modulation de la structure, localisation et fonction des récepteurs pour l’inositol...

Modulation de la structure, localisation et fonction des récepteurs pour

l’inositol trisphosphate: le rôle des interactions protéiques.

Jan B. Parys

K.U.LeuvenOrsay, 7 février 2003

(Clapham, 1995)

Intracellular Ca2+ homeostasis

Ca2+

Lumen of ER

Cytoplasm

Structure of the IP3R – the simplified view

• 4 subunits = 1 functional IP3R (homo- or heterotetrameric)

C

Lumen of ER

N

• each subunit can be divided in three parts:1. an IP3-binding domain (about 600 aa)2. a coupling domain (about 1500 aa)3. a channel domain (about 600 aa)

• Interactions of the monomers to form a tetramer

• Interactions of monomer/tetramer with associated proteins

- regulatory proteins CaM

- activatory proteins CaBP1

- kinases / phosphatases PKA, PP1

- anchor proteins homer

- determinants of the IP3R localization talin, ankyrin, 4.1N, …

- proteins regulated by the IP3R trp-channels

Protein interactions to consider:

ALL THESE INTERACTIONS MAY BE MODULATED,DEPENDING ON THE PHYSIOLOGICAL SITUATION

1) Inter- and intramolecular interactions with the N-terminal region of the IP3R(aa 1-225)

2) Dynamics concerning the intracellular localization of the IP3R

Why the N-terminal 225 amino acids of the IP3R1?

• Aa 1-225 act as a suppressor of IP3 binding

activity (Yoshikawa et al., 1999).

• Homer proposed to couple to aa 48-55

(Tu et al., 1998).

• CaM interacts with aa 1-159 (Adkins

et al., 2000).

What isthe

relationbetween

CaMandIP3R

?

A7r5 (Missiaen et al., 1999)

0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)

Cerebellum (Michikawa et al., 1999)

200 μM Ca2+ +10 μM CaM +20 μM CaM

Sf9 (Cardy & Taylor, 1998)

[3 H]I

P3 b

indi

ng (

%)

0

20

40

60

80

100

120

* * **

**

*

0 10

*

0 0.3 1 3 10 0 0.3 1 3 10

Effects of CaM on Ca2+ release:

Effects of CaM on IP3 binding:

CaM (μM)

Lbs-domains (Vanlingen et al., 2000)

Lbs-1 Lbs-2 Lbs-3

What isthe

relationbetween

CaMandIP3R

?

http://www.neuron.org/content/vol23/issue4/images/large/NEURO.23_4_799.9045.f3.jpeg


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)




[3 H]I

P3 b

indi

ng (

%)

0

20

40

60

80

100

120

* * **

**

*

0 10

*

0 0.3 1 3 10 0 0.3 1 3 10

CaM on Ca2+ release: INHIBITORY, Ca2+-DEPENDENT

CaM on IP3 binding: INHIBITORY, Ca2+-INDEPENDENT

CaM (μM)


Lbs-1 Lbs-2 Lbs-3

What isthe

relationbetween

CaMandIP3R

?


control ca cam ca cam cam1234 ca cam12340

20

40

60

80

100

Ca

2+ C

aM12

34

CaM

1234

Ca

2+ C

aM

Apo

CaM

Ca

2+

Con

trol

[3H

]IP3 b

indi

ng

(%

)

0.1 1 100

20

40

60

80

100

[3H

]IP3 b

indi

ng

(%

)

Lbs-1

Lbs-1 1-225

1

581226

581+HIS

+HIS

Effects of CaMon IP3 binding

CaM1234 (μM)

control ca cam ca cam cam1234 ca cam12340

20

40

60

80

100

Ca

2+ C

aM12

34

CaM

1234

Ca

2+ C

aM

Apo

CaM

Ca

2+

Con

trol

[3H

]IP3 b

indi

ng

(%

)

0.1 1 100

20

40

60

80

100

[3H

]IP3 b

indi

ng

(%

)

Lbs-1

Lbs-1 1-225

1

581226

581+HIS

+HIS

Effects of CaMon IP3 binding

CaM1234 (μM)

IC50 ~ 2 μM

Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154 309

+HIS

+HIS

+GST

Ca2+

EGTA

Localisation of the N-terminal CaM-binding site

CaM1234

Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154

+HIS

+HIS

+GST

A

B

C

E

D

F

1-5-10 1-5-10

1-5-8-14 70% IQ 76% IQ

53% IQ

A B C D E F-0.2

0.0

0.2

0.4

0.6

0.8

1.0

200 µM free Ca 2+

1 mM EGTA

309

• Band-shift experiments on non-denaturing gels

• Interaction with dansyl-CaM


Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154

+HIS

+HIS

+GST

A

B

C

E

D

F

1-5-10 1-5-10

1-5-8-14 70% IQ 76% IQ

53% IQ

A B C D E F-0.2

0.0

0.2

0.4

0.6

0.8

1.0

200 µM Ca2+

1mM EGTA

309

Inte

nsi

ty lo

ss (

1-B

/Bo)

Kd 0.1 μM Kd 1 μM


CaM or CaBP1 ?

Inhibitory

Activatory ?(Yang et al., 2002)

CaBP1

GSTGST

1-604GST

1-225GST

226-604

Binding of (activatory?) CaBP1to the same site?

A

B

C

E

D

F

1CaM CaM

159

CaBP1

GSTGST

1-604GST

1-225GST

226-604

Binding of (activatory?) CaBP1to the same site

A

B

C

E

D

F

1CaM CaM

159

CaBP1

CaB

P

1/1 1/2 1/4 1/5 1/6 1/81/3 CaB

P

Ratio of CaBP: peptide B

0 2 4 6 8 100.0

0.5

1.0

Band

in

ten

sity

Peptide B: CaBP

Ca2+

EGTA

1/10

Ca2+

EGTA

GS

T

GS

T-H

omer

1a

The 1225 suppressor domain interacts withHomer1a

1225

N-terminal fragment of the IP3R

CaMCaBP1

Homer

CC

Expression of IP3R11-225 inIP3R triple knockout cells

ko wt 1-225 IP3R1[3 H

]IP

3 bi

ndin

g (f

mol

/U I

P3R

)

IP3R11-225 is not functionally active

ko wt 1-225 IP3R1

0 200 400 600 800 1000 1200 1400

0

200

400

600

800

1000

1200

A2

31

87

1 µ

M I

P3

Dig

ito

nin

[Ca

2+]

(nM

)

Time (sec)

0 200 400 600 800 1000 1200 14000

200

400

600

800

25

µM

IP

3

A2

31

87

10

µM

IP

3

10

µM

IP

3

1 µ

M I

P3

1 µ

M I

P3

Dig

ito

nin

[Ca

2+]

(nM

)

Time (sec)

IP3 binding to IP3R11-225 is not sensitive to thimerosal

ko wt 1-225 IP3R1

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

+BME +Thim. +BME +Thim.

wt IP3R1 IP3R1 1-225

1225

GS

T

GS

T-

226

604

GS

T

GS

T-

226

604

Can 1225(suppressor)interact with

226604(IP3 binding domain) ?

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

+BME +Thim +BME +Thim

1225

GS

T

GS

T-

226

604

GS

T

GS

T-

226

604


Can 1225(suppressor)interact with

226604(IP3 binding domain) ?

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

[3 H]I

P3

bind

ing

(fm

ol/U

IP

3R)

+BME +Thim +BME +Thim

1225

BME Thim

GS

T

GS

T-

226

604

GS

T

GS

T-

226

604


The interactionbetween1225 &226604

is sensitive tothimerosal

1225

GS

T

Idem

+ IP

3

GS

T-2

26

604

Idem

+ A

dA

Idem

+ C

aM

Idem

+ C

aBP

1

The 1225 suppressor domain interacts withthe IP3-binding core:

role of Ca2+, calmodulin and CaBP1

1225226604

• CaM• CaBP1• Homer• Ca2+

• Thim.

• IP3

• Ca2+

?

N

C

Interactions between N- and C-termini of IP3R1 ?

N

C

Flag3 c-myc

GST 1225

GST 226604

GST 1604

GST

= affinity matrix

N

C

GST 1225

GST 226604

GST 1604

GST

Flag3 c-myc

Flag1 2170-2749 ~60 kDa

Flag2 2253-2749 ~54 kDa

Flag3 2170-2427 ~30 kDa

Flag3 2170-2407 ~28 kDa

Flag4 2407-2749 ~41 kDa

Flag4 2560-2749 ~26 kDa

TM1-6

TM1-4

TM(5)-6

M SOL GS

T

GS

T 1

225

GS

T

GS

T 1

225

GS

T 1

604

GS

T 22

660

4

GS

T 1

604

GS

T 22

660

4

Flag1

BME Thim

The interactions between N- and C-termini of the IP3R1 are not thimerosal sensitive

M GS

T

GS

T 1

22

5

GS

T

GS

T 1

22

5

GS

T 1

60

4G

ST

226

60

4

GS

T 1

60

4G

ST

226

60

4

SO

L

Flag1

The interactions between N- and C-termini of the IP3R1 are Ca2+ sensitive

EDTA Ca2+

1mM 25 M

GST 1225 GST 226604

FLAG2

FLAG3

FLAG4

FLAG3

FLAG4

Multiple interactions between N- and C-termini of IP3R11225226604

• CaM• CaBP1• Homer• Ca2+

• Thim.

• IP3

• Ca2+N

C

• Ca2+

CaM/CaBP1

CC

CaM/CaBP1

CC

CC

CaM/CaBP1

CC

• Interactions between monomers ?

Oligom.

Oligom.

CC

CaM/CaBP1

CC

IRBITHomer

Ankyrin

4.1NPP1

• Interactions between monomers ?• Interactions with other proteins ?

Regulation of localization ?Regulation of phosphorylation ?

Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells

IP3R1 IP3R3

Redistribution of IP3R1 after prolonged stimulation

Resting cells + AVP

AVP > 1hImipramineIP3-ester ThapsigarginCPA

[Ca2+]cyt

Redistribution during AVP treatment:time dependence

CytoplasmicIP3R1

localization

PerinuclearIP3R1

localization

AVPtreatment

Perinuclear localizationafter AVP wash-out

Structure of the endoplasmic reticulum

Resting cells +AVP

ER-targetedEYFP

PDI

SERCA localization and redistribution

Resting cells +AVP

0

20

40

60

80

100

Role of PKC

perinuclear IP3R1 cytoplasmic IP3R1

Percentages of cells with:C

ells

(%

)

Con

trol

Con

trol

AV

P

AV

P

AV

P+S

taur

.

AV

P+S

taur

.

TG+S

taur

.

TG+S

taur

.

AV

P+B

IM

AV

P+B

IM

OA

G

OA

G

Role of cytoskeleton

perinuclear IP3R1

Percentages of cells with:

Ce

lls (

%)

Con

trol

AV

P

AV

P+N

ocod

.

AV

P+T

axol

Noc

od.

Taxo

l

0

10

20

30

40

50

60

70

80

Resting cells

+AVP

Microtubular network

or +OAG

Role of vesicle trafficking

• Brefeldin A (2 μg/ml, 2h) complete IP3R1 redistribution

(29 ± 2 % cells with perinuclear localization)

• Cooling (15°C, 2h) inhibition of AVP-induced IP3R1 redistribution

(76 ± 3 % cells with perinuclear localization)

Conclusions (part 1)

The first 225 amino acids of IP3R1 contain important

regulatory sites, including for Ca2+-binding proteins as calmodulin and CaBP1, for homer as well as potentially for thimerosal.

Interactions occur between the N-terminal suppressor domain, the IP3-binding domain and the channel domain.

These interactions are under control of Ca2+, calmodulin/CaBP1 and thimerosal.

The role of additional interactions between monomers or with other proteins and the role of phosphorylation in this process will be investigated.

Conclusions (part 2) Various IP3R isoforms can have a different intra-

cellular distribution.

The localization of the IP3Rs is dynamic and change

with the physiological status of the cell.

This redistribution is dependent on PKC activation and on the microtubular network and is probably mediated by vesicle trafficking.

The results indicate a dynamic regulation of the structure and localization of the IP3R, which we

believe to be functionally relevant.Future work will focus on the understanding of the physiological importance of the various interactions.

Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN - Jan B. PARYS

IP3-team (Leuven, Belgium)

Zerihun ASSEFA

Geert BULTYNCK

Patrick DE SMET

Rafael A. FISSORE

Nael NADIF KASRI

Joelle NSIMIRE CHABWINE

Ilse SIENAERT

Karolina SZLUFCIK

Kristel VAN ACKER

Sara VANLINGEN

Marijke VAN MOORHEM

Esther VENMANS

Leen VERBERT

Elke VERMASSEN

IP3R1IP3R2IP3R3

RyR1RyR2RyR3

Intraluminal proteins

Calreticulin

Calsequestrin

Chromogranins A and B

Annexin VI

ERmembrane proteins

Triadin

Junctin

Kinase anchor proteins

Plasma membrane proteins

Trp’s DHPR

G-proteinsCytosolic proteins

CaM CaBPs

FKBP’s

SorcinS100

IRAG

Cytoskeletal proteins

Homer

Ankyrin

TalinVinculin

Alpha-actin

Myosin II

FKBP12-binding

siteIP3

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+Ca2+

FKBP12

NH2

COOH

SI

SIIPORE

SIII

PP

ATP

ATP

IP3R1 VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV

IP3R2 ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV

IP3R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV

RyR1 QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2 HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI

RyR3 QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL

Cytosol

Lumen of the store

Effects on RyR

(Rodney et al., 2001)

3614 3643

ApoCaM: Ca2+/CaM:

ACTIVATOR OF RYR1 INHIBITOR OF RYR1

Ca2+

http://www.jbc.org/content/vol276/issue3/images/large/bc0219781008.jpeg

IP3

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+Ca2+

CaM

CaM

NH2

COOH

SI

SII

CaM

PORE

SIII

Cytosol

Lumen of the store

PP

ATP

ATP

CaM-binding

sitesHigh-affinityCa2+-dependentTypes 1 and 2

Low-affinityCa2+-dependentNot in neuronalIP3R1 (SII+)

IP3R


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)




[3 H]I

P3 b

indi

ng (

%)

0

20

40

60

80

100

120

* * **

**

*

0 10

*

0 0.3 1 3 10 0 0.3 1 3 10

Effects of CaM on Ca2+ release:

Effects of CaM on IP3 binding:

CaM (μM)


Lbs-1 Lbs-2 Lbs-3


Discontinuous (aa 49-81 and 106-128).

Low affinity.

Ca2+ independent.

Involved in inhibition of IP3 binding.

Not involved in inhibition of IP3-induced Ca2+ release.

Other possible functions:Role in conformation of the IP3R ?

Tethering of CaM ?Binding site for CaM-like proteins ? CaBP-1?Protective effect ? Proteolysis? Oxidative stress?

N-terminal CaM-binding site of the IP3R

modulation de la structure, localisation et fonction des récepteurs pour l’inositol...

Documents