![Page 1: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/1.jpg)
Regulatory pathways acting oninositol trisphosphate receptor
localization and function
Jan B. Parys
K.U.Leuven Univ. AlbertaEdmonton20-22 July 2004
![Page 2: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/2.jpg)
(Clapham, 1995)
Intracellular Ca2+ homeostasis
Tetrameric structureGenes: 3Regulation by Ca2+
![Page 3: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/3.jpg)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
![Page 4: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/4.jpg)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
![Page 5: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/5.jpg)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
![Page 6: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/6.jpg)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
![Page 7: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/7.jpg)
N
C
IP3
PP1
CaM
CaBP
AKAP9
PP1 PKA
RACK1Gβ
HOMERCARP
IRBITTRP
mGluR 1a, 5ShankIP3RRyRCa2+ channels
HAP1A/Htt
ANKYRIN B
4.1N
Spectrin-actincytoskeleton
CASK and/orsyndecan-2
SIG-1R
CHROMOGRANIN A & B
Cyt c
Monomeric type 1 IP3R (IP3R1)
![Page 8: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/8.jpg)
1) Regulation of the IP3R by calmodulin
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
![Page 9: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/9.jpg)
1) Regulation of the IP3R by calmodulin
![Page 10: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/10.jpg)
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)
Effects of CaM on IP3-induced Ca2+ release:
![Page 11: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/11.jpg)
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)
Effects of CaM on IP3-induced Ca2+ release:200 μM Ca2+ +10 μM CaM
![Page 12: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/12.jpg)
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)
Effects of CaM on IP3-induced Ca2+ release:200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
![Page 13: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/13.jpg)
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)
Sf9 (Cardy & Taylor, 1998)
Effects of CaM on IP3-induced Ca2+ release:
Effects of CaM on IP3 binding:
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
![Page 14: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/14.jpg)
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)
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 IP3-induced Ca2+ release:
Effects of CaM on IP3 binding:
CaM (μM)
Lbs-domains (Vanlingen et al., 2000)
Lbs-1 Lbs-2 Lbs-3
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
![Page 15: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/15.jpg)
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)
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 IP3-induced Ca2+ release: Ca2+ dependent
Effects of CaM on IP3 binding: Ca2+ independent
CaM (μM)
Lbs-domains (Vanlingen et al., 2000)
Lbs-1 Lbs-2 Lbs-3
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
![Page 16: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/16.jpg)
N
C
IP3
CaM
High-affinityCa2+-dependentMutations ineffectiveNot in IP3R3
Low-affinityCa2+-dependentNot in neuronal IP3R1
Low-affinityCa2+-independentResponsible for effects on IP3
binding
Monomeric type 1 IP3R (IP3R1)
![Page 17: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/17.jpg)
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
Detailed analysis of the N-terminal CaM-binding site
![Page 18: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/18.jpg)
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
Detailed analysis of the N-terminal CaM-binding site
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
(Sienaert et al., 2002)
![Page 19: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/19.jpg)
A
B
C
E
D
F
1 159
Lbs-1 49-81 106-128
0
20
40
60
80
100
[3 H]I
P 3 bin
din
g t
ov
co
ntr
ole
(%
) controle 10 µM CaMcontrol ∆ B ∆ E
[3H
]IP3 b
indi
ng
CaM -+ ++- -
Both the B and the E sites are necessary for CaM binding
![Page 20: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/20.jpg)
Lbs-1 Lbs-1 N CaM Lbs-1 C Ca Lbs-1 N+C CaM Lbs-1 CaM
0
20
40
60
80
100
[3H]IP3 binding
control controle N-CaM C-CaM N+C-CaM CaM
0
20
40
60
80
45C
a2+
45Ca2+ flux
control
Both the N- and C-terminal parts of CaM are needed
CaM
C-CaMN-CaM
![Page 21: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/21.jpg)
CaM-like Ca2+-binding proteins
Inhibitory
Activatory ???
(Haeseleer et al., 2002)
![Page 22: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/22.jpg)
CaBP1
GSTGST
1-604GST
1-225GST
226-604
Binding of CaBP1 to the same site?
A
B
C
E
D
F
1CaM CaM
159
![Page 23: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/23.jpg)
CaBP1
GSTGST
1-604GST
1-225GST
226-604
Binding of CaBP1 to the same sitebut only to the domain B
A
B
C
E
D
F
1CaM CaM
159
CaBP1
![Page 24: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/24.jpg)
CaB
P1/1 1/2 1/4 1/5 1/6 1/81/3 C
aB
P
Ratio of CaBP: peptide B
0 2 4 6 8 100.0
0.5
1.0
Ban
d in
tens
ity
Peptide B: CaBP
Ca2+
EGTA
1/10
Ca2+
EGTA
CaBP binding is also Ca2+ independent
(Nadif Kasri et al.,2004)
![Page 25: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/25.jpg)
0.5 µM 1 µM 100 µM
Control
CaM
IICR is inhibited in vivo by CaM and CaM1234 …
Time (s)
ATP
600
400
200
Ca2
+i (
nM
)
100075050025000
CaM1234
![Page 26: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/26.jpg)
0.5µM 1µM 100µMATP
control
0 200 400 600 800 10000
Time (s)
1000
200
400
600
800
Ca2+
i (n
M)
… and by both short and long CaBP1
(Nadif Kasri et al., 2004)
sCaBP
lCaBP
![Page 27: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/27.jpg)
0.5µM 1µM 100µMATP
control
0 200 400 600 800 10000
Time (s)
1000
200
400
600
800
Ca2+
i (n
M)
… and by both short and long CaBP1and also by the Ca2+-insensitive CaBP1134
(Nadif Kasri et al., 2004)
sCaBP
lCaBP
CaBP134
![Page 28: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/28.jpg)
Suramin
CaM
In the presence of B In the presence of E
0 100 µM
Suramin interacts with the CaM-binding sites
0 100 µM
0.01 0.1 1 10
0
10
20
30
40
50
60
70
Ca2
+ re
lea
se
vs
A2
31
87
(%
)
IP3 (µM)
(Nadif Kasri et al., in press)
+suramin
aa 1-581
aa 226-581
![Page 29: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/29.jpg)
Calmodulin is not the Ca2+ sensor of the IP3R
(Nadif Kasri et al., in press)
L15 fibroblasts
![Page 30: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/30.jpg)
Model: IP3R structure is dependent on Ca2+
+ Ca2+
- Ca2+
Change in way that CaM,CaBP, … interacts
(Hamada et al., 2003)
![Page 31: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/31.jpg)
1) Dynamics concerning the intracellular localization of the IP3R
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
![Page 32: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/32.jpg)
Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells
IP3R1 IP3R3
(Vermassen et al., 2003)
![Page 33: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/33.jpg)
Redistribution of IP3R1 after prolonged stimulation
Resting cells + AVP
AVP > 1hPLC activationIP3-ester ThapsigarginCPA
[Ca2+]cyt
(Vermassen et al., 2003)
![Page 34: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/34.jpg)
Structure of the endoplasmic reticulum
SERCAPDIER-targeted
EYFP
Control
AVP
![Page 35: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/35.jpg)
Factors participating in IP3R redistribution
PKC activator OAG induction
Staurosporine inhibitionPKC inhibitors Bisindolylmaleimide inhibition
Gö-6876 inhibition
Drugs acting on Nocodazole inhibitionmicrotubuli Taxol inhibition
Action on vesicle Brefeldin A inductiontransport Cooling to 15 ºC inhibition
![Page 36: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/36.jpg)
Do other IP3R isoformsalso redistribute in a similar way?
IP3R3 in
16HBE14o-
cells
Control
Agonist (ATP) OAG PKC
TG Ca2+
![Page 37: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/37.jpg)
Agonist( )
IP3Rredistribution
IP3R redistribution is dependent on the cell status
![Page 38: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/38.jpg)
PKC-mediated phosphorylation of IP3R1
[32P]ATP
Anti-Ser-P
![Page 39: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/39.jpg)
PKC-mediated phosphorylation of IP3R1and IP3R3
[32P]ATP
Anti-Ser-P[32P]ATP
Anti-IP3R
![Page 40: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/40.jpg)
Anti-Ser-P
PKA-mediated phosphorylation of IP3R1stimulates PKC-mediated phosphorylation
![Page 41: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/41.jpg)
Ca2+ inhibits PKC-mediated phosphorylation of IP3R1
![Page 42: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/42.jpg)
Ca2+ and calmodulin differentiallyregulate PKC-mediated phosphorylation of
IP3R1 and IP3R3
![Page 43: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/43.jpg)
Con
trol
0.2
µg/m
l αC
T0.
4 µg
/ml α
CT
Phosphorylatedfragments
273 kDa225 kDa 210 kDa
130 kDa
40 kDa
Determination of the PKC phosphorylationsites on IP3R1 and IP3R3
Blot strippedand
reprobed
273 kDa225 kDa 210 kDa130 kDa
40 kDa
Anti-(1829-1848) Ab
PurifiedIP3R1
![Page 44: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/44.jpg)
N
C
IP3
Monomeric type 1 IP3R (IP3R1)
CaM
High-affinityCa2+-dependentMutations ineffectiveNot in IP3R3
Low-affinityCa2+-dependentNot in neuronal IP3R1
PKA/PKG phosphoryl.
Low-affinityCa2+-independentResponsible for effects on IP3
binding
![Page 45: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/45.jpg)
Potential physiological role ofPKC-mediated phosphorylation of IP3R1
![Page 46: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/46.jpg)
Potential physiological role ofPKC-mediated phosphorylation of IP3R1
CaMor
Preincubation
ACTIVATION
Ca2+
INHIBITION
Ca2+ CaM
PP
- function ?- interaction ?
PKC
![Page 47: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/47.jpg)
1) Dynamics concerning the intracellular localization of the IP3R
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
![Page 48: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/48.jpg)
During apoptosis, IP3R1 is cleavedto a 95 kDa C-terminal fragment
IP3R
95K
For expression in IP3R ko cells:
ACT.
IN.
![Page 49: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/49.jpg)
(Δ1-1891)IP3R1 expression does not reduce the amount of releasable Ca2+ from the ER
CN
CN
C
N
in the presence of Ca2+ex
![Page 50: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/50.jpg)
Caspase-3 mediated cleavage of IP3R1leads to an increase in ...
Caspase-3 activity Apoptosis
![Page 51: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/51.jpg)
Apoptosis-related increase in [Ca2+]i requires
caspase-3-mediated cleavage of IP3R1
IP3R-KO IP3R1casp
(1-1891)IP3R1(1-225)IP3R1WT-IP3R1
![Page 52: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/52.jpg)
Staurosporine Anti-chicken IgM
Caspase-3activation
IP3R1cleavage
Apoptosis
IP3-independentactivity
[Ca2+]i
Model: feedback of truncated IP3R on apoptosis in an IP3-independent way
![Page 53: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/53.jpg)
GENERAL CONCLUSIONS CaM and CaBP are prime negative regulators ofIP3R function in the presence of Ca2+, but are not the
Ca2+ sensor.
The localization of IP3Rs can be dynamically
regulated, depending on the physiological context.
The phosphorylation of the various IP3R isoforms
by PKC is differentially regulated by PKA, Ca2+, and CaM, which can contribute to the modulation of the spatio-temporal Ca2+ signals.
Caspase-3-mediated cleavage of IP3R1 leads to an
IP3-independent Ca2+ release, which promotes
apoptotic cell death.
![Page 54: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/54.jpg)
GENERAL CONCLUSIONS
Associated proteins and cellular factors
both contribute (in a direct or an indirect way)
to IP3Rs regulation by modulation
of their structure, function and/or localization.
![Page 55: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/55.jpg)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
![Page 56: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/56.jpg)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
![Page 57: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/57.jpg)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
![Page 58: Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004](https://reader035.vdocuments.us/reader035/viewer/2022070307/551a7221550346545e8b613e/html5/thumbnails/58.jpg)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)