Mapping of Calmodulin Binding Sites on the IP 3 R1 N. Nadif Kasri, I. Sienaert, J.B. Parys, G....
1
Mapping of Calmodulin Binding Sites on the IP 3 R1 N. Nadif Kasri, I. Sienaert, J.B. Parys, G. Callewaert, L. Missiaen and H. De Smedt Laboratory of Physiology, K.U.Leuven Campus Gasthuisberg, 3000 Belgium Introduction Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the inositol 1,4,5- trisphosphate receptor (IP 3 R). CaM inhibits IP 3 binding in both the presence and absence of Ca 2+ and IP 3 - induced Ca 2+ release (IICR) in the presence of Ca 2+ . Aim In this study we further charactarized the different CaM- binding sites on the IP 3 R1 in search for their role in the functioning of the intact IP 3 R1. We therefore used recombinant CaM and CaM1234, a Ca 2+ -insensitive mutant. Conclusion In this study we show the presence of two complex CaM-binding sites on the IP 3 R1. 1) In the N-terminal part we show the presence of a discontinuous Ca 2+ -independent CaM-binding site (aa P49-N81and aa E106-S128) that might be responsible for the inhibition of IP 3 binding. 2) In the regulatory domain we show that CaM-binding consists of overlapping Ca 2+ -independent and Ca 2+ - dependent CaM-binding sequences, with the Ca 2+ - independent sequence (aa L1554-R1585) located N- terminal of the Ca 2+ -dependent CaM-binding sequence (aa 1564-R1585). 3) It is conceivable that simultaneous binding to multiple CaM-binding sites is required for proper function on the intact IP 3 R1. Figure 2. Detailed analysis of CaM-binding properties of the N-terminal 1-159 amino acid region of IP 3 R1. (A) Map showing positions of synthetic peptides (A-F) used for binding experiments relative to the N-terminal 159 amino acid region of IP 3 R1. Partial consensus domains for CaM binding are indicated. (B) The increase in dCaM fluorescence emission at = 500 nm upon addition of 1 M peptide (A-F) in the presence or absence of Ca 2+ . Data for each peptide are shown as mean S.D. (n = 3). (C) The Ca 2+ -dependent CaM- binding curve of peptide B to dCaM, data in the presence of 50 M free Ca 2+ were fitted to a binding curve with K d 0.1 M. (D) CaM- binding curve of peptide E to dCaM in the presence or absence of Ca 2+ ; data in the presence of 1 mM EGTA are fitted to a binding curve with K d 1 M; in the presence of 50 M free Ca 2+ the estimated K d value was 1.5 M. Further analysis of the N-terminal 159 aa of the IP 3 R1 shows that two amino acid stretches, peptide B and E were able to bind to dansyl-CaM in a Ca 2+ -independent way. Same conclusions could be drawn from the band-shift experiments(data not shown). A B C E D F 1 159 1-5-10 1-5-10 1-5-8-14 70% IQ (site1) 76% IQ 53% IQ A F/F 0 500 A B C D E F 1,0 1,5 2,0 1 m M EGTA 50 µ M Ca 2+ B Fraction dCaM bound [Peptide B] (M) K d 0.1 µM C 0,0 0,5 1,0 1,5 2,0 2,5 3,0 0,0 0,2 0,4 0,6 0,8 1,0 1 mM EGTA 50 µM free Ca 2+ 0 1 2 3 4 5 6 0,0 0,2 0,4 0,6 0,8 1,0 [Peptide E] (µM) K d 1 µM Fraction dCaM bound D Figure 1 The effect of Ca 2+ , CaM and CaM1234 on binding to Lbs-1His, and Lbs-1His 1-225 (A) 3 HIP 3 binding to IP 3 -binding proteins purified on Ni-NTA (Qiagen) (Lbs-1His and Lbs-1His 1-225) was measured in the presence and absence of Ca 2+ (5 µM) and/or CaM/CaM1234 (10µM) and was expressed as the percentage in absence of these modulators (control). Binding was measured at pH 7.0 in the presence of 1 mM EGTA and 3.5 nM 3 HIP 3 . Data are expressed as the means S.E. of at least three experiments, consisting of independent triplicates. (B) 3 HIP 3 binding to purified Lbs-1His ( ) and Lbs-1His 1-225 ( ) in the presence of indicated concentrations of CaM1234 was expressed as a percentage of the binding measured in Ca 2+ -free buffer (1 mM EGTA, pH 7.0) without CaM1234. Curve fitting was done by Microcal TM Origin Version 6.0. (Northampton, MA) and yielded a EC 50 value of 1.7 M for Lbs-1His. (C) A scatchard analysis of IP 3 binding to Lbs-1His in the presence and absence of CaM is presented. Affinity purified Lbs-1His (1.5 g) was incubated with 3.5 nM [ 3 H]IP 3 at pH 7.0 and increasing concentrations of unlabeled IP 3 in the absence () or presence () of 10 M CaM. CaM and CaM1234 inhibit IP binding in both the presence and absence of Ca 2+ . co n tro l ca ca m ca ca m ca m 1 2 3 4 ca cam 1234 0 20 40 60 80 100 C a 2 + CaM1234 10 µM Ca M12 3 4 Ca 2+ CaM 10 µ M ap oCa M 5 µM Ca 2+ contro l [ 3 H]IP 3 binding (%) Lbs-1His Lbs-1 1-225His 1 581 W226 581 0 5 10 0.0 0.1 0.2 0.3 B/F Bound (nM) 0 .1 1 10 0 20 40 60 80 100 [ 3 H ]IP 3 b in d in g (% ) [C aM 1234] (µM ) EC 50 = 1.7µM A B C Results Figure 4.The effect of CaM and CaM1234 on the IP 3 -induced Ca 2+ release in permeabilized A7r5 cells. The IP 3 induced Ca 2+ release in efflux medium containing 6 mM BAPTA was calculated as the difference between the Ca 2+ release in the presence and that in the absence of IP 3 . Ca 2+ release was induced by 1 µM IP 3 in the absence or presence of 10 µM CaM or CaM1234 at different free [Ca 2+ ] Ca 2+ /CaM is required for inhibitory effects on IP 3 IICR while CaM1234 does not inhibit IICR in the same conditions as Ca 2+ /CaM . 13 18 31 25 Endoplasmic reticulum Cytosol CaM R1:LDSQVNNLFLKSHN-IV QKTA MNW RLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTV QRAA MGW RLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca 2+ /CaM Figure 5. Overview of the CaM binding sites on the IP 3 R. N-terminal: a discontinuous Ca 2+ - independent CaM binding site (P49-N81, E106-E128). Amino acids P49-N81 are highly conserved among the three isoforms. Regulatory domain: Complex site consisting of a high affinity Ca 2+ -dependent CaM-binding site and a low affinity Ca 2+ - independent CaM-binding site. No conserved amino acids between type 1 and 3 IP 3 R. IP 3 R1 Cyt1 C yt11 1 159 1499 1649 2749 IP 3 R1 Cyt1 C yt11 1 159 1499 1649 2749 + + + + not detectable + G ST-C yt11 + K d ~ 0.4 M + K d ~ 0.4 M + + not detectable not detectable G ST-Cyt1 1 m M EGTA 50 M Ca 2+ 1 m M EGTA 50 M Ca 2+ 1 m M EGTA 200 M Ca 2+ In the presence of: dansylC aM Binding to C aM 1234 Pulldow n of overlay 125 ICaM A ssay + + + + not detectable + G ST-C yt11 + K d ~ 0.4 M + K d ~ 0.4 M + + not detectable not detectable G ST-Cyt1 1 m M EGTA 50 M Ca 2+ 1 m M EGTA 50 M Ca 2+ 1 m M EGTA 200 M Ca 2+ In the presence of: dansylC aM Binding to C aM 1234 Pulldow n of overlay 125 ICaM A ssay Table 1. Different methods were used to assay both the Ca 2+ -dependent and Ca 2+ - independent CaM and/or CaM1234 interaction of IP 3 R1 fusion proteins GST-Cyt1 (aa 1-159) or GST-Cyt11 (aa 1499-1649). Figure 3. Detailed analysis of CaM-binding properties of the 1499-1649 amino acid region in the regulatory domain of IP 3 R1. (A) Map showing positions of synthetic peptides (G-J) used for binding experiments relative to the 1499-1649 amino acid region of IP 3 R1. Partial consensus domains for CaM binding are indicated. (B) The Increase in dCaM fluorescence emission at = 500 nm upon addition of 1 M peptide (G-J) in the presence or absence of Ca 2+ . (C) CaM-binding curve of peptide H to dCaM in the presence or absence of Ca 2+ ; data in the presence of 1 mM EGTA are fitted to a binding curve with K d 0.35 µM; in the presence of 50 M free Ca 2+ the estimated K d value was 0.5 µM. (D) The Ca 2+ - dependent CaM- binding curve of peptide I to dCaM, data in the presence of 50 M free Ca 2+ ; data are fitted to a binding curve with K d 0.125 µM. (E) The Ca 2+ -dependent CaM-binding curve of peptide J to dCaM, data in the presence of 50 M free Ca 2+ ; data are fitted to a binding curve with K d 0.075 µM. The CaM binding site of the regulatory domain of the IP 3 R1 contains both, a high affinity Ca 2+ -dependent and a low affinity (K d 0.5 µM) Ca 2+ -independent CaM binding sequence. Same conclusions could be drawn from the band- shift experiments(data not shown). ldsqvnnlflkshnivqkta ldsqvnnlflkshnivqktalmw rlsarnaar kshnivqktalmw rlsarna ar kshnivqktalmw rlsarna arrdsv laasrd 1499 164 9 72% 1-5-8- 14 60 % IQ 76 % IQ 1-5-8- 14 G H I J G H I J 1 .0 1 .5 2 .0 F /F 0 P ep tid es 1 m M EGTA 5 0 µ M Ca 2+ 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.2 0.4 0.6 0.8 1.0 F r a c t i o n d C a M b o u n d [Peptide H] (µM) 1 mM EGTA 50 µM free Ca 2+ K d ~ 0.35-0.5 µM 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 F r a c t i o n d C a M b o u n d [Peptide I] (µM) K d ~ 0.125 µM 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 F r a c t i o n d C a M b o u n d [Peptide J] (µM) K d ~ 0.075 µM A E D C B Different assays show the Ca 2+ -independent interaction of CaM with both GST-Cyt1 and GST-Cyt11 0 10 20 30 40 50 60 70 80 90 100 0.01 0.03 0.1 0.3 0.6 1 [Ca 2+ ](µM) Ca 2+ release(% / 2m in) 1 µM IP3 1 µM IP3/10 µM CaM 1 µM IP3/10 µM CaM 1234
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Transcript of Mapping of Calmodulin Binding Sites on the IP 3 R1 N. Nadif Kasri, I. Sienaert, J.B. Parys, G....
Mapping of Calmodulin Binding Sites on the IP3R1
N. Nadif Kasri, I. Sienaert, J.B. Parys, G. Callewaert, L. Missiaen and H. De Smedt
Laboratory of Physiology, K.U.Leuven Campus Gasthuisberg, 3000 Belgium
IntroductionCalmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the inositol 1,4,5-trisphosphate receptor (IP3R). CaM inhibits IP3
binding in both the presence and absence of Ca2+ and IP3-
induced Ca2+ release (IICR) in the presence of Ca2+.
Aim
In this study we further charactarized the different CaM- binding sites on the IP3R1 in search for their role in the functioning of the intact IP3R1. We therefore used recombinant CaM and CaM1234, a Ca2+-insensitive mutant.
Conclusion
In this study we show the presence of two complex CaM-binding sites on the IP3R1.
1) In the N-terminal part we show the presence of a discontinuous Ca2+-independent CaM-binding site (aa P49-N81and aa E106-S128) that might be responsible for the inhibition of IP3 binding.
2) In the regulatory domain we show that CaM-binding consists of overlapping Ca2+-independent and Ca2+-dependent CaM-binding sequences, with the Ca2+-independent sequence (aa L1554-R1585) located N-terminal of the Ca2+-dependent CaM-binding sequence (aa 1564-R1585).
3) It is conceivable that simultaneous binding to multiple CaM-binding sites is required for proper function on the intact IP3R1.
Figure 2. Detailed analysis of CaM-binding properties of the N-terminal 1-159 amino acid region of IP3R1.
(A) Map showing positions of synthetic peptides (A-F) used for binding experiments relative to the N-terminal 159 amino acid region of IP3R1.
Partial consensus domains for CaM binding are indicated. (B) The increase in dCaM fluorescence emission at = 500 nm upon addition of 1 M peptide (A-F) in the presence or absence of Ca2+. Data for each peptide are shown as mean S.D. (n = 3). (C) The Ca2+-dependent CaM-binding curve of peptide B to dCaM, data in the presence of 50 M free Ca2+ were fitted to a binding curve with Kd 0.1
M. (D) CaM-binding curve of peptide E to dCaM in the presence or absence of Ca2+ ; data in the presence of 1 mM EGTA are fitted to a binding curve with Kd 1 M; in the presence of 50 M free Ca2+ the
estimated Kd value was 1.5 M.
Further analysis of the N-terminal 159 aa of the IP3R1 shows that two amino acid stretches, peptide B and E were able to bind to dansyl-CaM in a Ca2+ -independent way. Same conclusions could be drawn from the band-shift experiments(data not shown).
A
B
C
E
D
F
1 1591-5-10 1-5-10
1-5-8-14 70% IQ (site1) 76% IQ
53% IQ
A
F/F
0 50
0
A B C D E F1,0
1,5
2,0
1 mM EGTA
50 µM Ca2+B
Fra
ctio
n d
CaM
bou
nd
[Peptide B] (M)
Kd 0.1 µMC
0,0 0,5 1,0 1,5 2,0 2,5 3,00,0
0,2
0,4
0,6
0,8
1,0
1 mM EGTA
50 µM free Ca2+
0 1 2 3 4 5 60,0
0,2
0,4
0,6
0,8
1,0
[Peptide E] (µM)
Kd 1 µM
Fra
ctio
n d
CaM
bou
nd
D
Figure 1 The effect of Ca2+, CaM and CaM1234 on binding to Lbs-1His, and Lbs-1His 1-225
(A) 3HIP3 binding to IP3-binding proteins purified on Ni-NTA (Qiagen) (Lbs-1His and Lbs-1His 1-225) was
measured in the presence and absence of Ca2+ (5 µM) and/or CaM/CaM1234 (10µM) and was expressed as the percentage in absence of these modulators (control). Binding was measured at pH 7.0 in the presence of 1 mM EGTA and 3.5 nM 3HIP3. Data are expressed as the means S.E. of at least three experiments, consisting of
independent triplicates. (B) 3HIP3 binding to purified Lbs-1His ( ) and Lbs-1His 1-225 ( ) in the presence of
indicated concentrations of CaM1234 was expressed as a percentage of the binding measured in Ca2+-free buffer (1 mM EGTA, pH 7.0) without CaM1234. Curve fitting was done by MicrocalTM Origin Version 6.0. (Northampton, MA) and yielded a EC50 value of 1.7 M for Lbs-1His. (C) A scatchard analysis of IP3 binding to Lbs-1His in the
presence and absence of CaM is presented. Affinity purified Lbs-1His (1.5 g) was incubated with 3.5 nM [3H]IP3 at
pH 7.0 and increasing concentrations of unlabeled IP3 in the absence () or presence () of 10 M CaM.
CaM and CaM1234 inhibit IP3 binding in both the presence and absence of Ca 2+.
control ca cam ca cam cam1234 ca cam12340
20
40
60
80
100
Ca
2+ C
aM12
34
10 µ
M C
aM12
34
Ca
2+ C
aM
10 µ
M a
poC
aM
5 µM
Ca
2+
cont
rol
[3 H]I
P3 b
indi
ng (
%)
Lbs-1His
Lbs-1 1-225His
1 581
W226 581
0 5 100.0
0.1
0.2
0.3
B/F
Bound (nM)
0.1 1 100
20
40
60
80
100
[3 H]I
P3
bin
din
g (
%)
[CaM1234] (µM)
EC50= 1.7µM
A B
C
Results
Figure 4.The effect of CaM and CaM1234 on the IP3-induced Ca2+ release in permeabilized A7r5 cells.
The IP3 induced Ca2+ release in efflux medium containing 6 mM BAPTA was calculated as the difference between the Ca2+ release in the presence and that in the absence of IP3. Ca2+ release was induced by 1 µM IP3 in the absence or presence of 10 µM CaM or CaM1234 at different free [Ca2+ ]
Ca2+/CaM is required for inhibitory effects on IP3 IICR while CaM1234 does not inhibit IICR in the same conditions as Ca2+/CaM .
13 18
3125
Endoplasmic reticulum
Cytosol CaM
R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLAR2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGGR3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA
R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGANR2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQGR3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD
Ca2+/CaM
Figure 5. Overview of the CaM binding sites on the IP3R.
N-terminal: a discontinuous Ca2+-independent CaM binding site (P49-N81, E106-E128). Amino acids P49-N81 are highly conserved among the three isoforms.
Regulatory domain: Complex site consisting of a high affinity Ca2+-dependent CaM-binding site and a low affinity Ca2+ -independent CaM-binding site. No conserved amino acids between type 1 and 3 IP3R.
IP3R1Cyt1 Cyt11
1 159 1499 1649 2749
IP3R1Cyt1 Cyt11
1 159 1499 1649 2749
++++not detectable
+GST-Cyt11
+
Kd ~ 0.4 M
+
Kd ~ 0.4 M
++not detectable
not detectable
GST-Cyt1
1 mM
EGTA
50 M
Ca2+
1 mM EGTA
50 M
Ca2+
1 mM EGTA
200 M
Ca2+
In the presence of:
dansyl CaMBinding toCaM1234Pull down ofoverlay125ICaM Assay
++++not detectable
+GST-Cyt11
+
Kd ~ 0.4 M
+
Kd ~ 0.4 M
++not detectable
not detectable
GST-Cyt1
1 mM
EGTA
50 M
Ca2+
1 mM EGTA
50 M
Ca2+
1 mM EGTA
200 M
Ca2+
In the presence of:
dansyl CaMBinding toCaM1234Pull down ofoverlay125ICaM Assay
Table 1. Different methods were used to assay both the Ca2+-dependent and Ca2+-independent CaM and/or CaM1234 interaction of IP3R1 fusion proteins GST-Cyt1 (aa 1-159) or GST-Cyt11 (aa 1499-1649).
Figure 3. Detailed analysis of CaM-binding properties of the 1499-1649 amino acid region in the regulatory domain of IP3R1.
(A) Map showing positions of synthetic peptides (G-J) used for binding experiments relative to the 1499-1649 amino acid region of IP3R1. Partial consensus domains for CaM binding are indicated.
(B) The Increase in dCaM fluorescence emission at = 500 nm upon addition of 1 M peptide (G-J) in the presence or absence of Ca2+. (C) CaM-binding curve of peptide H to dCaM in the presence or absence of Ca2+ ; data in the presence of 1 mM EGTA are fitted to a binding curve with Kd 0.35 µM; in the presence of 50 M free Ca2+ the
estimated Kd value was 0.5 µM. (D) The Ca2+- dependent CaM-
binding curve of peptide I to dCaM, data in the presence of 50 M free Ca2+; data are fitted to a binding curve with Kd 0.125 µM.
(E) The Ca2+-dependent CaM-binding curve of peptide J to dCaM, data in the presence of 50 M free Ca2+; data are fitted to a binding curve with Kd 0.075 µM.
The CaM binding site of the regulatory domain of the IP3R1 contains both, a high affinity Ca2+-dependent and a low affinity (Kd 0.5 µM) Ca2+-independent CaM binding sequence. Same conclusions could be drawn from the band-shift experiments(data not shown).
ldsqvnnlflkshnivqktaldsqvnnlflkshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaarrdsvlaasrd
1499 164972% 1-5-8-1460 % IQ
76 % IQ 1-5-8-14
GH
IJ
G H I J1.0
1.5
2.0
F/F
0
Peptides
1 mM EGTA
50 µM Ca2+
0.0 0.5 1.0 1.5 2.0 2.5 3.00.0
0.2
0.4
0.6
0.8
1.0
Fra
c tio
n d
CaM
bou
nd
[Peptide H] (µM)
1 mM EGTA50 µM free Ca2+
Kd ~ 0.35-0.5 µM
0.0 0.1 0.2 0.3 0.4 0.50.0
0.2
0.4
0.6
0.8
1.0
Fra
c tio
n d
CaM
bou
nd
[Peptide I] (µM)
Kd ~ 0.125 µM
0.0 0.1 0.2 0.3 0.4 0.50.0
0.2
0.4
0.6
0.8
1.0
Fra
c tio
n d
CaM
bou
nd
[Peptide J] (µM)
Kd ~ 0.075 µM
A
EDC
B
Different assays show the Ca2+ -independent interaction of CaM with both GST-Cyt1 and GST-Cyt11
0102030405060708090
100
0.01 0.03 0.1 0.3 0.6 1
[Ca2+
] (µM)
Ca
2+ r
elea
se (%
/ 2 m
in)
1 µM IP3
1 µM IP3/ 10 µM CaM
1 µM IP3/ 10 µM CaM1234
