Transcript Genomics of sensory systems - University of Maryland

Lecture 22 : Taste II
11/18/09
Today or Monday
 Final
project:
1 pg of background
Each taste modality relies on
different receptors
T1R2+T1R3
pH sensing
Cats don’t taste sweet
Search for T1R2 and T1R3
 Screen
cat BAC library
 Sequence
positive clones
Find complete T1R3 gene
T1R2 is a pseudogene - 247 bp
deletion + 5 stop codons
T1R3 expressed; T1R2 is not
Check other felines
Pseudogene common to several cat
lineages
X
X
X
Bitter receptors
Go 2006
Human
Mouse
Dog
Cow
Opossum
Chicken
Diversity of T2R receptors
Go 2006
Location of human T2R genes
Human genes
T2R genes in mouse and human
 Mouse
35 functional
6 pseudogenes
 Humans
25 functional
 Other
primates
11 pseudogenes
Human
OWM
NWM
Prosimians
Mouse
X marks
pseudogenes
A many : one
Human:mouse
B one : many
Human:mouse
C one : one
Human:mouse
Human variation in pseudogenes
T2R62
T2R2
Functional
Hosa - Homo sapiens
Patr - Pan troglodytes chimp
Gogo - Gorilla gorilla
Popy - Pongo pygmaeus orangutan
Hyag - Hylobates agilis gibbon
Mamu - Macaca mulatta rhesus
Caja - Callithrix jacchus marmoset
Ceap - Cebus apella capuchin
Convergence of pseudogenes
T2R64
T2R55
Hosa - Homo sapiens
Patr - Pan troglodytes chimp
Gogo - Gorilla gorilla
Popy - Pongo pygmaeus orangutan
Hyag - Hylobates agilis gibbon
Mamu - Macaca mulatta rhesus
Caja - Callithrix jacchus marmoset
Ceap - Cebus apella capuchin
Human sensitivity to PTC
Review
PTC

75% of humans
taste as bitter
Discovered in 1930’s
“Single” gene trait
 Originally thought
was Mendelian
recessive

AA, Aa = sensitive
aa = not sensitive

Probably 2 loci
Give people increasing concentrations of PTC
till they tasted bitter
x2
1
x2
2
x2
x2
8
9
x2
x2
3
x2
x2
4
x2
10
Increasing concentration
11
x2
5
x2
x2
6
7
13
14
x2
12
1M
Quantitative trait locus (QTL)
study
 Link
phenotype (sensitivity to PTC) to
genotype in Utah pedigree
 Found link to 4 Mb region on
chromosome 7
Are 9 T2R and 7 OR genes in this region
 Finer
scale
Genotyped 50 more SNPs in this region
Added broader diversity of taster and nontaster individuals
Mapping
PTC
taster
gene
Tas2R38 variations
PAV
AA V
AVI
P
A V
A
49
V I
262 296
European
49
3
47
EAsian
31
Taster
69
Non-taster
Phenotype is determined by cup # at which
tasted PTC
x2
1
x2
2
x2
x2
8
9
x2
x2
3
x2
x2
4
x2
10
Increasing concentration
11
x2
5
x2
x2
6
7
13
14
x2
12
1M
Genotype vs phenotype
Nontaster
Taster
Human origin of nontaster
Human
PAV /AAV/ AVI
Chimp PAV
Gorilla PAV
Orangutan
PAV
Macaque
PAV
Population haplotype frequencies
Non
Taster
Prevalence of genotypes
PAV/PAV
Taste
European
PAV=0.5
AVI=0.5
E Asian
PAV=0.7
AVI=0.3
W Asian
PAV=0.33
AVI=0.67
PAV/AVI
Taste
AVI/AVI
Not
Prevalence of genotypes
European
PAV/PAV
Taste
PAV/AVI
Taste
AVI/AVI
Not
0.25
0.5
0.25
0.49
0.42
0.09
0.11
0.44
0.45
PAV=0.5
AVI=0.5
E Asian
PAV=0.7
AVI=0.3
W Asian
PAV=0.33
AVI=0.67
Similarity of taste and olfactory
receptors
Matsunami and Amrein 2003
Similarity of taste and olfactory
receptors
Matsunami and Amrein 2003
Taste and VNO are related
V1R
T2R
V2R
T1R
Bitter taste transduction
Sheridan Nature
Biotech 2004
GPCR taste pathways
Sweet
Umami
Bitter
Receptor
T1R2/T1R3
T2R
G protein
GNAT3
Gustducin
PLC
AC/GC
IP3/Ca
cAMP/cGMP
T1R1/
T1R3
?
Effector
Messenger
?
?
GNAT3
Gustducin
PLC
PDE?
IP3/Ca
cAMP
Sour and salty
 Ionotropic
Salty - Na+
Sour - H+
channels
Salt detection likely a channel

Membrane responds
to Na+ gradient
RT Nao  K o
Vm 
ln
F
Nai  K i

Salt receptor
Humans have 2 salt detection
systems
Pathway 1
Pathway 2
(most imp)
Sodium
specificity
Na+ only
Na+, K+ and
NH4+
Sensitivity to
amiloride
Yes
No
Channel?
ENaC
??
Location
Anterior
Fungiform
Posterior
Circumvallate
Two salt pathways
Pathway 1
Pathway 2
Sodium
specificity
Na+ only
Na+, K+ and
NH4+
Sensitivity to
amiloride
Yes
No
Channel?
ENaC
Location
Anterior
TRPV
Vanilloid
Posterior
Capsicum
Capsaicin detected by vanilloid receptor ??
Sour detection
Approach
30,000 genes
Screen mouse
genome for genes
containing TM
Screen EST libraries
for genes which are
not common
5 genes
884
PCR genes in
papillae vs tongue
98
In situ hybridization
to identify ones
uniquely expressed
Pkd2L1 expressed in unique cell
types
If knockout Pkd2L2, sour
detection is lost
Knockout Pdk2L1 cells knocks
out sour detection
But is this the sour channel?
PkD are members of
the TrpP group
2nd lab also prove
Approach
 Assume
TRP channels are important in
taste
TRPM5 known to be channel in sweet,
umami and bitter
 Use
in situ hybridization
Screen taste papillae for all 33 TRP channels
TrpM - 40%
PKD1L - 20% of circumvallate
No other Trp’s label
Pkd2L1 and Pkd1L3 expressed
in papillae - in same cells
Transient transfection in HEK
cells - need both
Taste receptors