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Lecture 22 : Taste II

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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 Presentation
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Lecture 22 : Taste II Lecture 22 : Taste II 11/18/09
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Page 1: Lecture 22 : Taste II

Lecture 22 : Taste IILecture 22 : Taste II

11/18/09

Page 2: Lecture 22 : Taste II

Today or MondayToday or Monday

Final project:1 pg of background

Page 3: Lecture 22 : Taste II

Each taste modality relies on Each taste modality relies on different receptorsdifferent receptors

pH sensingT1R2+T1R3

Page 4: Lecture 22 : Taste II

Cats don’t taste sweetCats don’t taste sweet

Page 5: Lecture 22 : Taste II

Search for T1R2 and T1R3Search for T1R2 and T1R3

Screen cat BAC library

Sequence positive clones

Page 6: Lecture 22 : Taste II

Find complete T1R3 geneFind complete T1R3 gene

Page 7: Lecture 22 : Taste II

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

T1R3 expressed; T1R2 is notT1R3 expressed; T1R2 is not

Page 9: Lecture 22 : Taste II

Check other felinesCheck other felines

Page 10: Lecture 22 : Taste II

Pseudogene common to several Pseudogene common to several cat lineagescat lineages

X

X

X

Page 11: Lecture 22 : Taste II

Bitter receptorsBitter receptors

Page 12: Lecture 22 : Taste II

Go 2006Go 2006

Page 13: Lecture 22 : Taste II

HumanMouseDogCowOpossumChicken

Page 14: Lecture 22 : Taste II

Diversity of T2R receptorsDiversity of T2R receptors

Go 2006

Page 15: Lecture 22 : Taste II

Location of human T2R Location of human T2R genesgenes

Page 16: Lecture 22 : Taste II

Human genesHuman genes

Page 17: Lecture 22 : Taste II

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

HumanOWMNWMProsimiansMouse

X marks pseudogenes

A many : oneHuman:mouse

B one : manyHuman:mouse

C one : oneHuman:mouse

Page 19: Lecture 22 : Taste II

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

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

Human sensitivity to PTCHuman sensitivity to PTC

Page 22: Lecture 22 : Taste II

ReviewReview

Page 23: Lecture 22 : Taste II

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

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

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

Mapping Mapping PTC PTC

taster taster genegene

Page 27: Lecture 22 : Taste II

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

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

Genotype vs phenotypeGenotype vs phenotype

Nontaster

Taster

Page 30: Lecture 22 : Taste II

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

Population haplotype Population haplotype frequenciesfrequencies

Taster

Non

Page 32: Lecture 22 : Taste II

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

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

Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors

Matsunami and Amrein 2003

Page 35: Lecture 22 : Taste II

Similarity of taste and Similarity of taste and olfactory receptorsolfactory receptors

Matsunami and Amrein 2003

Page 36: Lecture 22 : Taste II

Taste and VNO are relatedTaste and VNO are related

T1R

V2R

T2R

V1R

Page 37: Lecture 22 : Taste II

Bitter taste transductionBitter taste transduction

Sheridan Nature Biotech 2004

Page 38: Lecture 22 : Taste II

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

Sour and saltySour and salty

Ionotropic channelsSalty - Na+

Sour - H+

Page 40: Lecture 22 : Taste II

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

Salt receptorSalt receptor

Page 42: Lecture 22 : Taste II

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

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

CapsicumCapsicum

Capsaicin detected by vanilloid receptor ??

Page 45: Lecture 22 : Taste II

Sour detectionSour detection

Page 46: Lecture 22 : Taste II

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

Pkd2L1 expressed in unique Pkd2L1 expressed in unique cell typescell types

Page 48: Lecture 22 : Taste II

If knockout Pkd2L2, sour If knockout Pkd2L2, sour detection is lostdetection is lost

Page 49: Lecture 22 : Taste II

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

PkD are members of the TrpP group

Page 51: Lecture 22 : Taste II

2nd lab also prove2nd lab also prove

Page 52: Lecture 22 : Taste II

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

Pkd2L1 and Pkd1L3 Pkd2L1 and Pkd1L3 expressed in papillae - in expressed in papillae - in

same cellssame cells

Page 54: Lecture 22 : Taste II

Transient transfection in Transient transfection in HEK cells - need bothHEK cells - need both

Page 55: Lecture 22 : Taste II

Taste receptorsTaste receptors


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