1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

357 Poster Chemosensory Molecular Genetics andVNO/PheromoneTHE G ENCODING GENE FAMILY OF THE MALARIAVECTOR MOSQUITO ANOPHELES GAMBIAE: EXPRESSIONANALYSIS AND IMMUNOLOCALIZATION OF AGGQ ANDAGGO IN FEMALE ANTENNAERuetzler M.R. 1 , Zwiebel L. 1 1 Biological Sciences, VanderbiltUniversity, Nashville, TNTo initiate a comprehensive investigation of chemosensory signaltransduction downstream of odorant receptors, we identify andcharacterize the complete set of genes that encode G-protein a subunitsin the genome of the malaria vector mosquito An. gambiae. Data isprovided on the tissue-specific expression patterns of 10 correspondingaga-transcripts in adult mosquitoes and pre-imago developmentalstages. Specific immunoreactivity in chemosensory hairs of femaleantennae provides evidence in support of the participation of a subset ofAgGaq isoforms in olfactory signal transduction in this mosquito. Incontrast, AgGao is localized along the flagellar axon bundle but isabsent from chemosensory sensilla, which suggests this G-protein asubunit does not participate in olfactory signal transduction359 Poster Chemosensory Molecular Genetics andVNO/PheromoneEXPRESSION OF GPR4, A PROTON SENSING GPCR, INHUMAN FUNGIFORM PAPILLAEHuque T. 1 , Lischka F.W. 1 , Breslin P.A. 1 , Feldman R.S. 2 , Spielman A.I. 3 ,Brand J.G. 1 1 Monell Chemical Senses Center, Philadelphia, PA;2 Dental Medicine, V.A. Medical Center, Philadelphia, PA; 3 New YorkUniversity, New York, NYThe molecular mechanisms underlying sourness remaincontroversial. Most proposed mechanisms postulate the involvement ofvarious ion channels. Recently, a proton sensing molecule named GPR4has been characterized which is a GPCR rather than an ion channel.Here we summarize our studies on the expression of GPR4 in humantaste tissue. Human fungiform papillae (HFP) were obtained from twosubjects who identified 15 mM citric acid as sour in both whole mouthand tongue tip tests. RTPCR of the pooled papillae confirmed theexpression of GPR4. Subsequently, the entire coding sequence of GPR4(1086 bp) was amplified from the pooled papillae. An open readingframe of 362 amino acids was identified, only two of which differedfrom the GenBank sequence. RTPCR of individual cells isolated fromHFP showed that GPR4 was expressed in a subset of taste cells. Twosour-abnormal subjects were studied, one of whom (ID # 62) identifiedthe sourness of 15 mM citric acid in a whole mouth test but not at thetongue tip. RTPCR of HFP from Subject 62 failed to detect the codingsequence of GPR4 after 50 cycles. The other sour-abnormal subject (ID# W) was unable to identify the sourness of citric acid, either at thetongue tip or in a whole mouth test, at concentrations up to 18 mM.RTPCR of HFP from Subject W failed to detect the coding sequence ofGPR4 after 50 cycles. Taken together, these initial data raise thepossibility of a role for GPR4 in human sour perception. Supported inpart by NSF Grant # 9816478 (TH); and NIH Grant P50DC0670 (PAB).358 Poster Chemosensory Molecular Genetics andVNO/PheromoneGENETIC ANALYSIS OF TONGUE SIZE AND TONGUEWEIGHT IN RECOMBINANT INBRED STRAINS OF MICEJan T.A. 1 , Reiner D.J. 1 , Peirce J.L. 1 , Li C.X. 1 , Boughter J.D. 1 , Lu L. 1 ,Williams R.W. 1 , Waters R.S. 1 1 Anatomy and Neurobiology, Universityof Tennessee Health Science Center, Memphis, TNLittle is known about the genetic factors underlying variability oftongue morphology. Quantitative trait locus (QTL) analysis is animportant methodology for mapping genes underlying differences inmorphology. QTL detection methods use a forward genetics approach,where a well-characterized phenotype is quantified in an effort toidentify a set of genes that are responsible for the variability of thephenotype. In the present study, the morphology of the mouse tonguewas examined in 18 recombinant inbred BXD strains. We measuredfour tongue dimensions that included apex to vallate (ApV), apex tomedian eminence (ApM), tongue width and tongue weight. Acorrelation analysis revealed that none of these measurements morethan modestly correlated with body weight, suggesting that the geneticfactors are largely independent of body size. Tongue weight correlatedwith tongue lengths of ApV and ApM at r 2 = 0.7 and r 2 = 0.8,respectively, and only modestly correlated with tongue width (r 2 = 0.5).Interestingly, the detected QTLs observed from residual regressionanalysis for ApV and ApM were different from those of tongue weight.We detected a QTL on chromosome 7 (LOD > 3.8) for both ApV andApM, while tongue weight showed two suggestive QTLs onchromosomes 9 (LOD = 4.0) and 16 (LOD = 3.8). Pair-scan analysisrevealed that the two suggestive QTLs affecting tongue weight werepurely additive in effect. These results are a first characterization ofgenetic variability in tongue morphology among BXD strains of mice.(Supported by NIH grant to RSW)360 Poster Chemosensory Molecular Genetics andVNO/PheromoneA NEWLY IDENTIFIED NEOHESPERIDINEDIHYDROCHALCONE BINDING SITE IN THE HUMANSWEET TASTE RECEPTOR OVERLAPS WITH ALLOSTERICMODULATOR BINDING SITES FOR CLASS 3 GPCRS.Winnig M. 1 , Bufe B. 1 , Kratochwil N. 2 , Slack J.P. 3 , Meyerhof W. 11 Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany; 2 Pharmaceuticals Division Chemistry,F. Hoffmann-La Roche Ltd, Basel, Switzerland; 3 Givaudan FlavorsCorp., Cincinnati, OHThe sweet inhibitor lactisole and the sweetener cyclamate share anoverlapping binding site in the seven-transmembrane section of theTAS1R3 subunit of the human sweet receptor (Jiang et al., 2005).Using heterologous expression and functional analysis of rat/humanreceptor chimeras we recently showed that the seven-transmembranesection of hTAS1R3 is crucial for the activation by the sweetenerneohesperidine dihydrochalcone (NHDC, Winnig et al 2005).Interestingly, lactisole competitively inhibits NHDC and cyclamateactivation. We therefore assumed that NHDC may share some residueswith the lactisole and cyclamate binding site. To test this hypothesis wetested 13 point mutants involved in lactisole and cyclamate bindingtowards NHDC. Indeed, 7 of them increased the EC50 of NHDC > 10fold. Modelling of the NHDC pharmacophore revealed 12 additionalpossible interaction sites. Mutational analysis showed that 10 of themclearly affected NHDC activation. Moreover, sequence alignments ofthe TAS1R3 seven-transmembrane section with other members oft theclass 3 GPCRs revealed that 40% of the amino acids involved in NHDCactivation overlap with known binding positions of allostericmodulators in other class 3 GPCRs. This allows us to predict additionalresidues in the TAS1R3 seven-transmembrane section that may beinvolved in the binding of other sweeteners.90