domain of human T1R3 to activate the receptor (13).…”

Structure and ligand recognition of class C GPCRs

“…In support of this hypothesis, Jiang et al identified 10 residues in the CRD of human T1R3 and the hinge region of T1R2 that play an important role in the effect of sweet proteins, such as brazzein [45] .…”

“…Aside from the orthosteric sites, there are at least eight allosteric sites that have been identified in taste receptors: the EDAM sites for IMP in T1R1-VFT [31] and for SE-2/SE-3 in T1R2-VFT [67] ; the allosteric agonist sites for sweet proteins in T1R3-CRD [45] , cyclamate in sweet receptor T1R3 7TM [70] , S807 in T1R1 7TM [31] and S819 in T1R2-7TM [31] ; the PAM site for cyclamate in the umami receptor T1R3-7TM [70] ; and the NAM site for lactisole in both the sweet and umami receptor T1R3-7TM [71] . These sites might represent potential targets for health-related products or drugs to treat diseases, such as hypertension or diabetes.…”

“…Based on this structure, the CRD forms an independent domain with a length of 40 Å, which physically separates the VFT and the 7TM. The CRD plays an important role in receptor activation of the mGlu receptors, CaS receptors and sweet taste receptors with subunit T1R3 [44,45] . In mGlulike receptors, a conserved disulfide bridge between the VFT and the CRD is required for the allosteric interaction between Figure 1.…”