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

2005; Elsevier BV; Volume: 280; Issue: 40 Linguagem: Inglês

10.1074/jbc.m505255200

ISSN

1083-351X

Autores

Peihua Jiang, Meng Cui, Baohua Zhao, Lenore Snyder, Lumie M.J. Benard, Roman Osman, Marianna Max, Robert F. Margolskee,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

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. 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 (1Gilbertson T.A. Damak S. Margolskee R.F. Curr. Opin. 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Sweet, bitter, and umami tastes are mediated in large part by G-protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCRs, G-protein-coupled receptors; TMD, transmembrane domain; VFTM, Venus flytrap module; h, human; HEK293E, human embryonic kidney 293E; m, mouse; TM, transmembrane. and their linked signaling pathways. Sour and salty tastes are thought to be mediated by direct effects on specialized ion channels (1Gilbertson T.A. Damak S. Margolskee R.F. Curr. Opin. Neurobiol. 2000; 10: 519-527Crossref PubMed Scopus (226) Google Scholar, 2Lindemann B. Nature. 2001; 413: 219-225Crossref PubMed Scopus (529) Google Scholar, 3Gilbertson T.A. Boughter Jr., J.D. Neuroreport. 2003; 14: 905-911Crossref PubMed Google Scholar). The detection of sweet taste is mediated by two GPCR subunits, T1R2 and T1R3, which are specifically expressed in taste receptor cells (4Hoon M.A. Adler E. Lindemeier J. Battey J.F. Ryba N.J. Zuker C.S. 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When expressed in vitro, T1R2 + 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) (10Nelson G. Hoon M.A. Chandrashekar J. Zhang Y. Ryba N.J. Zuker C.S. Cell. 2001; 106: 381-390Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar, 11Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1142) Google Scholar, 14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 15Jiang P. Cui M. Ji Q. Snyder L. Liu Z. Benard L. Margolskee R.F. Osman R. Max M. Chem. 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Senses. 2001; 26: 925-933Crossref PubMed Scopus (231) Google Scholar), members of a group that also includes T1R1 (a component of the umami taste receptor), metabotropic glutamate receptors, the calcium-sensing receptor, γ-aminobutyric acid type B receptors, and vomeronasal receptors. Like most other GPCRs, each class C receptor has a heptahelical transmembrane domain (TMD). Unlike other types of GPCRs, each class C GPCR has a characteristic extracellular domain composed of two parts: a “Venus flytrap module” (VFTM), which is involved in ligand binding, and a cysteine-rich domain, which contains nine highly conserved cysteines and which links the VFTM to the TMD (17Pin J.P. Galvez T. Prezeau L. Pharmacol. Ther. 2003; 98: 325-354Crossref PubMed Scopus (557) Google Scholar). A variable length intracellular C-terminal tail completes the class C receptor. Although the “canonical” ligand-binding site lies within the VFTM, additional ligand-binding sites are present elsewhere on T1R proteins and other class C receptors (14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 15Jiang P. Cui M. Ji Q. Snyder L. Liu Z. Benard L. Margolskee R.F. Osman R. Max M. Chem. Senses. 2005; 30: i17-i18Crossref PubMed Scopus (37) Google Scholar, 16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 18Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1121) Google Scholar, 19Binet V. Brajon C. Le Corre L. Acher F. Pin J.P. Prezeau L. J. Biol. 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U. S. A. 2004; 101: 14258-14263Crossref PubMed Scopus (430) Google Scholar). How does the sweet receptor detect and respond to so many chemically diverse compounds? We have shown previously that the cysteine-rich domain of human (h) T1R3 is essential for sweet receptor responses to sweet proteins, suggesting that these proteins bind here on the receptor (14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). It has also been shown that responses to aspartame depend on the canonical VFTM binding site within T1R2 and that lactisole (an inverse agonist) interacts with the TMD of hT1R3 (15Jiang P. Cui M. Ji Q. Snyder L. Liu Z. Benard L. Margolskee R.F. Osman R. Max M. Chem. Senses. 2005; 30: i17-i18Crossref PubMed Scopus (37) Google Scholar, 16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Xu H. Staszewski L. Tang H. Adler E. Zoller M. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14258-14263Crossref PubMed Scopus (430) Google Scholar). We show here that cyclamate also interacts with the TMD of hT1R3 within a potential binding pocket that overlaps with the proposed binding site of lactisole. Thus, there are at least three broadly defined potential binding domains on the heterodimeric sweet receptor, all of which appear capable of mediating its activation. In this study, we show that activation of the sweet receptor by cyclamate requires the human form of T1R3. Using human/mouse chimeric receptors, we have determined that it is the TMD of hT1R3 that specifies responsiveness to cyclamate. From additional chimeras and mutants, we have identified several residues within the TMD of hT1R3 that account in large part for the species-specific response to cyclamate. From molecular models of the predicted binding pocket within the TMD and systematic alanine-scanning mutagenesis, we have identified additional residues involved in sweet receptor responses to cyclamate. Interestingly, certain of these mutations altered responsiveness to both cyclamate (agonist) and lactisole (inverse agonist). Our experimental results support our computationally derived molecular model of cyclamate docked into the TMD binding pocket of T1R3, suggesting that molecular models of the TMDs of T1R proteins and other family C receptors may be generally useful for probing active state conformations of these receptors. Materials—Cyclamate and d-tryptophan were obtained from Sigma and, unless otherwise noted, used at a concentration of 10 mm in the cell-based assays. Chimeras and Mutants—Most of the T1R3 chimeras and mutants used here have been reported previously (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Site-directed mutagenesis or overlapping PCR was used to generate additional mutants of hT1R3. All mutants were confirmed by sequence analysis. Expression of wild-type hT1R3 and the alanine substitution mutants was examined by immunofluorescence confocal microscopy using an antibody generated against the N-terminal region of hT1R3 (6Max M. Shanker Y.G. Huang L. Rong M. Liu Z. Campagne F. Weinstein H. Damak S. Margolskee R.F. Nat. Genet. 2001; 28: 58-63Crossref PubMed Scopus (471) Google Scholar, 14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). All mutants examined reached the cell surface; no significant differences in cell-surface expression were observed between these mutants and wild-type hT1R3 (supplemental Fig. 1). Cell Culture and Calcium Imaging—Human embryonic kidney 293E (HEK293E) cells were cultured, maintained, and transfected as described previously (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). All transfections were done in triplicate, and all experiments were repeated at least twice. Cells were seeded in 6-well plates at a density of 8 × 105 cells/well. After 24 h, the cells were transiently transfected with plasmids encoding T1R proteins and Gα16-gust44 (a chimeric G protein and subunit containing the last 44 amino acids of gustducin). After an additional 24 h, the cells were trypsinized and reseeded onto polylysine-coated 96-well assay plates (Corning Inc.) at a density of 40,000 cells/plate in low glucose medium supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and 1× GlutaMAX-I (Invitrogen). After another 24 h, the cells were washed once with Dulbecco's phosphate-buffered saline and then loaded with 75 μl of 3 μm Fluo-4 (calcium-sensing dye; Molecular Probes, Inc.) in Dulbecco's phosphate-buffered saline for 2 h. The cells were washed twice with Dulbecco's phosphate-buffered saline and assayed using a FlexStation II (Molecular Devices Corp.). Fluorescence changes (excitation at 488 nm, emission at 525 nm, and cutoff at 515 nm) were monitored after addition of Dulbecco's phosphate-buffered saline supplemented with 2× tastants. For each response trace, the data were acquired at 2-s intervals; samples were added at 30 s; and scanning was continued for an additional 150 s. Data Analysis—Calcium mobilization was quantified as the change of peak fluorescence (ΔF) over the base-line level (F). As described previously (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), data are expressed as the mean ± S.E. of the ΔF/F value from three independent samples. The analysis was done automatically using an in-house written SAS program. The data are presented as ΔF/F of three independent measurements. Curve-fitting and generation of bar graphs was done with the GraphPad Prism 3 program. Homology Modeling and Molecular Docking—Homology modeling of hT1R3 based on sequence alignment to rhodopsin was as described (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). To dock cyclamate into the T1R3 TMD, the geometry of cyclamate (cyclohexyl sulfamate) was fully optimized by the ab initio quantum chemistry method at the HF/6-31G* level, followed by a single point calculation with the polarized continuum model solvation method to obtain the electrostatic potentials using the Gaussian 98 package (25Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Baboul A.G. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Gonzalez C. Challacombe M. Gill P.M.W. Johnson B.G. Chen W. Wong M.W. Andres J.L. Head-Gordon M. Replogle E.S. Pople J.A. Gaussian 98 Revision A.x. Gaussian Inc., Pittsburgh, PA1998Google Scholar). The CHELPG charge-fitting scheme (26Breneman C.M. Wiberg K.B. J. Comput. Chem. 1990; 11: 361-373Crossref Scopus (4094) Google Scholar) was then used to calculate partial charges for cyclamate. The missing force field parameters for cyclamate were obtained from similar parameters taken from charmm27 or QUANTA (Accelrys Software Inc., San Diego, CA). Cyclamate was docked into the pocket of the TMD of hT1R3 using the automatic docking program AutoDock Version 3.0.5 (27Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9366) Google Scholar). This program uses a powerful Lamarckian genetic algorithm method for conformational sampling and docking. The docked conformations of cyclamate were analyzed by the cluster analysis of AutoDock. The final docked conformation was selected from the total conformations based upon compatibility with results from mutagenesis, followed by some manual adjustments of the positions of cyclamate and the side chains of the hT1R3 TMD before employing model refinement by molecular dynamics simulations. The same protocol of molecular dynamics simulations was used as described previously (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The α-carbon atoms of the helices were restricted by 1.0 kcal/mol/Å2 harmonic restraint force. The Sweet Taste of Cyclamate Requires the TMD of hT1R3—Although cyclamate tastes sweet to humans, mice do not detect it as sweet or show preference for it (28Richards R.K. Taylor J.D. O'Brien J.L. Duescher H.O. J. Am. Pharm. Assoc. 1951; 40: 1-6Abstract Full Text PDF Scopus (18) Google Scholar, 29Bachmanov A.A. Tordoff M.G. Beauchamp G.K. Chem. Senses. 2001; 26: 905-913Crossref PubMed Scopus (205) Google Scholar). To determine whether this differential responsiveness to cyclamate's sweetness is mediated by the human versus mouse forms of the sweet receptor, we expressed the human and mouse sweet receptors by transient transfection in HEK293E cells along with Gα16-gust44 and then monitored activation (calcium mobilization) by indicator dye using d-tryptophan as a positive control (Fig. 1). As expected, the human receptor (hT1R2 + hT1R3) responded to cyclamate, whereas the mouse (m) receptor (mT1R2+ mT1R3) did not; both receptors responded to the d-tryptophan control. To determine whether one or both human T1R subunits are required for responses to cyclamate, we expressed mismatched pairs of human and mouse T1R2 and T1R3 subunits and then tested for responses to cyclamate. One mismatched pair (hT1R2 + mT1R3) did not respond to cyclamate, but did respond to d-tryptophan (Fig. 1), indicating that hT1R3 is required for receptor sensitivity to cyclamate. The other mismatched pair (mT1R2 + hT1R3) is nonfunctional in this assay (14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and did not respond to cyclamate or d-tryptophan (Fig. 1) or to several other sweet compounds (14Jiang P. Ji Q. Liu Z. Snyder L.A. Benard L.M. Margolskee R.F. Max M. J. Biol. Chem. 2004; 279: 45068-45075Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), precluding us from determining in this way whether hT1R2 is also required for sweet receptor sensitivity to cyclamate. Using human/mouse chimeras, we show below that hT1R2 is not required for receptor sensitivity to cyclamate (Fig. 2C).FIGURE 2The hT1R3 TMD determines responsiveness to cyclamate. A, the schematic diagram shows chimeras between hT1R3 and mT1R3 (swapped at the extracellular domain-TMD boundary of the receptors). Mouse portions are colored gray. C-Rich, cysteine-rich domain. B, hT1R2 and T1R3 chimeras were expressed by transient transfection in HEK293E cells along with Gα16-gust44. The receptor-expressing cells were then assayed by calcium mobilization for their responses to cyclamate (10 mm) and d-tryptophan (10 mm). Receptors with their TMD from mT1R3 (h.1-547.mT1R3 and h.1-567.mT1R3) did not respond to cyclamate, whereas receptors with their TMD from hT1R3 (mT1R3.h.548-852 and mT1R3.h.568-852) were fully responsive to cyclamate. All transfections were done in triplicate; each experiment was repeated two to three times. C, transfection and assays of responses to cyclamate and d-tryptophan were as described for B. Responses to cyclamate did not require the human form of T1R2: receptors containing the TMD from hT1R3 (mT1R3.h.568-852) responded to cyclamate equally well whether paired with mT1R2 or hT1R2 (see B). The responses of the human sweet receptor are shown for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify the portion of hT1R3 required for sensitivity to cyclamate, we tested the responses to cyclamate of heterodimeric receptors composed of hT1R2 plus human/mouse chimeras of T1R3 in which varying portions of hT1R3 were substituted with the complementary portions of mT1R3 (Fig. 2A). As described above, receptor responses to d-tryptophan were used as a positive control for receptor activity. Heterodimers of hT1R2 plus T1R3 chimeras containing most or all of the extracellular region of hT1R3 coupled to the TMD and C-terminal tail of mT1R3 (i.e. h.1-547.mT1R3 and h.1-567.mT1R3) responded to d-tryptophan, but not to cyclamate. In contrast, heterodimers of hT1R2 plus T1R3 chimeras containing most or all of the extracellular domain of mT1R3 coupled to the TMD and C-terminal tail of hT1R3 (i.e. mT1R3.h.548-852 and mT1R3.h.568-852) responded to both d-tryptophan and cyclamate (Fig. 2B). These results indicate that sweet receptor responses to cyclamate require the TMD and/or C-terminal tail of hT1R3. To determine whether the human form of T1R2 is required for sweet receptor responsiveness to cyclamate, we examined the responses of the heterodimeric receptor formed by mT1R2 and mT1R3.h.568-852 (Fig. 2C). Unlike the nonfunctional pairing of mT1R2 and hT1R3 (Fig. 1), mT1R2 can function in combination with the mT1R3.h.568-852 chimera (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The heterodimer of mT1R2 and mT1R3.h.568-852 responded to d-tryptophan and cyclamate (as well as to several other sweeteners (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar)). Thus, hT1R2 is not required for responsiveness to cyclamate, indicating that the hT1R3 component of the heterodimeric receptor is necessary and sufficient for responsiveness to cyclamate. The combined results of Fig. 2 (B and C) demonstrate that only the TMD and C-terminal tail portion of T1R3 need to be from human for the sweet receptor to respond to cyclamate. Extracellular Loop 3 and Transmembrane (TM) Helix 7 of hT1R3 Are Required for Responsiveness to Cyclamate—To identify the portion(s) of the TMD and/or C-terminal tail of hT1R3 required for responses to cyclamate, we examined several additional human/mouse chimeras of T1R3 in combination with hT1R2. This set of chimeras contain the VFTM of hT1R3 along with varying portions of the TMD and C-terminal tail of hT1R3 coupled to the complementary portion of mT1R3 (Fig. 3A). All heterodimers formed by coexpressing hT1R2 plus these chimeras responded to d-tryptophan (Fig. 3B). However, only one heterodimer responded to cyclamate (i.e. hT1R2/h.1-812.mT1R3): this chimera contains the VFTM and entire TMD of hT1R3, but with the C-terminal tail of mT1R3. Heterodimers of hT1R2 with T1R3 chimeras in which mouse sequences replaced any portion of the TMD failed to respond to cyclamate. Thus, one or more differences between the human and mouse sequences within the TMD affect responsiveness to cyclamate. That hT1R2/h.1-812.mT1R3 responded to cyclamate but hT1R2/h.1-787.mT1R3 did not indicates that responsiveness to cyclamate requires human-specific residues between amino acids 787 and 812 of hT1R3, corresponding to extracellular loop 3 and TM helix 7. As noted below, additional residues within the TMD also contribute to sweet receptor responsiveness to cyclamate. Substitutions of Phe-7305.43 in TM Helix 5 and Arg-790ex3 in Extracellular Loop 3 Selectively Affect hT1R3 Responses to Cyclamate—We had previously observed the importance of TM helices 5 and 7 and extracellular loop 3 of hT1R3 in determining human-specific sensitivity of the sweet receptor to the inverse agonist lactisole (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). For our study of the interaction of lactisole with the sweet receptor, we had generated a series of hT1R3 mutants in which the human-specific residues in these regions had been replaced with their mouse counterparts (16Jiang P. Cui M. Zhao B. Liu Z. Snyder L.A. Benard L.M. Osman R. Margolskee R.F. Max M. J. Biol. Chem. 2005; 280: 15238-15246Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). We tested the responses to cyclamate and d-tryptophan of heterodimers of hT1R2 with these T1R3 mutants (Fig. 4). All mutant-containing heterodimers responded to d-tryptophan. Of the four human-to-mouse replacement mutants in TM helix 5, only the F7305.43L mutant showed diminished responsiveness to cyclamate (Fig. 4A). Of the eight replacement mutants in extracellular loop 3 and TM helix 7, only R790ex3Q showed an altered response to cyclamate: the response to cyclamate was completely absent in this mutant (Fig. 4B). Cyclamate and d-tryptophan dose-response curves were obtained for F7305.43L and R790ex3Q (Fig. 4C). In comparison with wild-type hT1R3, the F7305.43L mutant showed a pronounced righ

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