receptor-receptor interaction:
DESCRIPTION
Receptor-receptor interaction: A critical mechanism for the regulation of neurotransmitter receptor function. Fang Liu MD, PhD Centre for Addiction and Mental Health University of Toronto. Neurotransmitter Receptors:. Ionotropic: ligand-gated ion channels. Open ion channels—4-5 subunits - PowerPoint PPT PresentationTRANSCRIPT
Receptor-receptor interaction:
A critical mechanism for the regulation of neurotransmitter receptor function
Fang Liu MD, PhD
Centre for Addiction and Mental Health
University of Toronto
Neurotransmitter Receptors:
Ionotropic: ligand-gated ion channels
Open ion channels—4-5 subunits
Fast neuro-transmission
e.g. for NMDA, AMPA and GABA-A receptors
Metabotropic: G-protein coupled receptors
Activate G proteins
7 transmembrane domains
Couple to many effectors: e.g. adenylyl cyclase
Slow neuro-transmission
-
e.g. for dopamine receptors
PKA, PKC, PTK…
Regulation of neurotransmitter receptors function
The classic pathway: phosphorylation—PKA, PKC…
Direct protein-protein interaction
GAPDH and GluR2 interaction are essential in AMPA receptor-mediated excitotoxicity
Calcineurin
nNOS
Ca2+Na2+
NO
MitoO2
-ONOO-
H2O2
IκBNFκB
DAG
IP3Ca2+
CaM
NMDArNMDAr
AMPArmGlur
PLC
The AMPA Receptor
* Fast synaptic transmission
* neurotoxicity
GluR1/2 in hippocampus
GluR2 Subunit
I III IVII
NH2
COOH
NSF, PICK1, GRIP1/2…
NARP, N-cadherin
* Impermeable to Ca2+
* Interact with intracellular/extracellular proteins
Affinity purification:
Use specific region of one protein to “pull down” another protein from
? ? ? ?
X
a pool of proteins
GST
GSTGST
GST
1 MVKVGVNGFG RIGRLVTRAA FSCDKVDIVA INDPFIDLNY MVYMFQYDST51 HGKFNGTVKA ENGKLVINGK PITIFQERDP VKIKWGDAGA EYVVESTGVF101 TTMEKAGAHL KGGAKRVIIS APSADAPMFV MGVNHEKYDN
SLKIVSNASC151 TTNCLAPLAK VIHDNFGIVE GLMTTVHAIT ATQKTVDGPS
GKLWRDGRGA201 AQNIIPASTG AAKAVGKVIP ELNGKLTGMA FRVPTPNVSV VDLTCRLEKP251 AKYDDIKKVV KQAAEGPLKG ILGYTEDQVV SCDFNSNSHS STFDAGAGIA301 LNDNIVKLIS WYDNEYGYSN RVVDLMAYMA SKE
GluR1 NT
GSTGluR2 NT
GAPDH
GST-GluR2 NTGST
GST-GluR2 CT
GAPDH
37.1
GluR1 NTGST
GluR2 NT
GluR2-NT interacts with GAPDH
GAPDH(glyceraldehyde-3-phosphate dehydrogenase)
GAPDH
NITRIC OXIDE
APOPTOSIS
NEURONAL DISORDERS
VIRAL PATHOGENESIS
PROSTATED CANCER
ENDOCYTOSIS
MICROTUBULE BUNDLING
PHOSPHOTRANFERSE
TRANSLATIONAL REGULATION
NUCLEAR RNA EXPORT
DNA REPLICATION
DNA REPAIR
GAPDH Function:
GluR2 NT1-
3-1
GluR2 NT1-
3-2
GST
GAPDH
GluR2 NT3
GST
GluR2 NT2
GluR2 NT1
GST
GluR2 NT1-
1
GluR2 NT1-
2
GluR2 NT1-
3
GluR2 NT1-
4
GluR2 NT1-
5
I III IVII
NH2
COOH
GAPDH
GluR2-NT1-3-2 interacts with GAPDH
D2
?
?
?
Co-immunoprecipitation
Agonist promotes GAPDH-GluR2 complex formation
ExtractIP
IgG GluR2
GAPDH
Contro
l
Glutam
ate
GluR2 NT1-
3-2
+Glutam
ate
IP:GluR2
HEK293T Cells
GAPDH
GluR2
GAPDH
IP:GluR2
CTRLKA
GluR2 NT1-
3-2
+KA
Hippocampal Neurons
GluR2
NT Fresh Media
GAPDH
Tubulinlysates
AMPARAMPAR+GlutCM
NB B IgG ExtractsIP: GluR2
GAPDHIgG
IP: GAPDH
CM Fresh Media
GAPDH
Extracellular GAPDH-GluR2 complex formation
Fra
ctio
n D
ead
***
KA
GluR2N
T1-3-
2
+KA
0
20
40
60
80
100
120
(Per
cen
t +
KA
)Glu
R2NT1-
3-2
Gluta
mat
e
GluR2N
T1-3-
2
+Gluta
mat
e
0
20
40
60
80
100
120
Fra
ctio
n D
ead
(Per
cen
t G
luta
mat
e)
***
GluR2-GAPDH coupling in AMPA receptor-mediated toxicity
Cells Neurons
*
Contro
l
Glutam
ate0
20
40
60
80
100
120
Cel
l Sur
face
Glu
R2
(Per
cent
Con
trol
) *
0
20
40
60
80
100
120
Cel
l Sur
face
GA
PD
H(P
erce
nt C
ontr
ol)
Contro
l
Glutam
ate
GluR2N
T1-3-
2
+Glutam
ate
#
Contro
l
Glutam
ate0
20
40
60
80
100
120
Cel
l Sur
face
GA
PD
H(P
erce
nt C
ontr
ol)
Contro
l
Glutam
ate0
20
40
60
80
100
120
Cel
l Sur
face
GA
PD
H(P
erce
nt C
ontr
ol)
GluR1/3GluR1/2GluR1/2
GAPDH-GluR2 complex co-internalization
Contro
lKA
GluR2 NT1-
3-2
+KA
GluR2
GAPDH
LaminB1Contro
lKA
GluR2 NT1-3-2
+KA
GluR2 and GAPDH Nuclear Translocation
Biotinlylated GluR2
Biotinlylated GAPDH
Contro
l
Glutam
ate
Siah1 s
iRNA
+Glutam
ate
GluR2
GAPDH
LaminB1
GluR2/GAPDH Nuclear Translocation depends on GluR2-Siah1 interaction
GluR2 CT
GluR2 NT
GAPDHGST
Extract
Anti-Siah1 Anti-Siah1
GluR2 CT-1
GSTExtra
ct
GluR2 CT-2
GluR2 CT-3
GluR2 CT-4
GluR2
GAPDH
LaminB1
Contro
l
Glutam
ate
GluR2 CT-4
+Glut
amat
e
GST-GluR2NTGST
GST-GAPDH
WB: anti-p53
GST-GluR2NT
GAPDH
GST
p53-GST (μg): 0 0.05 0.5 5
GST (ug): 0.5
-
- - - -
GAPDH
IgGIP: p53
GluR2NT1-3-2:
Glutamate: -
- - -
-+++
P53 and GAPDH form complex in nucleus
0 10µM
0
20
40
60
80
100
120
*
Fra
ctio
n D
ead
(Per
cent
Con
trol
)
PFT-
GluR1/2
GAPDH2-2-1:
Glutamate:0
25
50
75
100
125
**
Fra
ctio
n D
ead
(Per
cent
Con
trol
)
- -
- -
+ +
+ +
0 10µM0
20
40
60
80
100
120
Fra
ctio
n D
ead
(Per
cent
Con
trol
)
GluR1/3
PFT-
p53-GAPDH coupling in AMPA receptor-mediated toxicity
GST-GAPDH2GST
GST-GAPDH1
GST-GAPDH2-2-1
GST-GAPDH2-2-2
GST-GAPDH2-1
GST-GAPDH2-2GST
p53
GAPDH
CTRL
IgGIP:GAPDH
GAPDH2-2-1
Control
pSer46
p53
GluR1/2GluR1/2+GAPDH2-2-1
GlutamateContro
l
Glutamate
P53 and GAPDH coupling enhances p53 expression and phosphorylation
anti-p53
GST
Disruption of GAPDH-GluR2 interaction prevents/rescues neuronal death from ischemia in rat hippocampalCA1 regionafter transient cerebral ischemia
B
DG
CA1
CA3
Area of InterestHPC
A
C
D
E
F
G
H
SO
SP
SR
Sham
Ische
mia
Post-P
eptid
e
Pre-pe
ptide
Post-S
cramble
Pre-Scra
mble
***
##
###
0
20
40
60
80
100
120
Neu
ron
al S
urv
ival
(Per
cen
tage
of
Sh
am)
Sham Pep (post) Ischemia
IP:GluR2IgG
GAPDH
GluR2
IP:p53IgG
Sham Pep (post) Ischemia
p53
GAPDH
Enhanced GAPDH-GluR2/GAPDH-p53 interaction in ischemia
Sham0
20
40
60
80
100
120
Ne
uro
na
l S
urv
iva
l(P
erc
en
t S
ha
m)
5 days 10 days
Ischemia
Peptide (Post)
*** ***
#
##
Sham
Isch
emia
2 hours
6 hours
0
20
40
60
80
100
120
***
#
Ne
uro
na
l S
urv
iva
l(P
erc
en
t S
ha
m)
#
Ischemia
Peptide (Post)
Protective effect of GluR2NT1-3-2 in ischemia
Ischemia Pep(Pre) Pep(post)
0
200
400
600
1 2 3 4 5 6 7 8
Stereotactic Coordinates(mm)
Infa
rct
Are
a (
mm
)2 Ischemia Scram
Pep(Pre) Pep(post)
Ne
uro
log
ica
l S
co
res 2hrs after MCAO 24hrs after MCAO
Isch
emia
Scram
(post
)Pep
(Pre
)
Pep(p
ost)
0
2
4
6
8
10
12
*
*
Disruption of GAPDH-GluR2 interaction prevents/rescues neuronal death from focal ischemia (MCAo)
Ischemia
Peptide (post)
0
50
100
150
200
250
3 days
Infa
rct
vo
lum
e (
mm
3)
5 days
*
**
0
50
100
150
200
250
300
3 0.3 0.03 0.001in
farc
t v
olu
me
(m
m3)
***
*
Peptide dose (nmol/g)
Ischemia
Peptide (Post)
***
Protective effect of GluR2NT1-3-3 in focal ischemia (MCAo)
Summary
I III IVII
NH2
COOH
GAPDH
Extracellular
Intracellular
I III IVII
NH2
COOH
GAPDH
Extracellular
Intracellular
AMPA activation
Nucleus
P53
GAPDH
pGluR2
Siah1
Clinical ApplicationUltimate goal:
PEPTIDE
Acknowledgement
Dr. Shupeng LiMs Ming Wang
Dr. Yu-tian WangDr. Lin Pei Dr. Stephen FergusonDr. DongXu Zhai Dr. Sheng Chen
CAMHCIHR pop grantBioDiscovery MaRS