1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

361 Poster Chemosensory Molecular Genetics andVNO/PheromoneTASTE RECEPTORS FOR GLUTAMATE IN HUMANFUNGIFORM PAPILLAEMariam R. 1 , Boucher Y. 2 , Wiencis A. 3 , Bézirard V. 4 , Pernollet J. 4 ,Trotier D. 5 , Faurion A. 5 , Montmayeur J. 6 1 University of Paris7, Jouy enJosas, France; 2 Université Paris 7, Paris, France; 3 CSG-CNRS/INRA/UB, Dijon, France; 4 INRA, Jouy en Josas, France;5 CNRS/INRA, Jouy en Josas, France; 6 Centre National de la RechercheScientifique, Dijon, FranceMolecular and behavioural experiments in rodents suggest thatseveral candidate receptors might be involved in glutamate tastedetection. Truncated isoforms of metabotropic glutamate receptors,namely Taste-mGluR4 and Taste-mGluR1, have been found in tastebuds together with a heterodimer receptor (TAS1R1 and TAS1R3).Interindividual variability in the sensitivity to MSG [Lugaz et al.,Chem. Senses 2002] in humans led us to study candidate receptorexpression in human fungiform papillae, both by RT-PCR and immunohistochemistry.Our data indicate that mGluR4, TAS1R1 and TAS1R3are present in human fungiform taste buds. Potential variations of thesequences in the genes coding for TAS1R1 and TAS1R3 wereexamined in 215 subjects. Sequencing of the 6 exons encompassing thecoding region of TAS1R1 and TAS1R3 respectively uncovered threesingle-nucleotide polymorphisms (SNPs) in TAS1R1 and five SNPsdistributed throughout the coding sequence in TAS1R3. Two of 3 SNPsin TAS1R1 and 4 of 5 SNPs in TAS1R3 lead to an amino acidsubstitution. The prevalence of each SNP was evaluated and will bepresented. Additionally, Mendelian transmission of each SNP leading toan amino acid substitution was studied in 25 families. These resultsrepresent a first step towards understanding the genetic factorsunderlying interindividual variability of sensitivity for MSG in humans.362 Poster Chemosensory Molecular Genetics andVNO/PheromonePOSITIONAL CLONING APPROACH TO IDENTIFICATIONOF THE SUCROSE OCTAACETATE AVERSION (SOA) LOCUSBosak N.P. 1 , Theodorides M.L. 1 , Beauchamp G.K. 1 , Bachmanov A.A. 11 Monell Chemical Senses Center, Philadelphia, PASucrose octaacetate (SOA) tastes bitter to humans and has anaversive taste to some mice and other animals. While some mice avoidSOA, others do not, which depends on allelic variation of a single locus,Soa. A dominant Soa a allele produces the taster phenotype (i.e., SOAavoidance); recessive alleles Soa b and Soa c produce the nontaster anddemitaster phenotypes, respectively. We use a positional cloningapproach to identify a gene corresponding to the Soa locus. Ourprevious studies have shown that the Soa locus resides withinapproximately 5-Mb region on chromosome 6. This region contains anumber of genes encoding G protein-coupled receptors from the T2Rsfamily that are proposed to be bitter receptors and therefore arecandidate genes for the Soa locus. Currently, we conduct a highresolutionmapping of the Soa locus. We created a dense set of markersthroughout the Soa region, produced a large (>1,000 mice) crossbetween strains with different Soa alleles, and are genotyping thesemice to find recombinations that shorten the critical Soa interval. Weexpect to reduce the genomic segment encompassing Soa to < 100 kband thus exclude many of the T2R genes from the list of candidates forSoa.363 Poster Chemosensory Molecular Genetics andVNO/PheromoneMOLECULAR MODELING OF SWEET TASTE RECEPTORSCui M. 1 , Jiang P. 1 , Max M. 2 , Margolskee R.F. 3 , Osman R. 1 1 Physiology& Biophysics, Mount Sinai School of Medicine, New York, NY; 2 MountSinai School of Medicine, New York, NY; 3 Neuroscience, Mount SinaiSchool of Medicine, New York, NYThe heterodimer of T1R2 and T1R3 is a broadly acting sweet tastereceptor responsive to natural sugars, artificial sweeteners, D-aminoacids, and sweet-tasting proteins. T1Rs are characterized by a largeextracellular Venus flytrap model (VFTM), which is linked by acysteine rich domain (CRD) to the 7-TM-domain (TMD). Althoughcrystal structures are not available for the sweet taste receptor, usefulhomology models can be developed based on appropriate templates.The VFTM, CRD and TMD of T1R2 and T1R3 have been modeledbased on the crystal structures of metabotropic glutamate receptor type1, tumor necrosis factor receptor, and bovine rhodopsin, respectively.We have used homology models of the sweet taste receptors, moleculardocking of sweet ligands to the receptors, and directed mutagenesis ofthe receptors to identify potential ligand binding sites of the sweet tastereceptor. Financical support from National Institute Health Grant1R03DC007721-01(M.C.), and 1R01DC006696-01A2 (M.M.).364 Poster Chemosensory Molecular Genetics andVNO/PheromonePROBING THE ASPARTAME BINDING SITE OF HUMANT1R2Maillet E. 1 , Cui M. 2 , Jiang P. 1 , Ahmed F. 1 , Zhao B. 1 , Osman R. 2 ,Margolskee R.F. 1 , Max M. 1 1 Neuroscience, Mount Sinai School ofMedicine, New York, NY; 2 Physiology & Biophysics, Mount SinaiSchool of Medicine, New York, NYThe heterodimer of T1R2 + T1R3 is a broadly acting sweet tastereceptor responsive to natural sugars, small molecule artificialsweeteners and sweet tasting proteins. Certain compounds are sweet tohumans but not rodents; this species-specificity can be replicated invitro by expressing the human or mouse T1R2 + T1R3 heterodimers.We had previously used human/mouse mismatched and chimericreceptors and directed mutagenesis to map the sweet receptor´s sites ofinteraction with the sweet protein brazzein (interacts with the cysteinerich domain of T1R2), the sweetener cyclamate and the inverse agonistlactisole (both bind within the transmembrane domain of T1R3) (Jianget al. 2004, 2005ab). Li and colleagues (Li et al. 2002) had determinedthat the extracellular “Venus Fly Trap Domain” (VFTM) of humanT1R2 was required for a human-type response to the dipeptidesweetener aspartame. Based on models of T1R2 with aspartame dockedto the receptor (Cui et al. 2005) we have predicted residues of theVFTM likely to be critical for the interaction with aspartame and otherdipeptide sweeteners. We have used chimeric receptors and mutants toanalyze the interaction of aspartame, neotame and other dipeptidesweeteners with the canonical ligand binding site in the VFTM ofT1R2. From this functional analysis we have tested and refined ourmolecular models of the aspartame-T1R2 VFTM interaction. Ourvalidated model provides a biophysical explanation for why neotame isa much more potent sweetener than aspartame. Supported by NIDCDGrants DC007721 (MC), DC007984 (PJ), DC006696 (MM),DC003055 and DC03155 (RFM).91