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J. Biol. Chem., Vol. 280, Issue 40, 34296-34305, October 7, 2005

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

Peihua Jiang, Meng Cui, Baohua Zhao, Lenore A. Snyder, Lumie M. J. Benard¶, Roman Osman, Marianna Max, and Robert F. Margolskee¶1

From the Departments of Neuroscience and Physiology and Biophysics and the ^¶Howard Hughes Medical Institute, Mount Sinai School of Medicine, New York, New York 10029

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.

Received for publication, May 12, 2005 , and in revised form, August 1, 2005.

^* This work was supported in part by National Institutes of Health Grants 1F32 DC007021-01 (to P. J.), 1R03 DC007721-01 (to M. C.), DC003055 and DC003155 (to R. F. M.), and DC006696 (to M. M). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., Dept. of Neuroscience, P. O. Box 1065, Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029. Tel.: 212-659-8611; Fax: 212-849-2599; E-mail: bob{at}inka.mssm.edu.

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