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