the bhlh-pas protein arnt - effects on er signalling
TRANSCRIPT
Biological Effects of Dioxin
• Toxic Effects: Wasting, lymph atrophy, endometriosis, etc
• Teratogenic Effects
• Tumor Promotion
• Enzyme Induction
• Interference with Hormonal signaling (EDC)
The bHLH-PAS proteins
bHLHPAS DomainBABABABA
Dioxin Receptor, AhRArntSim
Per
Metabolism of xenobiotics
CNS growth/differentiation
Regulation of circadian rhythm
Partner for AhR and HIF-1
Examples:
DimerizationDNA- and
hsp90 binding
Ligand- andhsp90 binding
Ligand BindingDimerizationTCDDARNT
ARNT
hsp90
DNA Binding, TransactivationNUCLEUSCYTOPLASM
hsp90
Activation process of the AhR
AhR Nuclear Translocator: ARNT
• Obligatory partner for DR and HIF-1• Ligand DR-ARNT dimers bind to xenobiotic
response elements (XREs) of target genes • ARNT is constitutively expressed protein• Nuclear localization• 3 isoforms identified
ARNT isoforms
bHLH PAS1 PAS2
mARNT-1791 aa
mARNT-2712 aa
95% 78%
• ARNT-1: ubiquitously expressed
• ARNT-2: in developing brain, kidney, heart
• bMAL/ARNT-3: brain, muscle
bMAL/mARNT-3632 aa
63% 39%
57%
34%
A/B C D EAF-1 DBD Hinge LBD AF-2
Ligand regulate receptors like steroid receptors, thyroid
hormaone receptors, retinoid acid receptors and Vitamin D
Orpan receptors
Nuclear receptor superfamily
Nuclear receptor signaling
NRHSP 90 Complex
+ligand
NR+
HSP 90 Complex
HREcofactors
transcription
Gene promoter
Coactivators: SRC family, CBP/p300, PBP, PCAF…
Corepressors: N-CoR, SMRT, DAX-1, SHP…
Nuclear receptor co-factors
A B
p23
hsp90XAP-2
A B
A B
p23
hsp90
DR
HIF-1Other nuclear receptors?Other transcription factors?
ARNT
ARNT regulates multiple pathways
E2 Tam- -
pCMV-ARNT
pSG5-Flag-ER
pSG5-Flag- - - - +
+ + + -
+ + + +
IP:anti-Flag ARNT
ARNTWCEs: 10% of input
ER and ARNT interact
Western: anti-ARNT
E2 Tam
pCMV-ARNT
pSG5-ER
pSG5- + - - -
- + + +
+ + + +
IP:ER mAb ARNT
WCEs: 10% input
ER and ARNT interact
Western: anti-ARNT
ARNT
- -
ARNT stimulates ER activity
ARNT TIF-2
HeLa/3xERE-TATA-luc
050
100150200250
300350400450500
3xERETATA
pSG5ERa
ERa +ARNT
ERa +ARNT
ERa +ARNT
ERa +TIF-2
ERa +TIF-2
ERa +TIF-2
(-)E2 10 nMTam 1 uM
ARNT stimulates ER activity
ARNT TIF-2
HeLa/3xERE-TATA-luc
0
200
400
600
800
1000
1200
1400
1600
1800
2000
3xERETATAluc
ERb ERb +ARNT
ERb +ARNT
ERb +ARNT
ERb +TIF-2
ERb +TIF-2
ERb +TIF-2
(-)E2 10 nMTam 1 uM
200400600
ER-LBDARNTARNTER-LBD
Tam2E-ERER−ΔABARNTARNT10002000300040002E- 50010001500
ERER−ΔABARNTARNT2E-ARNT interacts
with the LBD of ER/
The C-Terminal domain of ARNT is required for co-activation
ER250500750
-ARNTARNT 603ARNT 407
C E2-5001000150020002500
ERβ-ARNTARNT 603ARNT 407
D E2-ERβ250-ARNTΔbHLHΔPASERα
500-ARNTΔbHLHΔPAS500750A E2-
100015002000B E2-
TCDD and 3-MC inhibit ERs transcriptional activity in HC11 cells
0
20
40
60
80
100
120
- TCDD 3-MC TCDD 3-MC TCDD 3-MC TCDD 3-MC
% Activity
E2 DPN PPT
Antiestrogens inhibit 3-MC induced transcriptional activity
0
20
40
60
80
100
120
140
160
180
- E2 3MC 3MC+ICI 3MC+4OHT
ERa
ERb
HC11-ERE
0
20
40
60
80
100
120
(-) E2 10 nM Tam 1 uM ICI 100 nM
% Activity
DMSO
TCDD
Hypoxia
Hypoxia+TCDD
Hypoxia and TCDD inhibit ER transcriptional activity in HC11 cells
E2+
TCDDE2NH
IgG
-ER
TCDD
Input
Activation of the AhR inhibits recruitment of ARNT to E2 regulated promoters
ARNT
IgG
-ARNT
Input
E2+TCDD
E2NH TCDD
c-Fos Lactoferrin
ERE
UTR
ERE
SUMMARY
• ARNT interacts with ligand-activated ER and ER
• ARNT stimulates both ER and ER transcriptional activity
• ARNT is recruited to the E2 regulated promoters in an ligand-dependent manner
A B
p23
hsp90XAP-2
A B
A B
p23
hsp90
DR
HIF-1
ER/
Other nuclear receptors?Other transcription factors?
ARNT
ARNT regulates multiple cellular pathways