Sweetness and Sweeteners - American Chemical Society

Chapter 4 T1R2, T1R3, and the Detection of Sweet Stimuli

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 2, 2015 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch004

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Stephan Vigues , Jeanette R. Hobbs , Yiling Nie , Graeme L. Conn , and Steven D. Munger 1*,

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Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 20 Penn Street, Baltimore, M D 21201 Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom 2

G protein-coupled receptors (GPCRs) play a central role in the detection of chemosensory cues, including sweet-tasting stimuli. To fully understand the basis of stimulus sensitivity and selectivity in chemosensory systems, it is essential to characterize the structural basis of receptor-ligand interactions. Efforts to express chemosensory receptors at levels suitable for detailed structure-function studies have met with limited success. We have developed a novel strategy for expressing and purifying functional domains of T1R taste receptors. Using these purified proteins in concert with spectroscopic analyses, we determined that each of the two subunits of the heteromeric T1R2:T1R3 sweet taste receptor binds sugar stimuli, though with different affinities and distinct conformational changes. Furthermore, a T1R3 variant associated with reduced sweet taste sensitivity in mice exhibits reduced affinities for sugars. These results provide fundamental new insights into the function of the sweet taste receptor and an important new strategy for studying the receptor basis of taste.

© 2008 American Chemical Society

In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Introduction Taste stimuli elicit five basic perceptual qualities in humans: sweet, bitter, sour, salty, and umami (IS).The ability to detect and discriminate taste stimuli is essential for health and survival, as taste stimuli convey important information about the nutritional value and quality of food. Mammals use a small number of G protein-coupled receptors (GPCRs) to detect sweet-, umami- and bitter-tasting stimuli (4). A l l classes of sweet-tasting stimuli, including natural sugars (e.g., sucrose, glucose), synthetic sweeteners (e.g., sucralose, saccharin, aspartame, cyclamate), sweet-tasting amino acids (e.g., D-phenylalanine, D-tryptophan) and sweet proteins (e.g., monellin, thaumatin, brazzein) activate a heteromeric G P C R comprised of the T1R2 and T1R3 subunits (5,6). T1R3 also combines with the T1R1 receptor to form an umami taste receptor sensitive to some L-amino acids, including L-glutamate (5,7). T1R2:T1R3 sweet taste receptors are expressed in a unique subset of taste receptor cells within taste buds of the tongue and palate. T1R receptors are Class C GPCRs, a group that includes metabotropic glutamate receptors (mGluRs) and γ-aminobutyric acid type Β receptors ( G A B A R s ) (8). This class of G P C R is distinguished by a long extracellular N terminal domain (NTD) containing a venus-flytrap module ( V F T M ) motif. The V F T M of mGluRs and G A B A R s contains the orthosteric ligand binding site for these receptors (8,9). However, it remained unclear if the same were true for the TIRs. Modeling studies of the T1R2 and T1R3 NTDs, based on the crystal structures of the mGluRl N T D , suggested that small molecule sweeteners could bind to both subunits (10). This prediction was consistent with in vitro activity assays and in vivo behavioral experiments indicating that homomeric T1R3 receptors can function as a low efficacy sweet receptor, though the heteromeric T1R2:T1R3 receptor is required for full activity (11). Several elegant studies using a combination of human-rodent T1R2 and T1R3 chimeras, along with stimuli that are sweet to humans but not preferred by rodents, added another layer of complexity (12-14): these studies provide strong evidence that the T1R2:T1R3 receptor contains multiple allosteric binding sites. For example, the T1R2:T1R3 receptor requires the N T D of human T1R2 to respond to the dipeptide sweeteners aspartame and neotame (14), the transmembrane domain of human T1R3 to respond to cyclamate (12,14), and the cysteine-rich linker region of human T1R3 to respond to brazzein (13). A sweet taste receptor with multiple binding sites provides a parsimonious explanation for the broad responsiveness of the receptor to such varied stimuli (15). However, none of these studies could directly address whether small sweet stimuli, including natural sugars that are preferred by both humans and rodents, bind to the NTDs of T1R2 and/or T1R3. Attempts to answer this question have been hampered by two technical limitations. First, mammalian chemosensory receptors, including the T1R taste B

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67 receptors, have been difficult to obtain in quantities suitable for biochemical or structural studies. Since T1R receptors are expressed in few taste receptor cells and at very low levels, isolation of a single chemosensory receptor type from gustatory tissues is not feasible. TIRs also express poorly in heterologous expression systems, possibly due to the absence of a critical chaperone or coreceptor (4,16,17). Therefore, optimization of in vitro expression and purification protocols for obtaining large quantities of functional T1R proteins or protein domains is required. Second, the low potency of most sweeteners, including natural sugars, suggests a low binding affinity that makes conventional binding assays problematic. Therefore, new approaches are needed that can quantify binding to TIRs and can decouple this binding from subsequent receptor activation events such as intra- and intersubunit interactions and G protein activation. Here we discuss a new strategy for the expression, purification and characterization of T1R ligand binding domains and its application in dissecting the roles of T1R2 and T1R3 in the recognition of sweet stimuli (78,19).

Expression and Purification of the N-terminal Ligand Binding Domains of T1R2 and T1R3 We used two strategies to express and purify the NTDs of the C57BL/6J variants of mouse T1R2 and T1R3 (18,19). In the first, the N T D of T1R3 (lacking a putative signal sequence and the cysteine-rich domain (20); Figure 1) was cloned into the IMPACT™ expression vector p T X B l (New England Biolabs, Ipswich, M A ) and expressed in BL21-CodonPlus(DE3)-RIL E. coli (Stratagene, L a Jolla, C A ) after induction with isopropyl-P-Dthioglycopyranoside (IPTG). Expression from this vector results in the production of a T1R3 N T D fusion protein containing an intein-chitin binding domain (CBD) affinity tag at the C-terminus. After sonication, the fusion protein was purified by chitin affinity chromatography. Cleavage of the T1R3NTD protein from the C B D was induced by reduction with dithiothreitol, and the purified receptor protein eluted in a highly purified form. The second strategy entailed expressing either the T1R2 or T1R3 N T D as a C-terminal fusion to the E. coli maltose binding protein (MBP). The fusion construct was built in the pET21a expression vector (New England Biolabs) and expressed in B L 2 1 CodonPlus(DE3)-RIL E. coli as above. The MBP-tagged proteins were purified by amylose affinity chromatography followed by anion exchange chromatography, and the M B P fusions left uncleaved to enhance stability of the T1R NTDs. In each case, we obtained highly purified T1R N T D proteins that were stable in solution.



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Figure 1. TIRs have seven transmembrane helices linked to a large extracellular N-terminal domain NTD (black) by a short cysteine-rich linker.

T1R2 and T1R3 N-Terminal Domains Bind Sugars To quantify ligand binding to T1R NTDs, we used steady-state fluorescence spectroscopy to monitor ligand-dependent changes in steady-state intrinsic tryptophan fluorescence in the receptor proteins (18,19). We measured the interaction of two natural sugars (glucose and sucrose) and one synthetic sweetener (the chlorinated sugar sucralose) with MBP-T1R2NTD, M B P T1R3NTD and T1R3NTD. For each protein, these ligands induced a dosedependent quenching of intrinsic tryptophan fluorescence (18,19) (Figure 2). In contrast, the umami stimulus L-glutamate and the sulfamate sweetener cyclamate (which binds to the transmembrane domains of human T1R3) had no effect (18). The tryptophan fluorescence of M B P alone, while quenched by maltose, was unaffected by sucrose or sucralose (18). Therefore, we conclude that glucose, sucrose and sucralose bind to the NTDs of both T1R2 and T1R3. Next, we determined Κ values for glucose, sucrose and sucralose binding to the T1R2 and T1R3 NTDs (18,19). T1R3NTD and MBP-T1R3NTD bound glucose and sucrose with nearly identical K& values (Figure 2 and Table I), again supporting the specificity of ligand binding to the N T D . A l l three sugars bound MBP-T1R2NTD, though with affinities somewhat different from those seen for the T1R3 N T D proteins. For example, sucralose exhibited a 20-fold lower Κ ά

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MBP-T1R2NTD

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T1R3NTD T1R3NTDI60T

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C change at nucleotide position 179 that resulted in an Ile-»Thr change at amino acid position 60 (I60T), was significantly associated with reduced sweetener preference across these strains. This amino acid change has been suggested to either affect ligand binding (29) or to interfere with the dimerization of T1R2 and T1R3 (20), but the basis of its effects on sweet taste sensitivity remained unclear. To address this question, we examined the impact of the I60T polymorphism on the ability of sweet ligands to bind T1R3NTD (18). Surprisingly, neither 5 m M glucose, sucrose or sucralose caused a shift in the S R C D spectra of T 1 R 3 N T D , (not shown) (18), suggesting that either the affinity or the efficacy of ligand binding had been altered in this protein. Titration of these ligands showed that the T1R3NTDI6OT protein continues to exhibit a dose-dependent quenching of intrinsic trytophan fluorescence (Figure 2). However, glucose, sucrose and sucralose, each bound T1R3NTDI6OT with a lower affinity than that for the C57BL/6J variant of T1R3NTD (Table I). These results provide a mechanistic basis for the reduced sweet taste preference of mice bearing the Sac nontaster allele. Higher resolution structural studies will be required to determine whether this residue participates directly in ligand binding or i f the mutation indirectly perturbs the binding pocket through a cascade of steric effects. 60T

Conclusions These studies offer a new approach to understanding the basis of receptor sensitivity and selectivity in sweet taste. The methodologies described here provide several advantages for dissecting the function of T1R taste receptors. First, ligand binding can be examined in the absence of other aspects of receptor activation, such as G protein coupling. Second, the contributions to binding of cooperative interactions between subunits binding can be examined by comparing results with homomeric receptors (as described here) and with heteromeric complexes (e.g., T1R2 + T1R3 NTDs). For example, glutamate can bind each subunit of the homomeric mGluR with the same affinity but induces a negative allosteric interaction as soon as the first subunit is bound (30). The G A B A R forms a heterodimer where only the G A B A R 1 subunit binds ligands at physiological concentrations, though the G A B A R 2 subunit also exerts cooperative effects (8). Third, binding sites can be mapped for ligands that are B

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Figure 3. Ligand binding induces distinct conformational changes for T1R2 and T1R3 NTDs. SRCD spectra of T1R3NTD (Λ, B) and MBP-T1R2NTD (C, D) in the absence (solid) or presence (dashed) of ligand.

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74 broadly preferred across species as the assay does not depend on receptor activation. Fourth, relatively low affinity interactions, even with K values in the millimolar range, can be quantified. Fifth, the highly purified state of the proteins eliminates potential non-specific ligand interactions. Sixth, the ability to express and purify functional ligand binding domains in large quantities will be necessary if their structures are to be determined by x-ray crystallography or other means. Some of this promise has already been realized. Using these approaches we have shown that both T1R2 and T1R3 NTDs bind sugar stimuli at physiologically relevant concentrations, indicating that both subunits play an important role in the detection of sweet stimuli. Furthermore, our studies provide a mechanistic link between sweet taste receptor function and taste behaviors. Future studies should provide interesting insights into the mechanisms of stimulus recognition by T1R taste receptors.


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Acknowledgements SRCD experiments were performed on beamlines U9B and U l l at the National Synchrotron Light Source, Brookhaven National Laboratory (supported by the United States Department of Energy). This work was funded by grants from the National Institute on Deafness and Other Communications Disorders (S.D.M; S.V.) and fellowship support from The Wellcome Trust (G.L.C.). The present affiliation for Y . N . is: Department of Physiology, U C L A , Los Angeles, C A 90095, USA.

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Sweetness and Sweeteners - American Chemical Society

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