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The Journal of Neuroscience, January 15, 2003, 23(2):403-415
Modulation of Type 1 Inositol (1,4,5)-Trisphosphate Receptor
Function by Protein Kinase A and Protein Phosphatase 1
Tie-Shan
Tang*,
Huiping
Tu*,
Zhengnan
Wang, and
Ilya
Bezprozvanny
Department of Physiology, University of Texas Southwestern Medical
Center, Dallas, Texas 75390
 |
ABSTRACT |
Type 1 inositol (1,4,5)-trisphosphate receptors
(InsP3R1s) play a major role in neuronal calcium
(Ca2+) signaling. The InsP3R1s are
phosphorylated by protein kinase A (PKA), but the functional
consequences of InsP3R1 phosphorylation and the mechanisms
that control the phosphorylated state of neuronal InsP3R1s
are poorly understood. In a yeast two-hybrid screen of rat brain cDNA
library with the InsP3R1-specific bait, we isolated the
protein phosphatase 1
(PP1). In biochemical experiments, we
confirmed the specificity of the InsP3R1-PP1
association and immunoprecipitated the InsP3R1-PP1 complex
from rat brain synaptosomes and from the neostriatal lysate. We also
established that the association with PP1 facilitates dephosphorylation
of PKA-phosphorylated InsP3R1 by the endogenous neostriatal
PP1 and by the recombinant PP1. We demonstrated that exposure of
neostriatal slices to 8-bromo-cAMP, dopamine, calyculin A, or
cyclosporine A, but not to 10 nM okadaic acid, promotes the
phosphorylation of neostriatal InsP3R1 by PKA in
vivo. We discovered that PKA activates and PP1 inhibits the activity of recombinant InsP3R1 reconstituted into planar
lipid bilayers. We found that phosphorylation of InsP3R1 by
PKA induces at least a fourfold increase in the sensitivity of
InsP3R1 to activation by InsP3 without shifting
the peak of InsP3R1 bell-shaped Ca2+
dependence. Based on these data, we suggest that InsP3R1
may participate in cross talk between cAMP and Ca2+
signaling in the neostriatum and possibly in other regions of the brain.
Key words:
inositol trisphosphate receptor; calcium signaling; dopamine; protein phosphorylation; yeast two-hybrid; planar lipid
bilayers
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Introduction |
Calcium ions
(Ca2+) are universal second messengers.
Changes in cytosolic Ca2+ concentration
influence most fundamental cellular processes in neuronal and
non-neuronal cells (Berridge, 1993
, 1998). Inositol 1,4,5-trisphosphate
(InsP3), a soluble compound generated by
enzymatic cleavage of the lipid phosphatidylinositol
4,5-bisphosphate after activation of phospholipase C (PLC), is a
second messenger used by many cell types to stimulate
Ca2+ release from intracellular
Ca2+ stores.
InsP3-induced Ca2+
release in these cells is supported by a highly specialized
Ca2+ channel, the inositol
(1,4,5)-trisphosphate receptor (InsP3R). Three
mammalian isoforms of InsP3R have been
identified, each with the unique expression pattern (for review, see
Furuichi et al., 1994; Taylor et al., 1999). The three mammalian
InsP3R isoforms share 60-70% amino acid
homology, but the differences in their functional properties are poorly
understood (for review, see Thrower et al., 2001). The type 1 InsP3R (InsP3R1) is a
predominant isoform in the CNS. Targeted deletion of
InsP3R1 gene in mice induces ataxia and
epileptic seizures, followed by a premature death (Matsumoto et al.,
1996), highlighting the importance of InsP3R1 for
brain function.
InsP3R1s are subjected to multiple levels of
regulation in cells (Bezprozvanny and Ehrlich, 1995). Binding of
InsP3 triggers the InsP3R1
channel opening. The activity of InsP3R1 is under feedback control by cytosolic Ca2+; at low
Ca2+ concentrations,
Ca2+ acts as a coactivator of the
InsP3R1, and at high
Ca2+ concentrations, the
InsP3R1 is inhibited by
Ca2+ (Iino, 1990; Bezprozvanny et al.,
1991; Finch et al., 1991; Kaznacheyeva et al., 1998). The activity of
InsP3R1 is allosterically potentiated by ATP
(Ferris et al., 1990; Iino, 1991; Bezprozvanny and Ehrlich, 1993). The
InsP3R1 is also one of the major substrates of
protein kinase A (PKA) phosphorylation in the brain (Walaas et al.,
1986; Supattapone et al., 1988; Maeda et al., 1990; Danoff et al.,
1991; Ferris et al., 1991a; Haug et al., 1999; Pieper et al., 2001). PKA can phosphorylate InsP3R1 at two sites, S1589
and S1755 (Danoff et al., 1991; Ferris et al., 1991a; Haug et al.,
1999; Pieper et al., 2001). Both sites are located in the coupling
domain of the InsP3R1 (Furuichi et al., 1994),
and PKA phosphorylation is likely to affect
InsP3R1 function. However, functional
consequences of neuronal InsP3R1 phosphorylation
by PKA remain controversial. An activation (Volpe and Alderson-Lang,
1990; Nakade et al., 1994; Wojcikiewicz and Luo, 1998) or an inhibition
(Supattapone et al., 1988; Cameron et al., 1995) of
InsP3R1 by PKA was observed using Ca2+ flux measurements.
What are the mechanisms that control phosphorylation of
InsP3R1 by PKA in the brain? What are the
functional consequences of InsP3R1
phosphorylation by PKA? Is modulation of neuronal
InsP3R1 function by PKA physiologically relevant?
Here we address some of these questions. In a yeast two-hybrid screen
of rat brain cDNA library with the
InsP3R1-specific bait, we isolated a cDNA of
protein phosphatase 1 (PP1). In a series of biochemical and electrophysiological in vitro experiments, we analyzed the
importance of InsP3R1-PP1 association for
control of InsP3R1 phosphorylation by PKA and
modulation of InsP3R1 activity. In addition, we
characterized the phosphorylation of InsP3R1 by
PKA during neostriatal dopaminergic signaling in vivo. Our
results suggest that InsP3R1 may play a role in
the cross talk between cAMP and Ca2+
signaling pathways in the neostriatum (Greengard et al., 1999) and
possibly in other regions of the brain.
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Materials and Methods |
Yeast two-hybrid methods. The C-terminal regions of
rat InsP3R1 (Mignery et al., 1990) (amino acids
Q2714-A2749), rat InsP3R2 (Sudhof et al., 1991)
(amino acids Q2666-H2701), and rat InsP3R3 (Blondel et al., 1993) (amino acids Q2641-R2670) were amplified by PCR
and cloned into pLexN vector to yield IC1, IC2, and IC3 baits. Mutant
and truncated versions of IC1 bait were generated by PCR and verified
by sequencing. The yeast two-hybrid screen of rat brain cDNA library in
pVp16-3 vector (3 × 105 independent
clones; gift from Dr T. Südhof, University of Texas Southwestern Medical Center, Howard Hughes Medical Institute, Dallas,
TX) with IC1 bait was performed according to published procedures (Hata
et al., 1996). Coding sequences of mouse PP1
and human PP1
were
amplified by PCR from expressed sequence tags (ESTs)
(GenBank accession numbers BF179322 and BG389563) and subcloned
into pVp16-3 prey vector. The liquid yeast two-hybrid assays were
performed as described previously (Maximov et al., 1999).
In vitro binding assay. RIGLLGHPPHMNVNPQQPA (RIGL-V1,
2731-2749 of rat InsP3R1), RLGFLGSNTPHENHHMPPH
(RLGF-V2, 2683-2701 of rat InsP3R2), and
RLGFVDVQNCMSR (RLGF-V3, 2658-2670 of rat
InsP3R3) peptides were synthesized and coupled to
N-hydroxysuccinimide (NHS)-activated Sepharose
according to the manufacturer's (Amersham Biosciences, Uppsala,
Sweden) instructions. The rat PP1 was cloned into
hemagglutinin (HA)-pCMV5 vector (Maximov et al., 1999),
expressed in COS7 cells by DEAE-dextran transient transfection, and
solubilized in the extraction buffer A [1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
137 mM NaCl, 2.7 mM KCl,
4.3 mM
Na2HPO4, 1.4 mM
KH2PO4, 5 mM EDTA, 5 mM EGTA, and
protease inhibitors]. The HA-PP1-containing extract was clarified
by 20 min of centrifugation (100,000 × g in TL-100)
and incubated with RIGL-V1, RLGF-V2, and RLGF-V3 Sepharose beads for 16 hr at 4°C. Beads were washed with 40 bead volumes of the extraction
buffer A, and attached proteins were sequentially eluted with 1 bead
volume of 1 M NaCl and then 1 bead volume of 1%
SDS. Samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies.
Immunoprecipitations. The RT1 baculovirus encoding the
SI(
)/SII(+) splice variant of rat InsP3R1
(Mignery et al., 1990) has been described previously (Tu et al., 2002).
The RT1
C baculovirus encoding rat InsP3R1
truncated at position G2736 was generated using the Bac-to-Bac system
(Invitrogen, San Diego, CA) as described previously (Tu et al., 2002).
High Five (Invitrogen) or Sf9 (American Type Culture Collection,
Manassas, VA) insect cells were infected with high titer
(>108 colony-forming units/ml) stocks of
RT1 and RT1C baculoviruses as described previously (Tu et al.,
2002). At 48 hr after infection, the insect cells were solubilized in
the extraction buffer A. Rat PP1, mouse PP1, and human PP1
were amplified by PCR from ESTs, cloned into HA-pCMV vector (Maximov
et al., 1999), expressed in COS7 cells by DEAE-dextran transient
transfection, and solubilized in the extraction buffer A. Extracts from
Sf9 cells and COS7 cells were clarified by centrifugation (100,000 × g in TL-100), mixed together, and immunoprecipitated for
2 hr at 4°C with anti-HA monoclonal antibodies (mAbs) attached to
protein G-agarose beads. mAb against InsP3R1 was
used as a positive control. The amount of precipitated
InsP3R1 was quantified by
[3H]InsP3 binding
as described previously (Kaznacheyeva et al., 1998). Glutathione
S-transferase (GST), GST-IC1, GST-IC2, and GST-IC3
fusion proteins (in pGEX-KG; Amersham Biosciences) were expressed in
BL21 cells, purified on glutathione beads as described previously
(Maximov et al., 1999), and added to immunoprecipitation reactions at a
concentration of 200 µg/ml. Cortex rat brain synaptosomes and rat
neostriatum homogenates were prepared according to published procedures
(Jones and Matus, 1974; Nishi et al., 1997; Maximov et al., 1999) and
verified by Western blotting with anti-postsynaptic density 95 (PSD95)
and anti-dopamine and cAMP-regulated phosphoprotein (DARPP)-32
polyclonal antibodies, respectively. The synaptosomes and neostriatum
homogenates were solubilized in extraction buffer A, clarified by
centrifugation (100,000 × g in TL-100), and
immunoprecipitated with anti-InsP3R1 T443
polyclonal antibodies attached to protein A-Sepharose beads. The
precipitate was analyzed by Western blotting with mAbs against PP1.
In vitro dephosphorylation assay. Recombinant RT1 and
RT1C were precipitated from insect cell extracts with the
anti-InsP3R1 polyclonal antibodies (T443 or
cytl3b2, respectively) attached to protein A-Sepharose beads and
phosphorylated as described previously (Wojcikiewicz and Luo, 1998).
Briefly, precipitated RT1 or RT1C was washed three times with the
ice-cold phosphorylation buffer (120 mM KCl, 50 mM Tris, pH 7.2, 0.3 mM
MgCl2, 0.1% Triton X-100) and resuspended in the
phosphorylation buffer. The phosphorylation reaction was initiated by
addition of 5 µCi [-32P]ATP, 5 µM ATP, and 10 U of PKA bovine heart catalytic
subunit in 200 µl volume; continued for 1 hr at 30°C; and stopped
by addition of 1.3 ml of ice-cold phosphorylation buffer containing 2 mM ATP. The beads were pelleted, washed two times
with the dephosphorylation buffer I (50 mM NaCl,
50 mM Tris, pH 7.2, 0.7 mg/ml BSA, 3.3 mM caffeine, 0.15 mM
MnCl2, 1.0 mM DTT), and
resuspended in 100 µl of the dephosphorylation buffer I. The
dephosphorylation reactions were initiated by addition of 0.1 U of
rabbit recombinant PP1 or 2 × 10
4 U of human recombinant PP1 (both
from Calbiochem, La Jolla, CA), incubated at 30°C for 0-40 min, and
stopped by addition of 5 mM EDTA.
The rat neostriatum homogenate prepared as described previously (Nishi
et al., 1999) was used as a source of endogenous PP1 activity (nsPP1).
The dephosphorylation reactions with nsPP1 were performed in
dephosphorylation buffer II (50 mM Tris-Cl, 50 mM NaCl, pH 7.2, 0.1 mM EGTA, 1 nM
okadaic acid, 1 mM DTT, 0.7 mg/ml BSA, 3.3 mM
caffeine). The nsPP1 dephosphorylation reactions were initiated by
addition of 4 µg of striatal homogenate and stopped by addition of
1.4 ml of ice-cold dephosphorylation buffer II, brief centrifugation,
and rapid (within 3 min) addition of equal volume of 2× SDS-gel
loading buffer. Resulting samples were boiled for 5 min, separated by
SDS-electrophoresis on 8% polyacrylamide gel, and analyzed by
phosphoimaging (Bio-Rad, Richmond, CA). GST/GST-IC1 (200 µg/ml),
DARPP-32/pDARPP-32 (0.2 µM), or PP1 inhibitor-2 (Inh2) (0.2 µM) were added to dephosphorylation reactions as
indicated in Results.
Neostriatal InsP3R1
back-phosphorylation. The neostriatum of adult rats was dissected
(Nishi et al., 1997), chopped into small slices (~1-2 × 1-2
mm) in ice-cold, oxygenated (95%O2-5%
CO2) Krebs-HCO3 buffer,
aliquoted, washed, and preincubated in 5 ml of fresh Krebs-HCO3 buffer at
30°C under constant oxygenation for 60 min, with a single change of
medium. The neostriatum slices were then placed into fresh
Krebs-HCO3 buffer
containing 20 µM IBMX and treated with
8-bromo-cAMP (8-Br-cAMP), dopamine, cyclosporine A, calyculin A,
or okadaic acid as indicated in Results. After the drug
treatment, the pieces were collected, homogenized, and solubilized in
the extraction buffer A containing 0.5 mM
Na3VO4. The extracts were
clarified by centrifugation (100,000 × g in TL-100),
and protein concentration in lysates was determined by Bio-Rad assay.
The equal amounts of protein from each lysate were used for
immunoprecipitation with anti-InsP3R1 T443
polyclonal antibodies attached to protein A-Sepharose beads. The
precipitated InsP3R1s were phosphorylated
in vitro by the catalytic subunit of PKA in the presence of
[-32P]ATP and analyzed by
phosphoimaging as described above. When the neostriatal lysate was
dephosphorylated by PP1 before in vitro phosphorylation
by PKA, the measured content of the
32P-InsP3R1 band
(32PPP1
) was
interpreted as total InsP3R1 in the neostriatal sample. To calculate the fraction of InsP3R1 in
the PKA-phosphorylated state, the 32P
content of the InsP3R1 band at each data point
(32P-InsP3R1) was
normalized to the total InsP3R1 content, as
follows: pInsP3R1 = (32PPP1 32P-InsP3R1)/32PPP1.
Planar lipid bilayer experiments. Single-channel recordings
of recombinant RT1 or RT1C activity were performed as described previously (Tu et al., 2002) at 0 mV transmembrane potential using 50 mM Ba2+
dissolved in HEPES, pH 7.35, in the trans (intraluminal)
side as a charge carrier. The cis (cytosolic) chamber
contained 110 mM Tris dissolved in HEPES, pH
7.35, log ([Ca2+]) (pCa) 6.7 (0.2 mM EGTA plus 0.14 mM
CaCl2) (Bezprozvanny et al., 1991), and 3%
sucrose. InsP3R1s were activated by addition of 2 µM InsP3 (Alexis) to the
cis chamber. The cis chamber contained 0.5 mM MgATP or 0.3 mM
MgCl2 plus 0.1 mM
Li4ATPS as indicated in Results. PKA
bovine heart catalytic subunit was diluted in 110 mM Tris/HEPES, pH 7.35, containing 0.2 mM ruthenium red to 2 U/µl. Rabbit recombinant
PP1 was diluted in 110 mM Tris/HEPES, pH 7.35, containing 0.2 mM ruthenium red and 0.2 mM MnCl2 to 1 U/µl. One
microliter of PKA or PP1 stocks was added directly to the bilayer
without stirring. The phosphorylation/dephosphorylation reactions were
stopped 1 min after PKA/PP1 addition by stirring the solution in the
cis chamber for 30 sec. Stirring resulted in a 3000-fold
reduction of PKA/PP1 concentration (1 µl in 3 ml dilution),
greatly reducing the rate of InsP3R1
phosphorylation/dephosphorylation in the bilayer. In
Ca2+-dependence experiments, the free
Ca2+ concentration in the
cis-chamber was controlled in the range of 10 nM (pCa 8) to 10 µM (pCa
5) by a mixture of 1 mM EGTA, 1 mM HEDTA, and variable concentrations of
CaCl2. The resulting free
Ca2+ concentration was calculated by using
a program described by Fabiato (1988). InsP3
dependence was measured by consecutive addition of
InsP3 to the cis chamber from 1 mM stock. All additions
(InsP3, ATP, CaCl2) were to
the cis chamber from the concentrated stocks, with at least
30 sec of stirring of solutions in both chambers. The
InsP3R1 single-channel currents were amplified
(OC-725; Warner Instruments, Hamden, CT), filtered at 1 kHz with a
low-pass eight pole Bessel filter, digitized at 5 kHz (Digidata 1200;
Axon Instruments, Foster City, CA), and stored on computer hard drive
and recordable optical disks.
For off-line computer analysis (pClamp 6; Axon Instruments)
single-channel data were filtered digitally at 500 Hz; for presentation of the current traces, data were filtered at 200 Hz. Evidence for the
presence of two to three functional channels in the bilayer was
obtained in the majority of experiments. The number of active channels
in the bilayer was estimated as a maximal number of simultaneously open
channels during the course of an experiment (Horn, 1991). The open
probability of closed level and first and second open levels was
determined by using half-threshold crossing criteria (t 
2 msec) from the records lasting at least 2.5 min. The
single-channel open probability (Po)
for one channel was calculated using the binomial distribution for the
levels 0, 1, and 2, assuming that the channels were identical and
independent (Colquhoun and Hawkes, 1983). To construct
InsP3 and Ca2+
dependence curves for the InsP3R1 in control and
PKA-phosphorylated states, the determined values of
Po were averaged across several independent experiments at each InsP3 or
Ca2+ concentration. For
InsP3-dependence experiments, the averaged values
of Po are presented as mean ± SE
(n = number of independent experiments) and fit by the
following equation: Po
(InsP3) = Pmax (InsP3)n/[(InsP3)n + kInsP3n)],
modified from Lupu et al. (1998), where
Pmax is a maximal Po value, n is a Hill
coefficient, and kInsP3 is the
apparent affinity of InsP3R1 for
InsP3. For
Ca2+-dependence experiments, the averaged
values of Po are presented as
mean ± SE (n = number of independent experiments)
and fit by the following bell-shaped equation:
Po
(Ca2+) = 4Pm
kn(Ca2+)n/[(kn + [Ca2+]n)(Kn + [Ca2+]n)],
modified from Bezprozvanny et al. (1991), where
Pm is a parameter proportional to the
maximal Po value, n is a
Hill coefficient, k is the apparent affinity of the
Ca2+ activating site, and K is
the apparent affinity of the Ca2+
inhibitory site. The fitting procedure used in this study differs from
the procedure used in our previous studies (Bezprozvanny et al., 1991;
Kaznacheyeva et al., 1998; Lupu et al., 1998; Nosyreva et al., 2002; Tu
et al., 2002) in that Po values in the
present study were not normalized to the maximal
Po before averaging and fitting.
Because Po values were not normalized,
Pm is equal to maximal
Po when k = K. If k 
K, Pm is
proportional (and higher) than maximal
Po.
Materials. The following mAbs were used: anti-HA for HA.11
(Covance), anti-InsP3R1 (Calbiochem), and
anti-PP1 mAb (Transduction Laboratories, Lexington, KY). The following
polyclonal antibodies were used: C-terminal
anti-InsP3R1 T443 (Kaznacheyeva et al., 1998),
N-terminal anti-InsP3R1 cytl3b2 (gift from J. Parys, Ku Leuven, Belgium) (Sipma et al., 1999), anti-PSD95
(gift from T. Südhof), and anti-DARPP-32 (Cell Signaling
Technologies). Protein G-agarose beads were supplied by Santa Cruz
Biotechnology (Santa Cruz, CA); protein A-Sepharose beads and
[-32P]ATP were obtained from Amersham
Biosciences; rabbit recombinant PP1, human recombinant PP1,
DARPP-32, pDARPP-32, PP1 inhibitor-2, calyculin A, and okadaic acid
were obtained from Calbiochem, and InsP3 was
supplied by Alexis. PKA bovine heart catalytic subunit, Li4ATPS, and all other reagents are from Sigma
(St. Louis, MO).
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Results |
InsP3R1 specifically binds PP1
Each of three mammalian InsP3R isoforms
contains a unique cytosolic C-terminal tail preceded by a highly
conserved region (Fig. 1a). To
search for the InsP3R1-specific neuronal binding partners, we performed a yeast two-hybrid screen of rat brain cDNA
library with the IC1 bait (amino acids Q2714-A2749 of rat InsP3R1) (Fig. 1a) and isolated the
full-length clone of PP1. When the corresponding regions of
InsP3R2 (IC2) and InsP3R3
(IC3) (Fig. 1a) were tested in a liquid yeast two-hybrid
assay, we found that PP1 did not bind IC2 and only weakly associated
with IC3 (Fig. 1b). Three isoforms of PP1 are expressed in
mammalian brain, each with a unique expression pattern (da Cruz e Silva
et al., 1995). In a yeast two-hybrid assay, IC1 associated with PP1
but not with PP1 or PP1 (Fig. 1b). No interaction of
IC2 or IC3 baits with PP1 or PP1 was detected in our yeast
two-hybrid experiments (Fig. 1b). Thus, the association
appears to be specific for the InsP3R1-PP1
pair.
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Figure 1.
InsP3R1 specifically binds PP1 in a
yeast two-hybrid assay. a, Alignment of C-terminal
regions of the InsP3R1, InsP3R2, and
InsP3R3. The IC1 fragment was used as a bait in the yeast
two-hybrid screen. The position of the
2731RIGL2734 motif in the IC1 bait is
indicated by a bar above the sequence. The domain
structure of the InsP3R C-terminal region [constant
(C)-RXGX-variable
(V)] is shown below the alignment.
b, Specificity of InsP3R-PP1 interactions.
IC1, IC2, and IC3 baits were tested for the strength of interactions
with PP1, PP1, and PP1 preys in liquid yeast two-hybrid
assays. The data are normalized to the strength of interaction for the
IC1-PP1 pair and are shown as mean ± SEM
(n 3). -gal,
-galactosidase.
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The PP1-targeting proteins share the R/K-V/I-X-F docking motif
(Greengard et al., 1999). A similar RIGL motif is present within IC1
sequence (Fig. 1a, indicated by a bar). However,
a similar RLGF motif is also present in IC2 and IC3 sequences (Fig.
1a), which are not strong PP1-binding partners (Fig.
1b). Where is a specific PP1-binding site in the IC1
sequence? To address this question, we performed a systematic analysis
of PP1 binding specificity by liquid yeast two-hybrid assay. From
sequence alignment of InsP3R isoforms, we
reasoned that the InsP3R C-terminal sequence
could be divided into a conserved (C) domain, RXGX motif, and variable (V) regions (Fig. 1a). A deletion of conserved domain had no
effect on IC1 association with PP1 (Fig.
2a), indicating that the RIGL motif and V1 variable domain (RIGL-V1) is sufficient for association with PP1. In contrast, the corresponding regions of IC2 and IC3 baits (RLGF-V2 and RLGF-V3) did not bind PP1 (Fig. 2a),
confirming a specificity of the interaction. To determine a role for
the RIGL motif in specific association with PP1, we generated a
series of IC1 bait point mutants and tested them in a yeast two-hybrid assay with PP1 prey. We found that in the context of the IC1 bait,
mutations of RIGL motif to RIGA or RAGL
had no apparent effect on the strength of interactions with PP1
(Fig. 2a, bold indicates mutated residues). In
fact, mutations of the RIGL motif to the RLGF
motif present in IC2 and IC3 baits or to the RIGF motif
corresponding to the "canonical" PP1 docking motif (Greengard et
al., 1999) resulted in approximately a twofold increase in the strength
of interaction with PP1 (Fig. 2a). Thus, presence of the
RIGL motif does not explain PP1 specificity for the IC1 bait.
Interestingly, the V1 variable region of IC1 bait alone is not
sufficient for association with PP1 (Fig. 2a). From these
results, we concluded that the association with PP1 requires the
RIGL motif and V1 variable region of IC1 (RIGL-V1, R2731-A2749), with
the specificity for IC1 conferred by the variable region. To further
confirm these findings, we coupled the peptides corresponding to the
RXGX motif and variable sequence of InsP3R1, InsP3R2, and InsP3R3 to
NHS-Sepharose and performed pull-down experiments with HA-tagged PP1
transiently expressed in COS cells (Fig.
3b). We found that the
InsP3R1-specific peptide (RIGL-V1) but not the
InsP3R2- or the
InsP3R3-specific peptides (RLGF-V2 and RLGF-V3)
formed a salt-sensitive complex with HA-PP1 (Fig. 2b).
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Figure 2.
PP1-binding motif in the InsP3R1
sequence. a, Analysis of PP1 binding specificity. IC1
(C-RIGL-V1, 2714-2749 of InsP3R1), RIGL-V1 (2731-2749 of
InsP3R1), RLGF-V2 (2683-2701 of InsP3R2),
RLGF-V3 (2658-2670 of InsP3R3), IC1 point mutants in the
RIGL motif (indicated in bold), and V1 (2736-2749 of
InsP3R1) baits were tested with PP1 prey in liquid yeast
two-hybrid assays. The data are normalized to the strength of
interaction for the IC1-PP1 pair and are shown as mean ± SEM
(n 3). -gal,
-galactosidase. b, HA-PP1 pull-down experiments
with RIGL-V1 (2731-2749 of InsP3R1), RLGF-V2 (2683-2701
of InsP3R2), and RLGF-V3 (2658-2670 of
InsP3R3) peptides. Fractions eluted from the beads by 1 M NaCl and SDS were analyzed by Western blotting with
anti-HA mAbs. The input lane on all three panels
contains  th of the COS cell lysate used for
pull-downs.
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Figure 3.
InsP3R1 binds PP1 in
vitro. a, Expression of full-length (RT1) or
truncated (RT1C) recombinant InsP3R1 in Sf9 cells was
analyzed by Western blotting with polyclonal antibodies directed
against the InsP3R1 N-terminal region (cytl3b2).
b, Expression of HA-tagged , , and PP1
isoforms in COS cells was analyzed by Western blotting with anti-HA
mAbs. c, GST, GST-IC1, GST-IC2, and GST-IC3 proteins
were expressed in BL21 Escherichia coli was purified on
glutathione beads and analyzed by Coomassie staining. d,
Analysis of InsP3R1-PP1 association in
vitro by immunoprecipitation. HA-tagged , , and PP1
isoforms were mixed with solubilized full-length (RT1) or truncated
(RT1C) recombinant InsP3R1 and precipitated with anti-HA
mAbs. GST, GST-IC1, GST-IC2, and GST-IC3 proteins were included in
the immunoprecipitation reactions at a 200 µg/ml concentration as
indicated. The amount of precipitated InsP3R1 was
quantified by [3H]InsP3 binding.
Anti-InsP3R1 mAbs (IP3R1Ab) were used as a
positive control; empty beads (protein G beads) were used as a negative
control.
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To further confirm a specific association between
InsP3R1 and PP1, we performed a series of
in vitro binding experiments. For these experiments,
full-length (RT1) and truncated (RT1C) rat
InsP3R1 were expressed in insect cells by
baculovirus infection (Fig. 3a) and solubilized in CHAPS.
The HA-tagged PP1, PP1, and PP1 were transiently expressed in
COS cells (Fig. 3b), solubilized in CHAPS, mixed with the
InsP3R1-containing lysates, and precipitated with
anti-HA antibodies. The amount of immunoprecipitated
InsP3R1 was quantified by
[3H]InsP3 binding
assay. We found that HA-PP1, but not HA-PP1 or HA-PP1,
efficiently precipitated the InsP3R1 (Fig.
3d). The ability of HA-PP1 to precipitate
InsP3R1 critically depended on the
InsP3R1 C-terminal region, because HA-PP1 did
not precipitate RT1C protein (Fig. 3d). In complementary
experiments, we found that GST-IC1, but not GST alone, GST-IC2, or
GST-IC3 proteins (Fig. 3c), effectively interfered with the
InsP3R1 precipitation by HA-PP1 (Fig.
3d). Thus, the most C-terminal region of the InsP3R1 is both necessary and sufficient for
specific association with PP1.
Do InsP3R1 and PP1 associate in
vivo? In the brain, PP1 is concentrated in postsynaptic spines
(Ouimet et al., 1995). The InsP3R1 is also
present in postsynaptic terminals (Sharp et al., 1993a,b). To establish
whether InsP3R1-PP1 complexes form in synaptic locations, we isolated cortical rat brain synaptosomes, extracted the obtained material in CHAPS, precipitated with the anti-InsP3R1 polyclonal antibody, and blotted
with the anti-PP1 mAb. We found that PP1 was precipitated by
anti-InsP3R1 antibodies but not by the preimmune
sera (Fig. 4a). In the brain,
the PP1 isoform is most enriched in the neostriatum region (da Cruz
e Silva et al., 1995). Are InsP3R1-PP1
complexes formed in the neostriatum? By following published procedures
(Nishi et al., 1997), we isolated the neostriatum region of the adult
rat brain and performed immunoprecipitation experiments. Similar to
experiments with the synaptosomes, PP1 was precipitated from the
neostriatum by anti-InsP3R1 antibodies but not by
the preimmune sera (Fig. 4b). Thus,
InsP3R1-PP1 complexes exist in synaptic
locations and in the neostriatum region of the brain. The PP1 mAbs
available to us do not discriminate between different PP1 isoforms, but based on the specificity of InsP3R1 interactions
in vitro (Figs. 1-3), it is likely that the observed
complexes correspond to InsP3R1-PP1.
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Figure 4.
InsP3R1 binds PP1 in
vivo. The InsP3R1 forms complexes with PP1 in brain
synaptosomes (a) and in the neostriatum
(b). The samples were precipitated with
anti-InsP3R1 polyclonal antibodies (T443) and blotted with
anti-PP1 mAbs. Preimmune sera (P/S) were used as a
negative control. The input lane on a and
b contains th of the lysate used for
immunoprecipitation. Quantification of PP1 band intensity suggests that
3.8% (synaptosomes) and 3.4% (neostriatum) of total PP1 is associated
with the InsP3R1.
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PP1 dephosphorylates PKA-phosphorylated InsP3R1
in vitro
The InsP3R1 is one of the major substrates
of PKA phosphorylation in the brain (Supattapone et al., 1988; Danoff
et al., 1991; Ferris et al., 1991a; Haug et al., 1999; Pieper et al.,
2001). In the neostriatum, PP1 and PKA play an antagonistic role
(Greengard et al., 1999). Can neostriatal PP1 dephosphorylate
InsP3R1? To answer this question, we performed a
series of in vitro dephosphorylation experiments. For these
experiments, InsP3R1 (RT1) was expressed in
insect cells by baculovirus infection (Fig. 3a),
immunoprecipitated, and phosphorylated in vitro by a
catalytic subunit of PKA in the presence of
[-32P]ATP. The
32P-InsP3R1 was
incubated for a variable amount of time with the rat neostriatal
homogenate. Rapid dephosphorylation of
32P-InsP3R1 by
neostriatal homogenate was observed (Fig.
5a). The dephosphorylation
assay was performed in the presence of 0.1 mM EGTA and 1 nM okadaic acid to inhibit PP2A, PP2B,
and PP2C activities (Nishi et al., 1999). Under these conditions,
dephosphorylation of InsP3R1 by neostriatal
homogenate was almost completely inhibited by Inh2 (Fig.
5c), confirming that the observed phosphatase activity corresponds to the activity of endogenous nsPP1.
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Figure 5.
PP1 dephosphorylates the InsP3R1
in vitro. a, Effects of GST
(top) and GST-IC1 (bottom) on the
32P-InsP3R1 dephosphorylation by endogenous
nsPP1 in vitro. For each sample, the time of incubation
with nsPP1 is indicated above the autoradiogram. The experiment was
repeated three times with similar results. b, The
normalized data from several independent experiments were averaged
together and plotted in semilogarithmic coordinates as mean ± SE
(n = 3) for experiments in the presence of GST
(open circles) and GST-IC1 (filled
circles). The rate of the dephosphorylation reaction is
determined from the slope of the straight line used to
fit the data. c, Summary of the
32P-InsP3R1 in vitro
dephosphorylation experiments. The rates of
32P-InsP3R1 dephosphorylation
(Deph) reactions determined as described for
b are shown as mean ± SE (n 3). DARPP-32 and pDARPP-32 indicate recombinant unphosphorylated and
PKA-phosphorylated forms of DARPP-32, respectively.
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The substrate specificity of PP1 is primarily determined by its
targeting subunits, such as spinophilin, neurabin, and
GM (Greengard et al., 1999). Is it possible that
the identified InsP3R1-PP1 association (Figs.
1-4) facilitates the InsP3R1 dephosphorylation by PP1? To test this hypothesis, we compared the rates of
32P-InsP3R1
dephosphorylation by nsPP1 in the presence of GST and GST-IC1 proteins
(Fig. 3c). We found that dephosphorylation of 32P-InsP3R1 by nsPP1
was significantly faster in the presence of GST than in the presence of
GST-IC1 (Fig. 5a). To quantify these data, the content of
the 32P-InsP3R1 band
at each time point was quantified by phosphoimaging (Fig.
5a) and normalized to time 0. When the normalized
32P-InsP3R1 content
was plotted versus time of incubation with nsPP1 in semilogarithmic
coordinates, the measured values could be fitted by the straight line
(Fig. 5b), the slope of which corresponds to the rate of the
32P-InsP3R1
dephosphorylation. On average, for our experimental conditions the
nsPP1 dephosphorylated
32P-InsP3R1 at a
rate of 0.13 ± 0.02 (n = 3)
min1 in the presence of GST and at a
rate of 0.054 ± 0.006 (n = 3) min1 in the presence of GST-IC1 (Fig.
5c).
To further analyze the specificity of InsP3R1
dephosphorylation by PP1, we performed a series of in vitro
dephosphorylation experiments with recombinant PP1. Similar to results
with endogenous nsPP1, we found that recombinant PP1 rapidly
dephosphorylates 32P-InsP3R1 (Fig.
5c). As with nsPP1, dephosphorylation of
32P-InsP3R1 by
PP1 was abolished by PP1 inhibitor 2 (Fig. 5c). Similar
to nsPP1, we observed a threefold reduction in the rate of
32P-InsP3R1
dephosphorylation by PP1 in the presence of GST-IC1 but not in the
presence of GST (Fig. 5c). A similar effect was caused by
truncation of the InsP3R1 C-terminal in RT1C
mutant (Fig. 5c). Similar to PP1, the PP1 isoform was
also able to dephosphorylate
32P-InsP3R1 in
vitro (Fig. 5c). The absolute rates of PP1- and PP1-mediated dephosphorylation of
32P-InsP3R1 are not
comparable, because different amounts of phosphatase activity were
added to the dephosphorylation reactions. Importantly, in contrast to
experiments with PP1, GST-IC1 had no effect on the rate of
32P-InsP3R1
dephosphorylation by PP1 (Fig. 5c). This result agrees with the inability of InsP3R1 and PP1 to form
a complex in yeast two-hybrid and biochemical assays (Figs.
1b, 3d). From the results shown on Figure
5c, we concluded that direct association of PP1 with the
InsP3R1 C-terminal enables efficient
dephosphorylation of
32P-InsP3R1 by
PP1. In the neostriatum, DARPP-32 plays a predominant role in
control of PP1 activity (Greengard et al., 1999). We found that the
PKA-phosphorylated form pDARPP-32, but not DARPP-32 itself, inhibited
32P-InsP3R1
dephosphorylation by PP1 (Fig. 5c).
PKA phosphorylation of neostriatal InsP3R1
in vivo
In the neostriatum, stimulation of D1 dopamine receptors causes an
increase in cAMP levels (Greengard et al., 1999). Are neostriatal InsP3R1s phosphorylated by PKA when cAMP is
elevated? To answer this question, we used the PKA back-phosphorylation
method to determine the fraction of neostriatal
InsP3R1 in the phosphorylated state
(pInsP3R1). By following published procedures
(Nishi et al., 1997), we isolated neostriatal slices from adult rat
brains and incubated them in the oxygenated Krebs media. For
back-phosphorylation experiments, the neostriatal
InsP3R1 was solubilized in CHAPS in the presence
of phosphatase inhibitors, immunoprecipitated with
anti-InsP3R1 antibodies, phosphorylated in
vitro by the catalytic subunit of PKA in the presence of
[-32P]ATP, separated by
electrophoresis, and analyzed by phosphoimaging. When the sample was
incubated with PP1 before in vitro phosphorylation by
PKA, the content of the
32P-InsP3R1 band was
the greatest (Fig. 6a,
PP1 lane). This value was interpreted as total
InsP3R1 in the neostriatal sample
(32PPP1). Without
preincubation with PP1, the content of the 32P-InsP3R1 band was
reduced by ~30% (Fig. 6a, Ctr lane). By using the normalization procedure described in Materials and Methods, we
determined that 37 ± 3% (n = 4) of
InsP3R1s in the neostriatum are in the
PKA-phosphorylated state in control conditions (Fig. 6b).
When neostriatal slices were incubated with 1 mM
8-Br-cAMP or 100 µM dopamine for 10 min, we
observed a drastic reduction in the content of the
32P-InsP3R1 band
(Fig. 6a, cAMP and Dop lanes). We
estimated that 8-Br-cAMP and dopamine increased the fraction of
PKA-phosphorylated InsP3R1 in the neostriatum to
86 ± 6% (n = 4) and 85 ± 8%
(n = 4), respectively (Fig. 6b).
Preincubation of neostriatal slices for 60 min with 5 µM cyclosporine A, a calcineurin inhibitor, or
400 µM calyculin A, a PP1/PP2A inhibitor,
increased the fraction of PKA-phosphorylated
InsP3R1 to 71 ± 4% (n = 4)
and 68 ± 3% (n = 4), respectively (Fig.
6a,b, CsA and CalA lanes). In
contrast, preincubation of neostriatal slices with 10 nM okadaic acid, a specific inhibitor of PP2A at
this concentration, had only a minor effect on the PKA-phosphorylated
state of InsP3R1 when compared with control
conditions (Fig. 6a,b, OA lane).
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Figure 6.
PKA phosphorylation of neostriatal
InsP3R1 in vivo. PKA back-phosphorylation of
neostriatal InsP3R1 after pretreatment with PP1
(PP1); in controls (Ctr); after 10 min of incubation
of neostriatal slices with 1 mM 8-Br-cAMP
(cAMP) or 100 µM dopamine
(Dop); or after 60 min of treatment with 5 µM cyclosporine A (CsA), 400 µM calyculin A (CalA), or 10 nM okadaic acid (OA). a,
Autoradiogram of a representative experiment. The data for calyculin A
and okadaic acid are taken from the different experiments.
b, Summary of neostriatal InsP3R1
back-phosphorylation experiments. The estimated fraction of
PKA-phosphorylated InsP3R1 in neostriatal slices (see
Materials and Methods) is shown as mean ± SE
(n = 4).
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To determine a dynamic of dopamine-induced
InsP3R1 phosphorylation in the
neostriatal slices, we used the PKA back-phosphorylation method at
different time points after application of 100 µM
dopamine. We found that the fraction of PKA-phosphorylated
InsP3R1 peaks 10-15 min after dopamine
application and returns to prestimulation levels within 30 min (Fig.
7a). Thus, similar to DARPP-32
(Nishi et al., 1997; Greengard et al., 1999), the dopamine-induced
phosphorylation of neostriatal InsP3R1 by PKA is
transient, although with a slower time course. The dopamine-induced
changes in the InsP3R1 phosphorylated state were
abolished by preincubation of neostriatal slices with 400 µM calyculin A, a PP1/PP2A inhibitor (Fig.
7b). In contrast, preincubation of neostriatal slices with
10 nM okadaic acid, a specific inhibitor of PP2A
at this concentration, had only a minimal effect on dopamine-induced
changes in neostriatal InsP3R1
phosphorylated state (Fig. 7c). From the obtained
pharmacological profile (Figs. 6, 7), we concluded that the
PKA-phosphorylated state of neostriatal InsP3R1
in the resting state and in response to stimulation with dopamine is
determined by the activity of PP1 and PP2B phosphatases but not by the
activity of PP2A phosphatase.
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Figure 7.
Dopamine induces transient phosphorylation of
neostriatal InsP3R1 by PKA. a, Time course
of changes in neostriatal InsP3R1 PKA-phosphorylated state
in response to application of 100 µM dopamine. Dopamine
was applied to neostriatal slices at time 0. At each time point, the
fraction of neostriatal InsP3R1 in the PKA-phosphorylated
state is shown as mean ± SE (n = 3)
(filled circles). b, c, The same
experiment as in a performed with slices exposed to 400 µM calyculin A (CalA, b)
(open circles) or 10 nM okadaic acid
(OA, c) (filled
squares) for 60 min before the application of dopamine.
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PKA activates and PP1 inhibits InsP3R1
What are the functional consequences of
InsP3R1 phosphorylation by PKA?
Ca2+ flux measurements used previously to
address this question provided conflicting answers (Supattapone et al.,
1988; Nakade et al., 1994; Cameron et al., 1995; Wojcikiewicz and Luo,
1998). To study modulation of the InsP3R1 by PKA
phosphorylation, we incorporated recombinant
InsP3R1 expressed in insect cells into planar
lipid bilayers by microsomal fusion (Tu et al., 2002). Addition of 2 µM InsP3 to the cytosolic
(cis) chamber induced InsP3R1 activity (Fig. 8a, second
trace), but the Po was only
5-10% (Fig. 8b). In the presence of 0.5 mM MgATP in the cis chamber, the
application of PKA catalytic subunit directly to the bilayer induced
immediate facilitation in channel activity (Fig. 8a,
third trace), with a Po of
phosphorylated channels in the range of 30-40% (Fig. 8b). Application of PP1 to the bilayer resulted in almost complete inhibition of channel activity (Fig. 8a, fourth
trace, b). Inactivation of
InsP3R1 by PP1 could be reversed by a
second application of PKA catalytic subunit (Fig. 8a,
fifth trace, b), which in turn could be
counteracted by the second application of PP1 (Fig. 8a,
sixth trace, b). Results similar to the
experiment shown in Figure 8a,b were obtained in three
independent experiments. No effect was observed if the catalytic
subunit of PKA was boiled before addition to the bilayer
(n = 5) or if 0.5 mM
Na2ATP was present in the cis chamber
instead of MgATP (n = 3). As an additional control, we
performed experiments with a nonhydrolysable ATP analog, ATPS. If
100 µM Mg-ATPS was present in the
cis chamber, the application of a catalytic subunit of PKA
to the bilayer resulted in InsP3R1 activation
that could no longer be reversed by PP1 or affected by a second
application of PKA (Fig. 8c). From our experiments, we concluded that under identical experimental conditions PP1-dephosphorylated InsP3R1s have low
Po (<2-3%), and PKA-phosphorylated InsP3R1s have much higher
Po (30-40%).
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Figure 8.
PKA and PP1 modulate InsP3R1
activity in planar lipid bilayers. a, PKA activates and
PP1 inhibits the recombinant InsP3R1 reconstituted into
planar lipid bilayers. Each trace corresponds to 10 sec of
current recordings from the same experiment. The experiment is
performed in the presence of pCa 6.7 and 0.5 mM MgATP in
the cis chamber. Additions of 2 µM
InsP3 to the cis chamber and PKA/PP1
directly to the bilayer are indicated. Similar results were obtained in
three independent experiments. b, The average
InsP3R1 Po is calculated for a 5 sec window of time and plotted for the duration of an experiment. The
times of InsP3, PKA, and PP1 additions are shown
above the Po plot. The same experiment was
used to generate a and b.
c, The InsP3R1 Po
plot for the experiment performed in the presence of 100 µM Mg-ATPS in the cis chamber. The
times of InsP3, PKA, and PP1 additions are shown
above the Po plot. Similar results were
obtained in three independent experiments.
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To test the importance of
InsP3R1-PP1 association for
InsP3R1 modulation by PKA phosphorylation, we
performed planar lipid bilayer experiments with RT1C mutants
expressed in Sf9 cells (Fig. 3a). We found that RT1C
mutants formed functional InsP3-gated channels,
which were modulated by PKA and PP1 in a manner similar to the
wild-type InsP3R1 (data not shown). To explain
these results, we reasoned that because of the high concentration of
PP1 added to the bilayer, the C-terminal PP1-docking site in the
InsP3R1 sequence is not important for functional
regulation of InsP3R1 in our in vitro
experiments. However, in vivo the concentration of PP1 is
much lower, and InsP3R1-PP1 association is
likely to play an important role in control of the
InsP3R1 PKA-phosphorylated state.
Mechanism of InsP3R1 activation by PKA
To obtain mechanistic insights into InsP3R1
activation by PKA, we evaluated effects of PKA phosphorylation on
Ca2+ and InsP3
dependence of recombinant InsP3R1 reconstituted
into planar lipid bilayers. In the first series of experiments, the activity of InsP3R1 was recorded at variable
Ca2+ concentrations in the presence of 2 µM InsP3 and 0.5 mM
Mg-ATP. With addition of InsP3, we observed two
distinct populations of InsP3R1. In some (6 of
14) experiments, the initial activity of InsP3R1
was low, with Po 
10%
("low-activity" channels). In other experiments (8 of 14), the
activity of InsP3R1 was much higher, with
Po ~30% ("high-activity"
channels). As described in the previous section, addition of PKA to
low-activity channels increased their Po to 30-40% (Fig. 8). Addition of
PKA to high-activity channels had very little or no effect on their
Po, but addition of PP1 reduced
their Po to levels of <10% (data not
shown). In in vitro back-phosphorylation experiments, we
determined that ~20% of recombinant InsP3R1 in
microsomes isolated from Sf9 cells are in the PKA-phosphorylated state
(data not shown), presumably because of activity of endogenous PKA present in Sf9 cells. Thus, we reasoned that high-activity channels
are likely to correspond to partially phosphorylated InsP3R1, and low-activity channels correspond to
unphosphorylated InsP3R1.
The experiments in the previous section (Fig. 8) were performed with
low-activity channels. In Ca2+-dependence
experiments, we compared the behavior of low-activity, high-activity,
and PKA-phosphorylated channels. In agreement with our previous
findings (Nosyreva et al., 2002; Tu et al., 2002), recombinant
high-activity InsP3R1 displayed bell-shaped
dependence on cytosolic Ca2+ with the peak
at pCa 6.65 (Fig. 9, open
circles). The parameters of the optimal fit
(Pm, n, k,
K) for each series of
Ca2+-dependence experiments are presented
in Table 1. Fit to the data using the modified bell-shaped equation
(see Materials and Methods) yielded the affinity of activating site
equal to 0.22 µM
Ca2+, the affinity of inhibitory site
equal to 0.21 µM
Ca2+, and the cooperativity coefficient of
1.31 (Fig. 9, smooth curve; Table 1). Recombinant
low-activity InsP3R1 displayed similar bell-shaped Ca2+ dependence with the peak
at pCa 6.55 (Fig. 9, open triangles). Fit to low-activity
data set yielded the affinity of activating site equal to 0.10 µM Ca2+, the
affinity of inhibitory site equal to 0.72 µM
Ca2+, and the cooperativity coefficient of
2.09 (Fig. 9, smooth curve; Table 1). When the same
experiment was performed with the InsP3R1 phosphorylated by PKA in bilayers (initially displaying low activity), we found that the PKA-phosphorylated InsP3R1 also
displayed bell-shaped Ca2+ dependence that
peaked at pCa 6.65 (Fig. 9, filled circles). For PKA-phosphorylated
InsP3R1, the fit yielded the affinity of activating site equal to 0.24 µM
Ca2+, the affinity of inhibitory site
equal to 0.21 µM
Ca2+, and the cooperativity coefficient of
1.32 (Fig. 9, smooth curve; Table 1). From these
experiments, we concluded that PKA phosphorylation induces only minor
changes in bell-shaped Ca2+ dependence of
the InsP3R1.
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Figure 9.
Effect of PKA on InsP3R1
Ca2+ dependence. The Po
of recombinant InsP3R1 was determined in the presence of 2 µM InsP3 and 0.5 mM MgATP at
cis (cytosolic) Ca2+ concentrations
in the range between 10 nM and 5 µM
Ca2+. Po values measured
in several independent experiments were averaged together at each
Ca2+ concentration as described in Materials and
Methods and shown as mean ± SE for low-activity
InsP3R1 (n = 2; open
triangles), high-activity InsP3R1
(n = 3; open circles), and
PKA-phosphorylated InsP3R1 (n = 3;
filled circles). The averaged data were fitted by the
bell-shaped equation modified from Bezprozvanny et al. (1991), as
explained in Materials and Methods. The parameters of the optimal fits
(smooth curves) are shown in Table 1.
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Table 1.
Parameters of the bell-shaped fit to the
Ca2+-dependence data obtained with control (low and high
activity) and PKA-phosphorylated InsP3R1
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In the next series of experiments, we analyzed effects of PKA on
InsP3 dependence of
InsP3R1. These experiments were performed in the
presence of 300 nM Ca2+ (pCa
6.7) and 0.5 mM MgATP on the cytosolic side of the bilayer. By adding increasing amounts of InsP3 to the
cis chamber, we determined that the apparent affinity of
high-activity InsP3R1 for
InsP3 (kInsP3)
is equal to 0.19 µM InsP3
(Fig. 10a, open
circles). The apparent affinity of low-activity
InsP3R1 could not be reliably determined because
of the extremely low Po of these
channels at low InsP3 concentrations (data not
shown). To determine the effect of PKA on the
InsP3 dependence of
InsP3R1, we started an experiment by addition of
100 nM InsP3. The
InsP3R1 activity at this concentration of
InsP3 was very low (Fig. 10b,
second trace), with a Po of
1-2% (Fig. 10c). Addition of PKA to the bilayer resulted
in dramatic activation of InsP3R1 (Fig.
10b, third trace), with the
Po increased to 30-40% (Fig.
10c). Increasing the InsP3
concentration from 100 nM to 2 µM did not result in additional
InsP3R1 activation (Fig. 10b,
traces 4-6, and c). Thus, PKA-phosphorylated
InsP3R1s are maximally activated by 100 nM InsP3, indicating that
the kInsP3 value for
PKA-phosphorylated InsP3R1 must be <50
nM InsP3. This estimate is
in contrast to the values measured for high-activity InsP3R1 in the absence of PKA treatment (Fig.
10a). From these experiments, we concluded that PKA
phosphorylation causes at least a fourfold increase in
InsP3R1 sensitivity to activation by
InsP3.
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Figure 10.
Apparent affinity of InsP3R for
InsP3 is increased by PKA phosphorylation.
a, The InsP3 dependence of
InsP3R1 in control conditions. The InsP3R1
Po values measured in several independent
experiments were averaged together at each InsP3
concentration as described in Materials and Methods and shown as
mean ± SE for high-activity InsP3R1
(n = 3; open circles). The averaged
data were fitted by the equation modified from Lupu et al. (1998), as
explained in Materials and Methods. The parameters of optimal fit
(smooth curve) yielded
kInsP3 = 0.19 µM;
n = 1.47; Pmax = 0.154. b, c, Effect of PKA on the InsP3R1
InsP3 dependence. b, Each trace
corresponds to 20 sec of recombinant InsP3R1 current
recordings from the same experiment. The experiment was performed in
the presence of pCa 6.7 and 0.5 mM MgATP in the
cis (cytosolic) chamber at InsP3 concentrations from
100 nM to 2 µM. Addition of PKA directly to
the bilayer increased the InsP3R1 activity (third
trace). The filter frequency is 200 Hz for all
traces shown. c, The InsP3R1
Po was calculated for a 5 sec window of time
and was plotted for the duration of an experiment. Changes in
InsP3 concentration (from 100 nM to 2 µM as indicated) in the cis chamber and
the time of PKA addition to the bilayer are shown by the bar
diagram. The data shown in b and
c are from the same experiment.
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Discussion |
Modulation of InsP3R1 by PKA and PP1 was
investigated in this study. The main conclusions of our study are as
follows: (1) the InsP3R1 specifically associates
with PP1 via the C-terminal region; (2) association with PP1
facilitates dephosphorylation of PKA-phosphorylated
InsP3R1; (3) the neostriatal
InsP3R1s are phosphorylated by PKA after exposure
of neostriatal slices to 8-Br-cAMP, cyclosporine A, calyculin A, but
not to 10 nM okadaic acid; (4) the neostriatal
InsP3R1s are transiently phosphorylated by PKA
after application of dopamine; (5) the dopamine-induced PKA
phosphorylation of neostriatal InsP3R1 is
affected by cyclosporine A but not by 10 nM okadaic acid;
(6) the InsP3R1s reconstituted into planar lipid
bilayers are activated by PKA and inhibited by PP1; (7)
phosphorylation of InsP3R1 by PKA does not shift the peak of InsP3R1 bell-shaped
Ca2+ dependence; (8) phosphorylation of
InsP3R1 by PKA induces at least a fourfold
increase in the sensitivity of InsP3R1 to
activation by InsP3. Implications of these
findings for InsP3R1 function and dopaminergic
signaling in the neostriatum are briefly discussed below.
Modulation of InsP3R1 activity by PKA
A number of previous biochemical studies analyzed phosphorylation
of InsP3R1 by PKA. The neuronal
InsP3R1 is one of the best-known substrates for
both endogenous and exogenous PKA (Walaas et al., 1986; Supattapone et
al., 1988; Maeda et al., 1990; Danoff et al., 1991; Ferris et al.,
1991b; Wojcikiewicz and Luo, 1998; Haug et al., 1999
TOP
ABSTRACT
Introduction
