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
Lecture 22 : Taste IILecture 22 : Taste II
11/18/09
Today or MondayToday or Monday
Final project:1 pg of background
Each taste modality relies on Each taste modality relies on different receptorsdifferent receptors
pH sensingT1R2+T1R3
Cats don’t taste sweetCats don’t taste sweet
Search for T1R2 and T1R3Search for T1R2 and T1R3
Screen cat BAC library
Sequence positive clones
Find complete T1R3 geneFind complete T1R3 gene
T1R2 is a pseudogene - 247 T1R2 is a pseudogene - 247 bp deletion + 5 stop codonsbp deletion + 5 stop codons
T1R3 expressed; T1R2 is notT1R3 expressed; T1R2 is not
Check other felinesCheck other felines
Pseudogene common to several Pseudogene common to several cat lineagescat lineages
X
X
X
Bitter receptorsBitter receptors
Go 2006Go 2006
HumanMouseDogCowOpossumChicken
Diversity of T2R receptorsDiversity of T2R receptors
Go 2006
Location of human T2R Location of human T2R genesgenes
Human genesHuman genes
T2R genes in mouse and T2R genes in mouse and humanhuman
Mouse35 functional 6 pseudogenes
Humans25 functional 11 pseudogenes
Other primates
HumanOWMNWMProsimiansMouse
X marks pseudogenes
A many : oneHuman:mouse
B one : manyHuman:mouse
C one : oneHuman:mouse
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
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
Human sensitivity to PTCHuman sensitivity to PTC
ReviewReview
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
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
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
Mapping Mapping PTC PTC
taster taster genegene
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
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
Genotype vs phenotypeGenotype vs phenotype
Nontaster
Taster
Human origin of nontasterHuman origin of nontaster
Human PAV /AAV/ AVI
Chimp PAV
Gorilla PAV
Orangutan PAV
Macaque PAV
Population haplotype Population haplotype frequenciesfrequencies
Taster
Non
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
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
Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors
Matsunami and Amrein 2003
Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors
Matsunami and Amrein 2003
Taste and VNO are relatedTaste and VNO are related
T1R
V2R
T2R
V1R
Bitter taste transductionBitter taste transduction
Sheridan Nature Biotech 2004
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
Sour and saltySour and salty
Ionotropic channelsSalty - Na+
Sour - H+
Salt detection likely a Salt detection likely a channelchannel
Membrane responds to Na+ gradient
€
Vm =RT
FlnαNao +KoαNai +K i
Salt receptorSalt receptor
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
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
CapsicumCapsicum
Capsaicin detected by vanilloid receptor ??
Sour detectionSour detection
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
Pkd2L1 expressed in unique Pkd2L1 expressed in unique cell typescell types
If knockout Pkd2L2, sour If knockout Pkd2L2, sour detection is lostdetection is lost
Knockout Pdk2L1 cells Knockout Pdk2L1 cells knocks out sour detectionknocks out sour detection
But is this the sour channel?
PkD are members of the TrpP group
2nd lab also prove2nd lab also prove
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
Pkd2L1 and Pkd1L3 Pkd2L1 and Pkd1L3 expressed in papillae - in expressed in papillae - in
same cellssame cells
Transient transfection in Transient transfection in HEK cells - need bothHEK cells - need both
Taste receptorsTaste receptors