title: Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3.
abstract: 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.
authors.affiliation: Department of Neuroscience and Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Peihua
authors.initials: P
authors.lastname: Jiang
authors.forename: Meng
authors.initials: M
authors.lastname: Cui
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
authors.forename: Lenore A
authors.initials: LA
authors.lastname: Snyder
authors.forename: Lumie M J
authors.initials: LM
authors.lastname: Benard
authors.forename: Roman
authors.initials: R
authors.lastname: Osman
authors.forename: Marianna
authors.initials: M
authors.lastname: Max
authors.forename: Robert F
authors.initials: RF
authors.lastname: Margolskee
mesh_terms: D000409:Alanine; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002678:Chimera; D003494:Cyclamates; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D003494:Cyclamates; D043562:Receptors, G-Protein-Coupled; C431926:taste receptors, type 1; D000409:Alanine