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