lecture 22 : taste ii
DESCRIPTION
Lecture 22 : Taste II. 11/18/09. Today or Monday. Final project: 1 pg of background. Each taste modality relies on different receptors. pH sensing. T1R2+T1R3. Cats don’t taste sweet. Search for T1R2 and T1R3. Screen cat BAC library Sequence positive clones. Find complete T1R3 gene. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/1.jpg)
Lecture 22 : Taste IILecture 22 : Taste II
11/18/09
![Page 2: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/2.jpg)
Today or MondayToday or Monday
Final project:1 pg of background
![Page 3: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/3.jpg)
Each taste modality relies on Each taste modality relies on different receptorsdifferent receptors
pH sensingT1R2+T1R3
![Page 4: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/4.jpg)
Cats don’t taste sweetCats don’t taste sweet
![Page 5: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/5.jpg)
Search for T1R2 and T1R3Search for T1R2 and T1R3
Screen cat BAC library
Sequence positive clones
![Page 6: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/6.jpg)
Find complete T1R3 geneFind complete T1R3 gene
![Page 7: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/7.jpg)
T1R2 is a pseudogene - 247 T1R2 is a pseudogene - 247 bp deletion + 5 stop codonsbp deletion + 5 stop codons
![Page 8: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/8.jpg)
T1R3 expressed; T1R2 is notT1R3 expressed; T1R2 is not
![Page 9: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/9.jpg)
Check other felinesCheck other felines
![Page 10: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/10.jpg)
Pseudogene common to several Pseudogene common to several cat lineagescat lineages
X
X
X
![Page 11: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/11.jpg)
Bitter receptorsBitter receptors
![Page 12: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/12.jpg)
Go 2006Go 2006
![Page 13: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/13.jpg)
HumanMouseDogCowOpossumChicken
![Page 14: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/14.jpg)
Diversity of T2R receptorsDiversity of T2R receptors
Go 2006
![Page 15: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/15.jpg)
Location of human T2R Location of human T2R genesgenes
![Page 16: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/16.jpg)
Human genesHuman genes
![Page 17: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/17.jpg)
T2R genes in mouse and T2R genes in mouse and humanhuman
Mouse35 functional 6 pseudogenes
Humans25 functional 11 pseudogenes
Other primates
![Page 18: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/18.jpg)
HumanOWMNWMProsimiansMouse
X marks pseudogenes
A many : oneHuman:mouse
B one : manyHuman:mouse
C one : oneHuman:mouse
![Page 19: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/19.jpg)
Human variation in Human variation in pseudogenespseudogenes
Hosa - Homo sapiens Hyag - Hylobates agilis gibbon Patr - Pan troglodytes chimp Mamu - Macaca mulatta rhesus Gogo - Gorilla gorilla Caja - Callithrix jacchus marmoset Popy - Pongo pygmaeus orangutan Ceap - Cebus apella capuchin
Functional
T2R62 T2R2
![Page 20: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/20.jpg)
Convergence of pseudogenesConvergence of pseudogenes
Hosa - Homo sapiens Hyag - Hylobates agilis gibbon Patr - Pan troglodytes chimp Mamu - Macaca mulatta rhesus Gogo - Gorilla gorilla Caja - Callithrix jacchus marmoset Popy - Pongo pygmaeus orangutan Ceap - Cebus apella capuchin
T2R64
T2R55
![Page 21: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/21.jpg)
Human sensitivity to PTCHuman sensitivity to PTC
![Page 22: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/22.jpg)
ReviewReview
![Page 23: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/23.jpg)
PTCPTC
75% of humans taste as bitterDiscovered in 1930’s
“Single” gene trait Originally thought
was Mendelian recessiveAA, Aa = sensitiveaa = not sensitive
Probably 2 loci
![Page 24: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/24.jpg)
Give people increasing concentrations of Give people increasing concentrations of PTC till they tasted bitterPTC till they tasted bitter
1 2 3 4 5 6 7
8 9 10 11 12 13 14
x2 x2 x2 x2 x2 x2
x2 x2 x2 x2 x2
x2
x2
1MIncreasing concentration
![Page 25: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/25.jpg)
Quantitative trait locus Quantitative trait locus (QTL) study(QTL) study
Link phenotype (sensitivity to PTC) to genotype in Utah pedigree
Found link to 4 Mb region on chromosome 7Are 9 T2R and 7 OR genes in this
region Finer scale
Genotyped 50 more SNPs in this regionAdded broader diversity of taster and
non-taster individuals
![Page 26: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/26.jpg)
Mapping Mapping PTC PTC
taster taster genegene
![Page 27: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/27.jpg)
Tas2R38 variationsTas2R38 variations
P
A49
A V
V I262 296
European EAsianP A V 49 31 TasterA A V 3A V I 47 69 Non-taster
![Page 28: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/28.jpg)
Phenotype is determined by cup # at Phenotype is determined by cup # at which tasted PTCwhich tasted PTC
1 2 3 4 5 6 7
8 9 10 11 12 13 14
x2 x2 x2 x2 x2 x2
x2 x2 x2 x2 x2
x2
x2
1MIncreasing concentration
![Page 29: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/29.jpg)
Genotype vs phenotypeGenotype vs phenotype
Nontaster
Taster
![Page 30: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/30.jpg)
Human origin of nontasterHuman origin of nontaster
Human PAV /AAV/ AVI
Chimp PAV
Gorilla PAV
Orangutan PAV
Macaque PAV
![Page 31: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/31.jpg)
Population haplotype Population haplotype frequenciesfrequencies
Taster
Non
![Page 32: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/32.jpg)
Prevalence of genotypesPrevalence of genotypes
PAV/PAVTaste
PAV/AVITaste
AVI/AVINot
European PAV=0.5
AVI=0.5
E Asian PAV=0.7
AVI=0.3
W AsianPAV=0.33AVI=0.67
![Page 33: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/33.jpg)
Prevalence of genotypesPrevalence of genotypes
PAV/PAVTaste
PAV/AVITaste
AVI/AVINot
European PAV=0.5
AVI=0.5
0.25 0.5 0.25
E Asian PAV=0.7
AVI=0.3
0.49 0.42 0.09
W AsianPAV=0.33AVI=0.67
0.11 0.44 0.45
![Page 34: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/34.jpg)
Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors
Matsunami and Amrein 2003
![Page 35: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/35.jpg)
Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors
Matsunami and Amrein 2003
![Page 36: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/36.jpg)
Taste and VNO are relatedTaste and VNO are related
T1R
V2R
T2R
V1R
![Page 37: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/37.jpg)
Bitter taste transductionBitter taste transduction
Sheridan Nature Biotech 2004
![Page 38: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/38.jpg)
GPCR taste pathwaysGPCR taste pathways
Sweet Umami Bitter
Receptor T1R2/T1R3 T1R1/T1R3
T2R
G protein GNAT3Gustducin
? GNAT3Gustducin
Effector PLCAC/GC
? PLCPDE?
Messenger
IP3/Ca
cAMP/cGMP
? IP3/Ca
cAMP
![Page 39: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/39.jpg)
Sour and saltySour and salty
Ionotropic channelsSalty - Na+
Sour - H+
![Page 40: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/40.jpg)
Salt detection likely a Salt detection likely a channelchannel
Membrane responds to Na+ gradient
€
Vm =RT
FlnαNao +KoαNai +K i
![Page 41: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/41.jpg)
Salt receptorSalt receptor
![Page 42: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/42.jpg)
Humans have 2 salt Humans have 2 salt detection systemsdetection systems
Pathway 1 Pathway 2 (most imp)
Sodium specificity
Na+ only Na+, K+ and NH4
+
Sensitivity to amiloride
Yes No
Channel? ENaC ??
Location AnteriorFungiform
PosteriorCircumvallate
![Page 43: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/43.jpg)
Two salt pathwaysTwo salt pathways
Pathway 1 Pathway 2
Sodium specificity
Na+ only Na+, K+ and NH4
+
Sensitivity to amiloride
Yes No
Channel? ENaC TRPVVanilloid
Location Anterior Posterior
![Page 44: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/44.jpg)
CapsicumCapsicum
Capsaicin detected by vanilloid receptor ??
![Page 45: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/45.jpg)
Sour detectionSour detection
![Page 46: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/46.jpg)
ApproachApproach
Screen mouse genome for genes containing TM
30,000 genes
Screen EST libraries for genes which are not common884
PCR genes in papillae vs tongue
In situ hybridization to identify ones uniquely expressed
98
5 genes
![Page 47: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/47.jpg)
Pkd2L1 expressed in unique Pkd2L1 expressed in unique cell typescell types
![Page 48: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/48.jpg)
If knockout Pkd2L2, sour If knockout Pkd2L2, sour detection is lostdetection is lost
![Page 49: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/49.jpg)
Knockout Pdk2L1 cells Knockout Pdk2L1 cells knocks out sour detectionknocks out sour detection
But is this the sour channel?
![Page 50: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/50.jpg)
PkD are members of the TrpP group
![Page 51: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/51.jpg)
2nd lab also prove2nd lab also prove
![Page 52: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/52.jpg)
ApproachApproach
Assume TRP channels are important in tasteTRPM5 known to be channel in sweet,
umami and bitter Use in situ hybridization
Screen taste papillae for all 33 TRP channelsTrpM - 40%PKD1L - 20% of circumvallateNo other Trp’s label
![Page 53: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/53.jpg)
Pkd2L1 and Pkd1L3 Pkd2L1 and Pkd1L3 expressed in papillae - in expressed in papillae - in
same cellssame cells
![Page 54: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/54.jpg)
Transient transfection in Transient transfection in HEK cells - need bothHEK cells - need both
![Page 55: Lecture 22 : Taste II](https://reader035.vdocuments.net/reader035/viewer/2022062301/56815931550346895dc66843/html5/thumbnails/55.jpg)
Taste receptorsTaste receptors