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

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. 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 + T1R3 recognizes and responds to many sweet ligands, including sugars, small molecule artificial sweeteners, and protein sweeteners (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar). T1R2 and T1R3 are subclass 3 G-protein-coupled receptors (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar, 3Hoon M. A. Adler E. Lindemeier J. Battey J. F. Ryba N. J. Zuker C. S. 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Other members of this subclass are metabotropic glutamate receptors (mGluRs), 1The abbreviations used are: mGluR, metabotropic glutamate receptor; BRET, bioluminescence resonance energy transfer; DPBS, Dulbecco's phosphate-buffered saline; ATD, amino-terminal domain; GFP, green fluorescent protein. calcium-sensing receptors, pheromone receptors, and other taste/olfactory receptors (T1R1, 5. 24 odor receptor) (8Pin J. P. Galvez T. Prezeau L. Pharmacol. Ther. 2003; 98: 325-354Crossref PubMed Scopus (555) Google Scholar). 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 (9Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1118) Google Scholar). Each protomer comprises LB1 and LB2 domains that form a clamshell-like structure with the ligand-binding domain lying between LB1 and LB2. The free-form I (open-openR) is thought to be in the resting state, whereas the free-form II (closed-openA) is thought to be the active state. Agonist binding stabilizes the active closed-openA 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 + T1R3 sweet receptor will also have open-open and open-closed forms and that small sweet compounds may stabilize the active form of T1R2 + T1R3 by binding within the cleft. However, unlike the mGluRs, the sweet receptor is heterodimeric with each monomer potentially playing a distinct role. The T1R2 protomer is the ligand-specifying component for many agonists (10Zhao G. Q. Zhang Y. Hoon M. A. Chandrashekar J. Erlenbach I. Ryba N. J. Zuker C. S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). Although T1R3 is shared by both the sweet receptor (T1R2 + T1R3) (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar) and the amino acid receptor (T1R1 + T1R3) (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 11Nelson G. Chandrashekar J. Hoon M. A. Feng L. Zhao G. Ryba N. J. Zuker C. S. Nature. 2002; 416: 199-202Crossref PubMed Scopus (1168) Google Scholar), and is essential for both sweet and umami taste (10Zhao G. Q. Zhang Y. Hoon M. A. Chandrashekar J. Erlenbach I. Ryba N. J. Zuker C. S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 12Damak S. Rong M. Yasumatsu K. Kokrashvili Z. Varadarajan V. Zou S. Jiang P. Ninomiya Y. Margolskee R. F. Science. 2003; 301: 850-853Crossref PubMed Scopus (501) Google Scholar), its physical role in taste signal detection/transmission is unknown. Six sweet tasting proteins, ranging in size from 6 to ∼22 kDa, have been discovered (13Faus I. Appl. Microbiol. Biotechnol. 2000; 53: 145-151Crossref PubMed Scopus (98) Google Scholar). The mass of these protein sweeteners makes them too large to fit within the presumptive small molecule-binding pocket, suggesting that they stabilize the active conformation of the sweet receptor in a different manner. Identifying the binding sites for protein sweeteners might provide insights into the molecular events leading to receptor activation. Monellin and brazzein are intensely sweet proteins the structures of which have been solved (14Caldwell J. E. Abildgaard F. Dzakula Z. Ming D. Hellekant G. Markey J. L. Nat. Struct. Biol. 1998; 5: 427-431Crossref PubMed Scopus (110) Google Scholar, 15Somoza J. R. Jiang F. Tong L. Kang C. H. Cho J. M. Kim S. H. J. Mol. Biol. 1993; 234: 390-404Crossref PubMed Scopus (88) Google Scholar) ; these two proteins are not structurally similar or similar to any of the other sweet proteins. We used the fact that the protein sweeteners (and also aspartame) are only perceived as sweet by Old World primates (16Hellekant G. Ninomiya Y. Danilova V. Physiol. Behav. 1997; 61: 829-841Crossref PubMed Scopus (70) Google Scholar, 17Danilova V. Hellekant G. Tinti J. M. Nofre C. J. Neurophysiol. 1998; 80: 2102-2112Crossref PubMed Scopus (62) Google Scholar, 18Danilova V. Danilov Y. Roberts T. Tinti J. M. Nofre C. Hellekant G. J. Neurophysiol. 2002; 88: 579-594Crossref PubMed Scopus (53) Google Scholar) to identify a region of the human sweet receptor that is necessary for it to respond to brazzein. Heterologously expressed human T1R2 (hT1R2) + human T1R3 (hT1R3) responds to monellin and thaumatin, but mouse T1R2 (mT1R2) + mouse T1R3 (mT1R3) does not (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar). Using mixed pairs of human and mouse T1Rs and mouse/human chimeric receptors we have determined that the residues required for a human-like response to monellin lie within the ATD of T1R2. hT1R2 + hT1R3 responds to brazzein, but hT1R2 + mT1R3 does not, indicating that residues in hT1R3 are required for receptor activity toward brazzein. We have located these human-specific residues within a small area in the cysteine-rich region of T1R3 (amino acids 536–545) ; this is the first case in which the cysteine-rich region of a G-protein-coupled receptor has been implicated in receptor function. Replacement of the cysteine-rich region of mouse T1R3 with the corresponding human segment allows hT1R2 + humanized mT1R3 to respond to brazzein. This same segment of T1R3 influences receptor activity toward monellin as well. Preparation of Chimeras and Point Mutations—Human T1R expression constructs were generated in the pEAK12 vector (Edge Biosystems) by genomic DNA-based methods. To subclone each gene into pEAK12 vector, an EcoRI Kozak cassette was introduced at the 5′ end before the start codon, and a NotI site was introduced at the 3′ end after the stop codon. Mouse T1R2 and T1R3 were cloned as reported (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 4Max 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 (469) Google Scholar). Gαl6 chimeras were generated by polymerase chain reaction (PCR) with mutagenic primers and cloned into pCDNA3. The five-residue carboxyl-terminal tail of Gαl6 was replaced by its counterpart from Gαgust (DCGLF) or Gαi3 (ECGLY). Construction of human/mouse chimeras of T1Rs was performed by PCR using overlapping primers (19Horton R. M. Hunt H. D. Ho S. N. Pullen J. K. Pease L. R. Gene (Amst. ). 1988; 77: 61-68Crossref Scopus (2648) Google Scholar). The integrity of all DNA constructs was confirmed by automated DNA sequencing. Point mutations in genes were made using the same overlapping PCR strategy. To construct pRluc-hT1R2 and pGFP-hT1R3, hT1R2 and hT1R3 were amplified by PCR using primers that removed the stop codons and introduced a novel NruI restriction site. The restriction fragments the hT1R2 or hT1R3 were into the or between the EcoRI and This or at the of the T1R To construct hT1R3 hT1R3 were with EcoRI and into the between the EcoRI and This at the of the hT1R3 were at in Dulbecco's with were and were into using T1R2 or T1R3 or their or chimeras were using of or at and vector at well. We that a signal it was used for all but the The were by or by of T1R3 and were were and or for a and for a and in Dulbecco's with and an were with for a or for a of 3 in Dulbecco's phosphate-buffered for at the were with and in of for a and for a was performed with of with was using an and of were performed using were at and at the was into the the response was for an and the was To the was as the of level of the to the was as the of between the level of the at which reached the after of and the each of T1R2 and the response to sweeteners was The of sweeteners was as monellin brazzein and sweeteners were for their structural was a from monellin, and were from and was from were with T1R2 + T1R3 or T1R3 and were an of in with and were with and for with with phosphate-buffered were in for and with for secondary were used to the were made within the extracellular domain of mT1R3 and hT1R3 and using S. Rong M. Yasumatsu K. Kokrashvili Z. Varadarajan V. Zou S. Jiang P. Ninomiya Y. Margolskee R. F. Science. 2003; 301: 850-853Crossref PubMed Scopus (501) Google Scholar). was in Using these we of mouse S. Rong M. Yasumatsu K. Kokrashvili Z. Varadarajan V. Zou S. Jiang P. Ninomiya Y. Margolskee R. F. Science. 2003; 301: 850-853Crossref PubMed Scopus (501) Google Scholar) and human taste receptor not were using an of in were using with pairs of and constructs of DNA of each pRluc-hT1R2 and after the were two with 1 of phosphate-buffered and into of phosphate-buffered of were into of a from To start the of was to each to a of resonance energy were performed using a The was as at at to at at for the construct expressed in the same of by expressed hT1R2 + hT1R3 has been shown to respond to many sweeteners, including monellin and (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar). To hT1R2 + hT1R3 responds as to brazzein, we expressed these receptors together and in with a chimeric with of the sweet hT1R2 + hT1R3 to all sweet compounds, including sucrose, and monellin hT1R2 + hT1R3 to brazzein with a strong response A and to to brazzein depends on we the to brazzein, as as other sweeteners, in with same species pairs of T1R2 + T1R3 hT1R2 + + or pairs hT1R2 + + the + mT1R3 receptor not respond to brazzein or the other human-specific sweet and Although monellin and the hT1R2 + mT1R3 brazzein This that hT1R3 is required for with hT1R2 + mT1R3 to the small molecule sweeteners but a response to monellin, suggesting that hT1R3 also to of the receptor to The response of receptors to the small molecule sweeteners was or by the of mT1R3 in of hT1R3 We response to any of the sweeteners in the + hT1R3 Although + hT1R3 not P. Z. L. A. L. M. J. F. and M. in and this receptor was located at the the receptor was not by any of the of activity from an to or from an to be by in the active in the ATD of T1R2 for to the extracellular or of hT1R2 were required for the response of the sweet receptor to monellin, we made a human/mouse the extracellular domain of hT1R2 to the mouse domain of residues corresponding to human a the residues from hT1R2 or hT1R3 are in the of the construct and the mouse of T1R3 at the 5′ or 3′ this hT1R2 residues and the 3′ end residues from The human/mouse was with mT1R3 and and for to the of sweeteners The response of mT1R3 + T1R2 was from that of mT1R3 + human indicating that the human-specific residues of hT1R2 required for the sweet receptor to respond to monellin all in the extracellular in the of T1R3 for to hT1R2 + mT1R3 not respond to brazzein that human-specific residues in hT1R3 are required for this This with the other human-specific sweeteners that hT1R2 + mT1R3 (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar, G. Q. Zhang Y. Hoon M. A. Chandrashekar J. Erlenbach I. Ryba N. J. Zuker C. S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). We that the human-specific residues in T1R3 required for to brazzein in the To the of hT1R3 essential for we generated human/mouse chimeras the extracellular domain the presumptive ligand-binding domain and the cysteine-rich region A and The T1R3 in combination with were for to brazzein and the of two and to brazzein to brazzein, monellin, and whereas to all The other constructs with 5′ hT1R3 not respond to brazzein but respond to all other sweeteners in the and in some large in to the small molecule that human-specific residues of hT1R3 between and are required for to brazzein. To hT1R3 residues of amino acid are required for to brazzein we a of chimeras the amino-terminal residues of hT1R3 were replaced by the corresponding residues from mT1R3 A and Chimeras that 3′ hT1R3 residues from to brazzein. Chimeras with 3′ from hT1R3 not respond to brazzein. that hT1R3 residues between and are required for to brazzein. To that only this of T1R3 be of human to a response to brazzein, we replaced the corresponding mT1R3 residues in this region with human residues and the activity of the construct in the of The response of the construct to brazzein and the other sweeteners in our was similar to that of hT1R3 the of this region in to brazzein. and for hT1R3 to region of hT1R3 are amino acids that from mT1R3 We each of these amino acids of hT1R3 to their mouse counterpart and for of to brazzein were with the and all to brazzein but to the small molecule sweeteners and to to brazzein, to monellin, and to the small molecule sweeteners The and of to brazzein by that is an essential determinant of the response of hT1R3 to brazzein. The on T1R3 to brazzein or other sweeteners The to brazzein, and to the other sweeteners that may be to efficacy of the receptor expression and activity for was and The of to all sweeteners were in with of with a in to monellin brazzein and to the protein sweeteners the small molecule sweeteners it that only and hT1R3 from mT1R3 in to brazzein. To of any of these amino acids brazzein on mT1R3 we each of these amino acids of mT1R3 to the human counterpart and for of the brazzein the of for the of hT1R3 to brazzein. The other mouse to human mutations in this segment not brazzein activity but have on the to other sweeteners in the implicating this region in at in to monellin and the small molecule sweeteners and on with the to brazzein, suggesting that other residues might also to the of hT1R3 to respond to brazzein. and mutations were made by mouse residues in mT1R3 with their corresponding human The were by and by and by and and by and A and only the to brazzein, but that also the a response or and To more the of mT1R3 we determined brazzein for mT1R3 with was to hT1R3 in its response to brazzein for and for with for brazzein, as by receptor activity but an and a response of efficacy of the for brazzein to Although mT1R3 with hT1R2 responds to monellin of it is with that shown by hT1R3 + hT1R2 of to monellin were also by mutations in this region. The that of these the to monellin to of was with of was more and of was and T1R3 to the of residues and of hT1R3 on receptor activity toward brazzein, monellin, and small molecule sweeteners, we made at these two A and with the of by or to brazzein the to monellin or the other sweeteners a response to brazzein and to monellin and the other sweeteners to all of the sweeteners in our The expression of these was with that of and the of hT1R2 + were from of hT1R2 + hT1R3 and of hT1R3 with or to all sweeteners but to some sweeteners on the and a in response to brazzein, whereas an response to brazzein and a small in its response to and were responsive to both monellin and brazzein was the of these to the small molecule sweeteners were of A similar activity was for hT1R2 + or + hT1R3 The of T1R3 to hT1R2 + hT1R3 responds to brazzein but hT1R2 + mT1R3 does not that brazzein the human form of T1R3. This was a in that other human-specific sweeteners only required T1R2 to be from human (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar, G. Q. Zhang Y. Hoon M. A. Chandrashekar J. Erlenbach I. Ryba N. J. Zuker C. S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). Using human/mouse chimeras of T1R3 with we determined that hT1R3 residues within the cysteine-rich region were required for to brazzein. and within this region of hT1R3 were shown to be for to brazzein. hT1R2 + humanized mT1R3 with human at these two the human-like response to brazzein. of at and of hT1R3 on of hT1R3 with or to brazzein. the activity toward this also to the other sweeteners suggesting that signal in may be in this The of a small to for and the activity with that it is the small size of the chain of that is important at this by the of or to the of shown by these the of affected to brazzein, it seems likely that this is on the binding site of brazzein, or The of hT1R3 in a receptor that was to all sweeteners is in a segment predicted to have a conformation M. 1998; PubMed Scopus Google Scholar). The is predicted to be and may be in a with in this region M. 1998; PubMed Scopus Google Scholar). A in more or and might the of the predicted and the conformation of this region in a that makes it to the signal the This that the cysteine-rich region may binding in the ATD to receptor in the domain to the of It is to such an for the cysteine-rich region of T1R3 in of T1R2 to be the ligand-binding monomer for (1Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar, 2Nelson 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 (1407) Google Scholar, G. Q. Zhang Y. Hoon M. A. Chandrashekar J. Erlenbach I. Ryba N. J. Zuker C. S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). mutations at (and at may two to the brazzein binding site and signal for all sweeteners in our mutations at the response to brazzein to monellin or to brazzein and monellin and hT1R3 also to the small molecule of hT1R3 by to mT1R3 at this makes the response of hT1R2 + hT1R3 to that of hT1R2 + indicating that this a role in of the human receptor + from of the mixed species receptor + activity of T1R3 from large at is not a for a because at this activity was by or suggesting that an is important for this The of to brazzein by some T1R3 at and that at some of the essential of brazzein with the receptor at a site distinct from the small molecule binding the brazzein and small molecule binding site to be in the of may not in part or in The of T1R2 on is to that brazzein, unlike the other human-specific sweet ligands, with T1R3 because it is the species of T1R3 monomer that to brazzein. we have determined that hT1R2 is with the humanized to brazzein suggesting that are human T1R2 residues that with brazzein not This that brazzein may with both T1R2 and T1R3. We and have the structure of brazzein to hT1R3 2002; PubMed Scopus Google Scholar) or hT1R2 not such may for the structure of We and for the of this and Hellekant for of brazzein. with