From the Division of Molecular Neurobiology,
Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo 108-8639, Japan, the § Laboratory for
Developmental Neurobiology, Brain Science Institute, Institute of
Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama
351-0198, Japan, and the
Calcium Oscillation Project,
International Cooperative Research Project (ICORP), Japan Science
and Technology Corporation, 3-14-4 Shirokanedai, Minato-ku, Tokyo
108-0071, Japan
Received for publication, October 3, 2002, and in revised form, January 9, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The inositol 1,4,5-trisphosphate
(IP3) receptors (IP3Rs) are
IP3-gated Ca2+ channels on intracellular
Ca2+ stores. Herein, we report a novel protein, termed
IRBIT (IP3R binding
protein released with inositol
1,4,5-trisphosphate), which interacts with type 1 IP3R (IP3R1) and was released upon
IP3 binding to IP3R1. IRBIT was purified from a
high salt extract of crude rat brain microsomes with IP3
elution using an affinity column with the huge immobilized N-terminal
cytoplasmic region of IP3R1 (residues 1-2217).
IRBIT, consisting of 530 amino acids, has a domain homologous to
S-adenosylhomocysteine hydrolase in the C-terminal and in
the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding
experiments showed the N-terminal region of IRBIT to be essential for
interaction, and the IRBIT binding region of IP3R1 was
mapped to the IP3 binding core. IP3 dissociated
IRBIT from IP3R1 with an EC50 of ~0.5
µM, i.e. it was 50 times more potent than
other inositol polyphosphates. Moreover, alkaline phosphatase treatment
abolished the interaction, suggesting that the interaction was
dualistically regulated by IP3 and phosphorylation.
Immunohistochemical studies and co-immunoprecipitation assays showed
the relevance of the interaction in a physiological context. These
results suggest that IRBIT is released from activated IP3R,
raising the possibility that IRBIT acts as a signaling molecule downstream from IP3R.
The hydrolysis of phosphatidylinositol 4,5-bisphosphate in
response to cell surface receptor activation leads to the production of
an intracellular second messenger, inositol 1,4,5-trisphosphate (IP3).1
IP3 mediates the release of Ca2+ from
intracellular Ca2+ storage organelles, mainly the
endoplasmic reticulum, by binding to its receptor (IP3R).
In these IP3/Ca2+ signaling cascades,
IP3R works as a signal converter from IP3 to
Ca2+ (1-3).
IP3R is a tetrameric intracellular IP3-gated
Ca2+ release channel (3, 4). There are three distinct types
of IP3R in mammals (5-7). Type 1 IP3R
(IP3R1) is highly expressed in the central nervous system,
particularly in the cerebellum (8, 9). Mouse IP3R1 is
composed of 2749 amino acids (5), and is divided into three
functionally distinct regions: the IP3-binding domain near the N terminus, the channel-forming domain with six membrane-spanning regions close to the C terminus, and the regulatory domain separating the two regions (10, 11). Deletion mutagenesis analysis of the
IP3-binding domain has shown that residues 226-578 of
IP3R1 are close to the minimum for specific and high
affinity ligand binding, thus assigned to the IP3 binding
core (12). The precise gating mechanism of IP3R triggered
by IP3 remains unclear, but IP3 binding induces
a substantial but as yet undefined conformational change, which may
cause channel opening (10). Besides this channel opening, such
IP3-induced conformational change has been assumed to be
responsible for degradation of IP3R (13, 14).
The increase in the cytoplasmic Ca2+ concentration
resulting from IP3R activation regulates the activities of
thousands of downstream targets that play key roles in many aspects of
cellular processes, including fertilization, development,
proliferation, secretion, and synaptic plasticity (1, 2, 15). To
control such a vast array of cell functions, Ca2+ signals
need to be precisely regulated in terms of space, time and amplitude
(2, 15). Such a complex regulation of Ca2+ signals has been
partly attributed to the diversity of IP3R isoform expression, assembly of heterotetrameric complexes of IP3R
isoforms, subcellular distributions of IP3R, and regulation
of IP3R by Ca2+ itself, ATP, and
phosphorylation (3, 4, 16). IP3R channels are also
regulated by their interacting proteins (4, 17), including calmodulin
(18, 19), FKBP12 (Refs. 20-22, but also see Refs. 23 and 24),
calcineurin (Refs. 21 and 25, but also see Refs. 23 and 24), ankyrin
(26-28), sigma-1 receptor (28), chromogranins A and B (29-31), IRAG
(32), Fyn (33), and BANK (34). Moreover, a family termed CaBP has been
shown to interact with IP3R in a
Ca2+-dependent manner, and to directly activate
IP3R in the absence of IP3 (35).
IP3R has also been demonstrated to be physically coupled to
its upstream or downstream signaling molecules by protein-protein interactions. For example, IP3R is coupled with group
1 metabotropic glutamate receptors (mGluRs) via the Homer family of
proteins (36) and with B2 bradykinin receptors
(B2Rs) by an unknown mechanism (37). Activations of mGluRs
and B2Rs lead to the production of IP3 in
proximity to IP3R, the result being efficient and specific signal propagation. Another example is Trp3, a candidate for plasma membrane Ca2+ channels regulated by intracellular
Ca2+ store depletion (capacitative calcium entry channels).
IP3R has been shown to interact with Trp3 directly, and to
activate it via a conformational coupling mechanism (38, 39). These
protein-protein interactions are supposed to regulate the
IP3/Ca2+ signaling pathway and contribute to
the specificity of intracellular Ca2+ dynamics.
To gain further insights into regulation of the
IP3/Ca2+ signaling pathway, we searched for
IP3R-binding proteins. In particular, we focused on
molecules that interact with IP3R in a manner regulated by
IP3, because such molecules may recognize the
conformational change in IP3R induced by IP3
binding, and/or may function as novel upstream or downstream signaling
molecules of IP3R. For this purpose, we used an affinity
column conjugated with the N-terminal 2217 amino acid residues of
IP3R1 containing most of the large cytoplasmic region of
the receptor molecule. By eluting bound proteins with IP3
from this affinity column, we identified a novel IP3R-binding protein, IRBIT
(IP3R binding protein
released with inositol 1,4,5-trisphosphate).
IRBIT bound to IP3R1 in vitro and in
vivo, and co-localized intensively with IP3R1.
Moreover, IRBIT was released from IP3R1 at a physiological
concentration of IP3. On the basis of these results, we
consider herein the role of IRBIT in IP3/Ca2+ signaling.
Preparation of IP3R1 Affinity Column--
The
cDNA encoding the N-terminal region of mouse IP3R1
(residues 1-225) was inserted into glutathione
S-transferase (GST) fusion vector pGEX-KG (40). The
GST-IP3R1 (1-225) fragment was subcloned into the
baculovirus transfer vector pBlueBac4.5 (Invitrogen). The 3'-region
downstream from the SmaI site of
GST-IP3R1-(1-225) was replaced with the
SmaI-EcoRI fragment of mouse IP3R1
(corresponding to residues 79-2217) to generate
GST-IP3R1-(1-2217) (termed GST-EL, for the
EcoRI Large fragment)
construct. GST alone was subcloned into pBlueBac4.5 as a control.
Sf9 cells were cultured in TNM-FH medium supplemented with 10%
fetal bovine serum at 27 °C. Recombinant baculoviruses carrying
GST-EL or GST were generated with the Bac-N-BlueTM
transfection kit (Invitrogen) according to the manufacturer's protocols. GST-EL and GST were expressed in 2 × 108
Sf9 cells by infecting recombinant baculoviruses at a
multiplicity of infection of 5, and incubating for 48 h. Cells
were harvested and stored at Purification and Partial Amino Acid Sequencing of
IRBIT--
Adult rat cerebella (~5 g) were homogenized in 45 ml of
homogenizer buffer (10 mM Hepes (pH 7.4), 320 mM sucrose, 2 mM EDTA, 1 mM
2-mercaptoethanol, and protease inhibitors) with a glass-Teflon homogenizer (950 rpm, 10 strokes), and the homogenate was centrifuged at 1,000 × g for 10 min. The supernatant (S1 fraction)
was centrifuged at 100,000 × g for 60 min to obtain
the cytosolic fraction (the supernatant) and the crude microsome (the
pellet). The crude microsome was homogenized in 25 ml of homogenizer
buffer containing 500 mM NaCl with a glass-Teflon
homogenizer (1,200 rpm, 10 strokes), incubated on ice for 15 min, and
centrifuged at 100,000 × g for 60 min to obtain the
high salt extract (the supernatant) and the stripped-crude microsome
(the pellet). The high salt extract was diluted five times with 10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.01% Brij 35, and protease inhibitors. The diluted
high salt extract was precleared with glutathione-Sepharose and loaded
onto a GST-EL affinity column equilibrated with binding buffer (10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, and 1 mM 2-mercaptoethanol). The GST
column was used as a control. The columns were washed with 20 column
volumes of binding buffer, and bound proteins were eluted with binding
buffer containing 50 µM IP3 (Dojindo) and
0.05% Brij 35. The eluted material was concentrated, separated by
SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and
stained with Coomassie Brilliant Blue. The 60-kDa protein band was
excised from the gel and digested with lysyl endopeptidase (Wako)
essentially according to the previously described method (41). The
polypeptides were separated by a C18 reversed-phase column
(µRPC C2/C18 SC 2.1/10, Amersham Biosciences) connected on a SMART
system (Amersham Biosciences). The amino acid sequence of each peptide
was determined by 494 procise protein sequencer (Applied Biosystems).
Two peptide sequences, N-YSFMATVTK-C and N-QIQFADDMQEFTK-C were obtained.
cDNA Cloning of IRBIT--
BLAST searches of two peptide
sequences derived from the 60-kDa protein against the non-redundant
data base revealed that these sequences match the sequence of a human
cDNA deposited in a patent (GenBankTM accession number
CAC09285). Based on the data bases of mouse expressed sequence tags
(accession number AW229870 and BE282170) homologous to this cDNA,
primers (5'-ATGTCGATGCCTGACGCGATGC-3' and 5'-GCGTGGTTCATGTGGACTGGTC-3')
were synthesized. cDNA of IRBIT was amplified by polymerase chain
reaction (PCR) using mouse cerebellum oligo(dT)-primed, first-strand
cDNA as a template. PCR product was cloned into pBluescript II
KS(+) (Stratagene) and sequenced. Sequences of three independent clones
were confirmed.
Preparation of Recombinant Proteins--
The cDNA encoding
the N-terminal region (residues 1-104) of IRBIT was subcloned into the
bacterial hexahistidine (His6) fusion vector pET-23a(+)
(Novagen) to generate the IRBIT-(1-104)-His6 construct.
The same cDNA was subcloned into the GST fusion vector pGEX-4T-1
(Amersham Biosciences) to generate the GST-IRBIT-(1-104) construct.
The cDNA fragments corresponding to the amino acid residues 1-225,
1-343, 341-923, 600-1248, 916-1581, and 1553-1943 of mouse
IP3R1 were inserted into pGEX-KG to generate the GST-Ia, GST-Iab, GST-IIab, GST-IIbIIIa, GST-IIIab, and GST-IV construct, respectively. Residues 1593-2217 of mouse IP3R1 were
inserted into pGEX-4T-1 to generate the GST-IV-Va construct. These
fusion proteins were expressed in Escherichia coli. GST-EL
was expressed in Sf9 cells as described above. Expressed
IRBIT-(1-104)-His6 was purified using ProBond resin
(Invitrogen). GST fusion proteins were purified using
glutathione-Sepharose. GST-IbIIa (residues 224-604 of mouse
IP3R1) and its site-directed mutants K508A and R441Q were
described previously (Ref. 42, GST-IbIIa was termed G224 therein).
Production of Affinity-purified Anti-IRBIT Antibody--
A
Japanese white rabbit was immunized with purified
IRBIT-(1-104)-His6 by subcutaneous injection with the
complete Freund's adjuvant at 14-day intervals. The anti-IRBIT
antisera was affinity-purified by passing serum from the immunized
rabbit over a GST-IRBIT-(1-104) column covalently coupled with
cyanogen bromide-activated Sepharose 4B (Amersham Biosciences), and
specific antibodies bound to the column were eluted with 100 mM glycine-HCl (pH 2.5).
Subcellular Fractionation and Immunoblotting--
Cerebrum,
cerebellum, heart, lung, liver, kidney, thymus, spleen, testis, and
ovary were dissected from the adult mouse and S1 fraction were obtained
essentially as described above. The cytosol, the crude microsome, the
high salt extract, and the stripped-crude microsome of mouse cerebellum
were obtained essentially as described above. Proteins with the amount
indicated were subjected to 10% SDS-PAGE and transferred onto
polyvinylidene difluoride membrane by electroblotting. After blocking,
membranes were immunoblotted with anti-IRBIT antibody (1 µg/ml) for
1 h at room temperature, followed by horseradish
peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences).
Immunoreactive bands were visualized with the enhanced
chemiluminescence detection system (Amersham Biosciences).
Generation and Transfection of Expression Constructs--
The
cDNA encoding full-length IRBIT was subcloned into the pcDNA3
(Invitrogen). The cDNA encoding full-length IRBIT or its deletion
mutants (residues 1-277, 1-104, and 105-530) were subcloned into the
pEGFP-C1 (Clontech) to generate green fluorescent
protein (GFP) fusion protein constructs. Mouse IP3R1
expression vector pBact-STneoB-C1 was described previously (43). COS-7
cells were cultured in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum, penicillin, and streptomycin at 37 °C. Transient transfections were performed using TransIT transfection reagents (Mirus) according to the manufacturer's instruction. Transfected cells
were processed for immunoblotting, pull-down experiments, or
immunostaining 2 days after transfection.
In Vitro Binding Experiments--
Mouse cerebellar cytosolic
fraction was diluted two times with 10 mM Hepes (pH 7.4),
200 mM NaCl, 2 mM EDTA, 1 mM
2-mercaptoethanol, and 0.02% Triton X-100. The high salt extract was
diluted five times with 10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.01%
Triton X-100. Diluted fractions (the final NaCl concentration of both
fractions was 100 mM) were incubated with 20 µg of GST-EL or GST for 2 h at 4 °C. After adding 10 µl of
glutathione-Sepharose and another 2-h incubation, the resins were
washed five times with wash buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM
2-mercaptoethanol, and 0.01% Triton X-100), and bound proteins were
eluted with 20 mM glutathione. Eluted proteins were
analyzed by Western blotting with anti-IRBIT antibody.
For dephosphorylation, the diluted high salt extract was incubated with
or without bacterial alkaline phosphatase (Toyobo) in the presence of 2 mM MgCl2 for 30 min at 37 °C after which 5 mM EDTA was added, and the sample was processed for
pull-down assay as described above.
For the dissociation experiments, IRBIT in the diluted high salt
extract was pulled down with GST-EL and washed as described above, and
resins were added in 100 µl of wash buffer containing IP3, inositol 4,5-bisphosphate (IP2) (Dojindo),
inositol 1,3,4,5-tetrakisphosphate (IP4) (Calbiochem),
inositol 1,2,3,4,5,6-hexakisphosphate (IP6) (Calbiochem),
or ATP (Amersham Biosciences) (0.1, 0.3, 1, 3, 10 µM,
each). After incubation on ice for 10 min, samples were centrifuged at
10,000 rpm for 1 min, and the supernatant was subjected to immunoblot
analysis with anti-IRBIT antibody or goat anti-GST antibody (Amersham
Biosciences). For quantitation, Alexa 680-conjugated goat anti-rabbit
IgG (Molecular Probes) was used as a secondary antibody. Intensity of
fluorescence of immunoreactive bands of IRBIT was measured using
Odyssey infrared imaging system (Aloka). Quantitative data (the
mean ± S.D. from at least three independent experiments) are
expressed as percentage of the amount of IRBIT in the 10 µM IP3 eluate.
For the determination of the IRBIT binding region and the critical
amino acids of IP3R1, the diluted high salt extract were processed for pull-down assay with 100 pmol of GST, GST-EL, GST-Ia, GST-Iab, GST-IbIIa, GST-IIab, GST-IIbIIIa, GST-IIIab, GST-IV, GST-IV-Va, K508A, or R441Q as described above and analyzed by Western
blotting with anti-IRBIT antibody.
For the determination of the IP3R1-interacting region of
IRBIT, COS-7 cells expressing GFP-tagged full-length IRBIT or its truncated mutants were lysed in lysis buffer (10 mM Hepes
pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.5% Nonidet P-40, and protease
inhibitors) for 30 min at 4 °C, followed by centrifugation (100,000 × g, 30 min). The supernatants were processed
for pull-down assay with GST-EL or GST as described above, and bound
proteins were subjected to immunoblot analysis with anti-GFP antibody
(Medical & Biological Laboratories).
Indirect Immunofluorescence and Confocal
Microscopy--
Transfected COS-7 cells grown on glass coverslips were
washed once in phosphate-buffered saline (PBS), fixed in 4%
formaldehyde in PBS for 15 min, permeabilized in 0.1% Triton X-100 in
PBS for 5 min, and blocked in PBS containing 2% normal goat serum for 60 min at room temperature. For washing out cytosolic proteins, transfected cells were washed once in PBS, permeabilized in ice-cold permeabilization buffer (80 mM PIPES, pH 7.2, 1 mM MgCl2, 1 mM EGTA, and 4%
polyethylene glycol) containing 0.1% saponin for 10 min on ice, and
washed twice with ice-cold permeabilization buffer before fixation.
Cells were then stained with rabbit anti-IRBIT antibody (1 µg/ml for
60 min at room temperature) and rat anti-IP3R1 antibody
18A10 (44) overnight at 4 °C. Following four 5-min PBS washes, Alexa
488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat
anti-rat IgG (Molecular Probes) were applied for 45 min at 37 °C.
Following four 5-min PBS washes, the coverslips were mounted with
Vectashield (Vector Laboratories) and observed under IX-70 confocal
fluorescence microscopy (Olympus) with a ×60 objective.
Immunoprecipitation--
Immunoprecipitation was performed as
described (45) with modifications. Adult mouse cerebellum was
homogenized in 10 volumes of 4 mM Hepes (pH 7.4), 320 mM sucrose, and protease inhibitors with a glass-Teflon
homogenizer. The homogenate was centrifuged at 800 × g
for 10 min, and the supernatant was subjected to another centrifugation
at 9000 × g for 15 min. The supernatant from the second centrifugation was solubilized in 1% sodium deoxycholate at
36 °C for 30 min, followed by adding 0.1 volume of 1% Triton X-100
in 50 mM Tris-HCl (pH 9.0), and the preparation was
centrifuged at 100,000 × g for 10 min. The supernatant
was incubated with 5 µl of protein G-Sepharose 4 fast flow (Amersham
Biosciences) for 2 h at 4 °C to clarify nonspecific binding to
the protein G beads. At the same time, 3 µg of rabbit anti-IRBIT
antibody, control rabbit IgG, rat anti-IP3R1 antibody 10A6
(46), control rat IgG, mouse anti-IP3R2 antibody KM1083
(47), or control mouse IgG was preincubated with 5 µl of protein G
beads for 2 h, and the protein G-antibody complex was spun down at
3,000 rpm for 2 min. The clarified supernatant was then added to the
antibody-bound protein G beads, and the mixture was incubated for
2 h at 4 °C. Beads were washed five times with 10 mM Hepes (pH 7.4), 100 mM NaCl, and 0.5%
Triton X-100 and analyzed by Western blotting with anti-IRBIT antibody,
mouse anti-IP3R1 antibody KM1112 (47), KM1083, or mouse
anti-IP3R3 antibody KM1082 (47).
Purification and cDNA Cloning of a Novel
IP3R-interacting Protein--
To identify
IP3R-interacting molecules, we used a GST fusion protein of
the N-terminal 2217 amino acids of mouse IP3R1 (GST-EL). This region is the large cytoplasmic portion of IP3R1
containing the IP3 binding domain and regulatory domain
(10, 11). GST-EL or GST was expressed using a baculovirus/Sf9
cell system and conjugated to glutathione-Sepharose. The extract with a
high salt buffer (containing 500 mM NaCl) from crude rat
cerebellar microsomes, which was thought to be enriched with
peripherally membrane-bound proteins, was loaded onto a
glutathione-Sepharose affinity column on which GST-EL or GST was
immobilized. To detect proteins that were dissociated from
IP3R in the presence of IP3, the proteins bound
to the affinity columns were eluted by addition of 50 µM IP3. A protein with a mass of about 60 kDa was detected in
the 50 µM IP3-eluate from the GST-EL column
(Fig. 1A), but not from the
GST column (data not shown). Two peptide sequences derived from the
60-kDa protein were determined. BLAST searches of non-redundant databases revealed that these two sequences matched the sequence of a
human cDNA deposited in a patent. On the basis of sequence information on mouse expressed sequence tags homologous to this cDNA, the cDNA of the 60-kDa protein was obtained by reverse
transcriptase-PCR from the mouse cerebellum. The predicted amino acid
sequence of the cloned cDNA revealed a protein composed of 530 amino acid residues (Fig. 1B), with a calculated molecular
mass of 58.9 kDa, which was close to its apparent molecular mass of 60 kDa estimated by SDS-PAGE (Fig. 1A). We designated the
60-kDa protein IRBIT.
Homology analysis of the deduced amino acid sequence of IRBIT revealed
the C-terminal region (residues 105-530) to be homologous (51%
identical, 74% similar) to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (EC 3.3.1.1.) (48) (Fig. 1,
C and D). An appendage of the N-terminal region
(residues 1-104) of IRBIT had no homology with reported proteins and
contained a serine-rich region (residues 62-103) (Fig. 1, B
and D). Motif searches of the IRBIT sequence revealed the
presence of a putative coiled-coil motif (residues 111-138) and a
putative NAD+ binding region (residues 314-344) (Fig. 1,
B and D). There were 17 potential phosphorylation
sites for protein kinases such as casein kinase II, PKC, PKA/PKG, and
tyrosine kinases, out of which seven sites were concentrated in the
N-terminal region (Fig. 1B). Neither putative
membrane-spanning regions nor signal sequences were found. Recently, a
mRNA expressed in dendritic cells was cloned from a human cDNA
library, and it was named DCAL (49), but its physiological function was
not addressed. The 100% identical amino acid sequences of IRBIT and
DCAL indicate that IRBIT is a mouse homologue of human DCAL.
Although IRBIT was homologous with S-adenosylhomocysteine
hydrolase, which catalyzes the reversible hydrolysis of
S-adenosylhomocysteine to adenosine and homocysteine,
recombinant IRBIT expressed in bacteria had no enzyme activities in the
hydrolysis direction, nor had any effects on the enzyme activity of
S-adenosylhomocysteine hydrolase (data not shown).
Tissue Distribution and Subcellular Localization of IRBIT--
We
generated an affinity-purified antibody against the N-terminal region
of the IRBIT (Fig. 1B, boxed). To confirm the
specificity of this antibody, we transfected the cDNA of IRBIT into
COS-7 cells, and the whole cells lysates obtained were analyzed by
immunoblotting with the anti-IRBIT antibody. As shown in Fig.
2A, the anti-IRBIT antibody
recognized only a single protein with a size of ~60 kDa. The
molecular mass of the exogenously expressed IRBIT (Fig. 2A, lane 1) was the same as that of the endogenous protein in
COS-7 (Fig. 2A, lane 3), confirming that the
cDNA clone encodes the full-length IRBIT protein. We examined the
expression of IRBIT in several mouse tissues by immunoblot analysis
with this anti-IRBIT antibody. IRBIT was detected ubiquitously,
with the highest expressions in the cerebrum and cerebellum (Fig.
2B).
Next, we investigated the subcellular distribution of IRBIT by
fractionation of the mouse cerebellum. IRBIT was present in both the
cytosolic and the crude microsome fraction (Fig. 2C, lanes 2 and 3, respectively). The crude microsome
fraction was further separated into a peripherally membrane-bound
fraction (the fraction from which IRBIT was originally purified) and a stripped-membrane fraction, with the aforementioned high salt buffer.
As shown in Fig. 2C, IRBIT in the crude microsome fraction was partially extracted with the high salt buffer (Fig. 2C,
lane 4). In contrast, IP3R1, which is an
integral membrane protein of the endoplasmic reticulum, was not
extracted (Fig. 2C, lower panel). These results
indicate IRBIT to be both a cytosolic and a peripherally
membrane-bound protein.
IRBIT in the High Salt Extract Interacted with IP3R1
and the N-terminal Region of IRBIT Was Essential for
Interaction--
IRBIT was present in both the cytosolic and the
peripherally membrane-bound fraction of the mouse cerebellum (Fig.
2C). We investigated whether IRBIT in these fractions
interacted with IP3R1 in vitro employing GST
pull-down techniques. The cytosol and high salt extracts from crude
mouse cerebellar microsomes were incubated with GST-EL or GST, and
binding of IRBIT to the recombinant proteins was analyzed by
immunoblotting with anti-IRBIT antibody. As shown in Fig.
3A, IRBIT in the high salt
extract interacted with GST-EL (Fig. 3A, lane 6),
but not with GST (Fig. 3A, lane 5). In contrast,
IRBIT in the cytosolic fraction did not interact with GST-EL (Fig.
3A, lane 3). The same result was obtained when
both fractions were dialyzed against the same buffer, indicating that
the difference was due neither to a difference in buffer composition
nor to excluded small molecules (data not shown). We speculated that
the difference might be attributable to a post-translational
modification of IRBIT such as phosphorylation. To test this
possibility, we treated the high salt extract with alkaline
phosphatase, a nonspecific phosphatase, followed by incubation with
GST-EL or GST. As shown in Fig. 3B, IRBIT in the high salt extract no longer interacted with GST-EL after phosphatase treatment (Fig. 3B, lane 6). This result raises the
possibility that phosphorylation of IRBIT may be necessary for
association with IP3R1, although the possibility that
phosphorylation of other proteins may regulate the interaction between
IRBIT and IP3R1 cannot be excluded.
To determine the region of IRBIT responsible for the interaction with
IP3R1, GST pull-down experiments were carried out using GFP-tagged deletion mutants of IRBIT (Fig.
4A). As shown in Fig. 4B, both GFP-IRBIT and GFP-IRBIT-(1-277) bound to GST-EL
efficiently (Fig. 4B, lanes 3 and 6,
respectively). Although GFP-IRBIT-(1-104) interacted with GST-EL, the
interaction was much weaker than those of GFP-IRBIT and
GFP-IRBIT-(1-277) (Fig. 4B, compare lanes 7 and 9 with lanes 1 and 3 and lanes
4 and 6). In contrast, GFP-IRBIT-(105-530), which
lacked the N-terminal region, and GFP alone did not interact with
GST-EL (Fig. 4B, lanes 12 and 15,
respectively). These results demonstrate the N-terminal region of IRBIT
to be essential for the interaction with IP3R1, and the
following ~170 amino acids containing a coiled-coil structure might
be important for stabilizing the interaction.
IRBIT Co-localized with IP3R1 on the Endoplasmic
Reticulum in Transfected COS-7 Cells--
To test whether IRBIT
interacts with IP3R1 in intact cells, IRBIT and
IP3R1 were co-expressed in COS-7 cells, and their
distribution was analyzed by confocal immunofluorescence microscopy.
IRBIT was diffusely distributed in the cytoplasm, with no
immunoreactivity in the nucleus (Fig.
5A). Because IRBIT was shown
to be present in both the cytosolic and the crude microsome fraction by
biochemical fractionation (Fig. 2C), we attempted to
visualize only the membrane-bound population of IRBIT. For this
purpose, we permeabilized plasma membranes of transfected COS-7 cells
with saponin and washed out cytosolic IRBIT prior to fixation. As shown
in Fig. 5B, in cells treated with saponin, localization of
IRBIT on the reticular structure was revealed (Fig. 5B,
left panels). The immunoreactivity of IRBIT extensively
overlapped with that of IP3R1 (Fig. 5B,
middle panels, and merged image right panels).
The staining pattern of IP3R1 was not altered by
permeabilization with saponin (data not shown). Since IRBIT expressed
alone showed a coarse distribution instead (data not shown), these
results indicate that IRBIT co-expressed with IP3R1
localized on the endoplasmic reticulum via the interaction with
IP3R1. IP3R1 was expressed in COS-7 cells to a
trace level, whereas type 2 IP3R (IP3R2) and
type 3 IP3R (IP3R3) were predominantly expressed (50, 51). These endogenous IP3Rs showed again a coarse, not a reticular, distribution in COS-7 cells both in a previous
report and in our hands (Ref. 52 and data not shown, respectively).
Furthermore, a complex of IRBIT and endogenous IP3R2/IP3R3 were revealed by
co-immunoprecipitation assay (data not shown). Taken together, these
findings support our idea that IRBIT interacted not only with
IP3R1 but also with IP3R2 and IP3R3 (see below).
When we transfected IP3R1 and GFP-IRBIT instead of IRBIT
and observed the fluorescence of GFP, essentially the same results were
obtained (Fig. 5, C and D). To confirm the
specificity of co-localization, we transfected GFP-IRBIT-(105-530),
which did not interact with GST-EL because of the lack of the
N-terminal region (Fig. 4), with IP3R1 into COS-7 cells. In
contrast to GFP-IRBIT, GFP-IRBIT-(105-530) was distributed in the
nucleus as well as the cytosol (Fig. 5E). IRBIT does not
harbor predicted nuclear localization signals, and the reason
GFP-IRBIT-(105-530) localized in the nucleus is unclear at present.
When the cytosolic population was washed out by permeabilization,
GFP-IRBIT-(105-530) localized only in the nucleus and did not
co-localize with IP3R1 (Fig. 5F). This
observation is consistent with biochemical results indicating the
N-terminal region of IRBIT to be necessary for binding to IP3R1 (Fig. 4B).
IRBIT Interacted with IP3R in Vivo--
To demonstrate
an in vivo association between IRBIT and IP3R in
native tissues, we performed co-immunoprecipitation experiments using
mouse cerebellum. Cerebellar lysates were immunoprecipitated with
anti-IRBIT antibody, and the immunoprecipitates were analyzed by
immunoblotting with anti-IP3R1, anti-IP3R2, or
anti-IP3R3 antibody. All three IP3R isoforms
were co-immunoprecipitated by anti-IRBIT antibody, but not control IgG
(Fig. 6A). In the reciprocal
experiments, immunoprecipitation of IP3R1 or
IP3R2 resulted in the co-precipitation of IRBIT (Fig. 6,
B and C). IRBIT was not detected in the
anti-IP3R3 precipitates, probably due to the inefficiency
of immunoprecipitation with anti-IP3R3 antibody (data not
shown). When we performed immunoprecipitation assay using lysates of
COS-7 cells transfected with IRBIT and IP3R3, in which most
IP3R3 forms homotetramers (51), IRBIT was shown to interact
with IP3R3 (data not shown). As for IP3R2,
essentially the same result was obtained (data not shown). These
results confirm IRBIT interacted with all IP3R isoforms
in vivo.
Physiological Concentration of IP3 Selectively
Dissociated IRBIT from IP3R1--
IRBIT was originally
identified in the GST-EL column eluate with 50 µM
IP3 (Fig. 1A), suggesting that IP3
disrupted the interaction between IRBIT and IP3R1. However,
50 µM is a relatively high concentration compared with
the physiological range of IP3, which was estimated to be a
few micromolar after stimulation (53). Thus, we examined the
dose-dependence of IP3 with which IRBIT was dissociated
from GST-EL, and its selectivity against other related inositol
polyphosphates. IRBIT in the high salt extract of crude mouse
cerebellar microsomes was pulled down with GST-EL, and eluted with
0.1-10 µM IP3, IP2, IP4, IP6, or ATP. As shown in Fig.
7A, IP3
dissociated IRBIT from GST-EL most efficiently in a
dose-dependent manner (Fig. 7Aa, lower
panel). We confirmed GST-EL to be undetectable in the
IP3 eluates (Fig. 7Aa, upper panel),
even with longer exposure (data not shown). The EC50 (the
concentration required for half-maximal dissociation of IRBIT from
GST-EL) was ~0.5 µM, which was within the physiological
IP3 concentration range (53) (Fig. 7B).
IP3 dissociated IRBIT from GST-EL about 50 times more
efficiently than other inositol polyphosphates (Fig. 7,
Ab-d and B). ATP, which has three phosphate
groups like IP3, did not dissociate IRBIT from GST-EL even
at 10 µM (Fig. 7, Ae and B). These
results indicate that IRBIT was dissociated from IP3R1
selectively within the physiological concentration range of
IP3.
IRBIT Interacted with the IP3 Binding Region of
IP3R1 and Lys-508 of IP3R1 Was Essential for
Interactions with Both IRBIT and IP3--
We investigated
which region, the IP3 binding region or the regulatory
region, of IP3R1 was necessary for the interaction with
IRBIT, using eight deletion mutants of IP3R1 constructed as
GST fusion proteins based on the domain structure of IP3R1 (54) (Fig. 8A). As shown in
Fig. 8B, GST-IbIIa (residues 224-604), which contains the
IP3 binding core region (residues 226-578) (12) bound to
IRBIT to the same extent as GST-EL. In contrast, other GST fusion
proteins, including GST-Iab and GST-IIab, did not interact with IRBIT.
Next, we performed a site-directed mutagenesis analysis to determine
the IP3R1 amino acids important for the interaction with
IRBIT. Lys-508 of IP3R1 was a critical amino acid residue
for IP3 binding (12), and substitution of Lys-508 of
GST-IbIIa with alanine (K508A) resulted in an enormous loss of
IP3 binding affinity (42). Conversely, R441Q, in which
Arg-441 of GST-IbIIa was substituted for Gln, had higher
IP3 affinity than GST-IbIIa (42). GST pull-down assays
using these recombinant proteins showed that IRBIT bound to GST-IbIIa
and R441Q to the same extent, but not to K508A (Fig. 8C).
Taken together, these results indicate that IRBIT binds to the
IP3-binding region of IP3R1 and that Lys-508 of
IP3R1 is required for the interaction with IRBIT as well as
IP3, supporting the observation that IP3 disrupts the interaction between IRBIT and IP3R1.
We screened IP3R1-binding proteins released from
IP3R1 in the presence of IP3 and identified a
novel protein, IRBIT, from a high salt extract of crude cerebellar
microsomes. IRBIT interacted with IP3R1 in vitro
and in vivo, and co-localized extensively with
IP3R1 in the endoplasmic reticulum in transfected cells. These results strongly suggest that IRBIT associates with
IP3R1 in basal states. Moreover, the physiological
concentration of IP3, but not of other inositol
polyphosphates, dissociated IRBIT from IP3R1. IRBIT bound
to the IP3 binding region of IP3R1, and Lys-508
of IP3R1 was essential for the interactions with both IP3 and IRBIT. These results suggest that IRBIT is released
from IP3R1 with IP3 produced in response to
extracellular stimuli. Although many IP3R-binding proteins
have been reported (18-39), IRBIT is the first molecule for which the
interaction with IP3R was shown to be regulated by
IP3.
IRBIT is composed of two regions, the N-terminal region (residues
1-104) essential for the interaction with IP3R1, and the C-terminal region (residues 105-530) homologous to
S-adenosylhomocysteine hydrolase (48). Crystallographic
studies (55, 56) and site-directed mutagenesis studies (57-60) have
determined amino acid residues of S-adenosylhomocysteine
hydrolase involved in substrate binding or NAD+ binding
(Fig. 1C). Although most of these residues were well conserved in IRBIT, we did not detect enzyme activity of recombinant IRBIT expressed in bacteria. We concluded that the IRBIT does not have
S-adenosylhomocysteine hydrolase activity, probably due to
substitution of amino acids important for substrate binding, such as
Leu-54, Phe-302, and His-353 of S-adenosylhomocysteine hydrolase (Fig. 1C), as discussed by another group (49).
Domains that are homologous to certain enzymes, but are catalytically inactive, such as the esterase domain of the neuroligin family (61) and
the carbonic anhydrase domain of receptor tyrosine phosphatase In vitro binding experiments and immunostaining studies
showed the N-terminal region of IRBIT to be essential, though not sufficient, for the interaction with IP3R1. The
IRBIT-binding region of IP3R1 was shown to be its
IP3 binding region, and Lys-508 of IP3R1, the
critical amino acid for IP3 binding, was required for this
interaction. Based on mutagenesis analysis, Yoshikawa et al.
(12) proposed that basic amino acid residues, including Lys-508,
contribute to form a positively charged pocket for binding to the three
negatively charged phosphate groups of IP3. This model
leads us to speculate that acidic or phosphorylated amino acid residues
in the N-terminal region of IRBIT may be involved in interaction with
the positively charged IP3-binding pocket of
IP3R1. This hypothesis is supported by the following
findings: 1) although IRBIT is a neutral protein (calculated pI of
6.48), its N-terminal region is relatively acidic (calculated pI of
4.98), 2) seven potential phosphorylation sites are concentrated in the N-terminal region of IRBIT, and phosphorylation was supposed to be
required for the interaction, 3) Lys-508 of IP3R1 was
essential for the interaction with IRBIT, 4) IP3 disrupted
the interaction, and 5) a high salt buffer disrupted the interaction
between IRBIT and GST-EL2 and
extracted IRBIT from crude microsomes, indicating that the interaction
is dependent on an electrostatic bond. Deletion mutagenesis results
also indicate that residues 105-277 of IRBIT, which contain a
coiled-coil region, contribute to the interaction. The crystal structure of the IP3 binding region of mouse
IP3R1 in the complex with IP3 was recently
resolved (63). IP3 bound to the positively charged cleft of
the IP3 binding region, and the side chain of Lys-508
formed the hydrogen bond with the phosphate group at the 5-position of
IP3 (63). Remarkably, the C-terminal region of the
IP3 binding domain containing Lys-508 (residues 437-604)
formed an armadillo repeat-like fold (63), which generally acts as a
protein-protein interaction motif (64). IRBIT may interact with
IP3R1 via this motif. However, the armadillo repeat-like fold is not sufficient for interaction, since GST-IIab (residues 341-923 of IP3R1) did not interact with IRBIT.
IRBIT was dissociated from IP3R1 selectively with
IP3 at an EC50 of ~0.5 µM. This
EC50 value is higher than the Kd of
purified IP3R1 for IP3 (Kd = 83-100 nM) determined by conventional IP3
binding assays (46, 65). This difference may be attributable to
different buffer conditions because the IP3 binding
affinity of IP3R depends strongly on pH and ionic strength
(66-68). Conventional IP3 binding assays were performed under optimal binding conditions, with a higher pH (8.0-8.3) and a
lower ionic strength (salt-free). Surface plasmon resonance biosensor
studies using the N-terminal region of IP3R1 (residues 1-604) demonstrated the Kd value determined under
near physiological conditions (pH 7.4 and 150 mM NaCl) to
be 336 nM (68), i.e. ~7.5-fold lower than the
affinity determined by the conventional IP3 binding assay
(69), and close to the EC50 (~0.5 µM)
required for the dissociation of IRBIT from GST-EL determined at pH 7.4 and 100 mM NaCl. Therefore, taken together with the findings that IRBIT bound to the IP3 binding region of
IP3R1 and that both IRBIT and IP3 were
dependent on Lys-508 of IP3R1 for the interaction, these
results indicate that IRBIT is released from IP3R1 upon
IP3 binding to IP3R1, probably via a
competitive mechanism.
Phosphorylation, as well as IP3, is considered to regulate
the interaction between IRBIT and IP3R. In vitro
binding experiments showed IRBIT extracted from the membrane fraction
with a high salt buffer to interact with IP3R1, whereas
IRBIT in the cytosolic fraction did not. The difference in the
phosphorylation state of IRBIT may account for this discrepancy,
because alkaline phosphatase treatment of the high salt extract
disrupted the interaction between IRBIT and IP3R1. IRBIT
has 17 potential phosphorylation sites, and seven of these sites are
concentrated in the N-terminal region, which is necessary for the
interaction with IP3R1. These findings raise the
possibility that the dephosphorylated form of IRBIT is free in the
cytosol, whereas the phosphorylated form is membrane-bound via the
interaction with IP3R1, although we could not rule out the
possibility that phosphorylation of other proteins may regulate the
interaction. We propose that the interaction between IRBIT and
IP3R1 is dualistically regulated by IP3 and,
either directly or indirectly, by phosphorylation. Further studies are
needed to determine whether or not the interaction is regulated by
direct phosphorylation of IRBIT.
Using the detector cell/capillary electrophoresis system, Luzzi
et al. (53) estimated intracellular IP3
concentrations before and after stimulation to be tens of nanomolar and
a few micromolar, respectively. Because the EC50 of
IP3 (~0.5 µM) required for the dissociation
of IRBIT from IP3R1 was between these concentrations, IRBIT
was assumed to be released from IP3R1 after IP3
production has been induced by extracellular stimuli. What is the
physiological significance of the dissociation of IRBIT from
IP3R1 after stimulation and what is the function of IRBIT?
We propose four possible roles of IRBIT. First, IRBIT may modulate the
channel activity of IP3R1. Recently, Yang et al.
(35) showed that CaBP family members can act as direct ligands of
IP3R. Interestingly, the CaBP-binding region of
IP3R was within its 600 N-terminal residues (35), which
also contain the IRBIT-binding region. Considering our preliminary data
showing that IRBIT does not directly modulate the channel activity of
IP3R,2 IRBIT may block the binding of CaBP to
IP3R1 and inhibit IP3-independent activation of
IP3R1. Second, IRBIT may regulate the stability of
IP3R. IP3-generating stimuli cause degradation
of IP3R (13, 14, 50, 70-72). Zhu et al. (13,
14) proposed that the conformational change in IP3R induced
by IP3 binding unmasks the putative sites that facilitate
ubiquitin conjugation, resulting in degradation of IP3R by
the ubiquitin/proteasome pathway (71, 72). Alternatively, dissociation
of IRBIT induced by IP3 binding may reveal the putative degradation signals or protease attack sites of IP3R.
Third, IRBIT may play the role of a linker molecule coupling
IP3R and other proteins to allow efficient signal
propagation. Proteins possibly linked with IP3R include
proteins whose activities are regulated by Ca2+ released
from IP3R, or plasma membrane receptors, analogous with mGluR (36) and B2R (37). Indeed, substantial amounts of
IRBIT were present in the stripped microsome fraction (Fig.
2C), which might represent IRBIT tightly bound to membrane
proteins other than IP3R. IP3 may disrupt these
complexes, resulting in desensitization of signals and/or translocation
of linked proteins. To identify molecules possibly coupled with
IP3R, we are now searching for IRBIT-interacting proteins.
Fourth, IRBIT may be a direct downstream signal transducer of
IP3R1. It has been thought that the only direct downstream
molecule of IP3R1 is the calcium ion, which acts on a wide
variety of target molecules. Besides a multifunctional and universal
second messenger like Ca2+, IP3R1 may utilize
IRBIT as a downstream signaling molecule with more restricted target
molecules than Ca2+. In this model, IRBIT released from
IP3-bound IP3R1 must be different (for example,
in terms of phosphorylation state) from IRBIT originally present in the
cytosol, because significant amounts of IRBIT already exist in the
cytosol in the basal state. In this respect, the model in which only
phosphorylated IRBIT binds to IP3R1 appears to be
reasonable. Screening of IRBIT-binding proteins may reveal the target
molecules of IRBIT.
Finally, the dissociation of IRBIT from IP3R in the
presence of IP3 is a feature which may be utilized for the
development of a new IP3 indicator based on fluorescence
resonance energy transfer (FRET). FRET occurs when two fluorophores are
in proximity and in the right orientation such that an excited donor
fluorophore can transfer its energy to a second, acceptor fluorophore
(73). Based on the cAMP-dependent dissociation of catalytic
and regulatory subunits of cAMP-dependent protein kinase,
Adams et al. (74) developed a fluorescent indicator for
cAMP. Similarly, Miyawaki et al. (75) reported a genetically
encoded Ca2+ indicator based on the
Ca2+-dependent interaction between calmodulin
and calmodulin-binding peptide. Although IP3 concentration
changes could be detected by monitoring translocation of the GFP-tagged
pleckstrin homology domain (76), a FRET-based IP3 indicator
has yet to be developed due to lack of suitable molecules.
IP3-dependent dissociation of IRBIT and
IP3R1 is a characteristic that can provide a new tool
allowing real-time imaging of the spatiotemporal dynamics of
IP3 concentrations in living cells, although further
studies focusing on the regulation of this interaction by
phosphorylation are needed.
In summary, we identified IRBIT, a novel IP3R1-interacting
protein, which was released from IP3R1 in the presence of
IP3. Further studies aimed at elucidating the function of
IRBIT, including the screening of IRBIT-interacting proteins, are
anticipated to provide important insights into
IP3/Ca2+ signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Frozen cells were suspended in
10 ml of 10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% Triton
X-100, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 2 µM pepstatin
A, and 10 µM E-64), and were homogenized with a
glass-Teflon homogenizer (1000 rpm, 10 strokes). The homogenate was
centrifuged at 20,000 × g for 30 min. The supernatant
was incubated with 3 ml of glutathione-Sepharose 4B (Amersham
Biosciences) for 3 h at 4 °C. After washing eight times with 40 ml of 10 mM Hepes (pH 7.4), 250 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.1%
Triton X-100, GST-EL or GST coupled with glutathione-Sepharose was
packed into columns and equilibrated with 10 mM Hepes (pH
7.4), 100 mM NaCl, 2 mM EDTA, 1 mM
2-mercaptoethanol, and 0.1% Triton X-100. About 5 mg of GST-EL was immobilized.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (68K):
[in a new window]
Fig. 1.
Purification and cDNA cloning of IRBIT.
A, SDS-PAGE of IRBIT (indicated by arrow)
purified from the high salt extract of crude rat cerebellar microsomes
with the GST-EL column. IRBIT was eluted from the column with 50 µM IP3. The eluted material was concentrated,
separated by SDS-PAGE on 10% gel and stained with Coomassie Brilliant
Blue. The bands above 200 kDa and at 100-200 kDa were mostly leaked
GST-EL and its degradation products, respectively because these bands
were recognized by various anti-IP3R1 antibodies.
B, the deduced amino acid sequences of IRBIT. Two digested
peptides obtained from the purified IRBIT are
bold-underlined. The serine-rich region is
dashed-underlined. The putative coiled-coil region is
double-underlined. The putative NAD+ binding
site is underlined. Putative phosphorylation sites for
casein kinase II (closed circles), PKC (open
circles), PKA/PKG (a closed square), and tyrosine
kinases (open squares) are indicated above the sequences.
The N-terminal region is boxed. C, sequence alignment of the
C-terminal region of IRBIT and rat S-adenosylhomocysteine
hydrolase (AHCY) (48). Identical residues (*) and similar
residues (:) are indicated. Residues involved in substrate binding and
NAD+ binding of S-adenosylhomocysteine hydrolase
are indicated by closed circles and open circles,
respectively. D, schematic representation of the structure
of IRBIT. NTR and CTR indicate the N-terminal
region and the C-terminal region, respectively. Serine-rich region
(SER), coiled-coil region (CC) and
NAD+ binding site (NAD) are indicated.
View larger version (41K):
[in a new window]
Fig. 2.
Tissue distribution and subcellular
fractionation of IRBIT. A, Western blot analysis of
exogenously expressed and endogenous IRBIT. COS-7 cells were
transiently transfected with IRBIT (lane 1) or mock control
(lanes 2 and 3), and the whole cell lysates were
analyzed by Western blotting with anti-IRBIT antibody. In lane
3, 10× amounts of the lysate were loaded as compared with those
in lanes 1 and 2. B, tissue
distribution of IRBIT. S1 fractions (2 µg of total protein) of adult
mouse tissues were analyzed by Western blotting with anti-IRBIT
antibody. C, subcellular fractionation of the mouse
cerebellum. S1 fraction (lane 1) of mouse cerebella was
centrifuged at 100,000 × g to obtain the cytosolic
fraction (lane 2) and the crude microsomes (lane
3). The crude microsomes were extracted with the high salt buffer
containing 500 mM NaCl and centrifuged at 100,000 × g to obtain the peripherally membrane-bound fraction
(lane 4) and the stripped-crude microsomes (lane
5). Upper, each fraction (1 µg of total protein) was
analyzed by Western blotting with anti-IRBIT antibody.
Lower, each fraction (0.2 µg of total protein) was
analyzed by Western blotting with anti-IP3R1
antibody.
View larger version (48K):
[in a new window]
Fig. 3.
IRBIT in the high salt extract but not in the
cytosol interacted with IP3R1 in vitro.
A, mouse cerebellar cytosolic fraction (lanes
1-3) and the high salt extract of crude microsomes (lanes
4-6) were incubated with GST-EL (lanes 3 and
6) or GST (lanes 2 and 5). Bound
proteins were pulled down with glutathione-Sepharose, eluted with
glutathione, and analyzed by Western blotting using anti-IRBIT antibody
(upper panel). GST-EL and GST pulled down with
glutathione-Sepharose were visualized by staining with Coomassie
Brilliant Blue (lower panel). B, the high salt
extract of crude mouse cerebellar microsomes was incubated without
(lanes 1-3) or with (lanes 4-6) alkaline
phosphatase prior to pull down with GST-EL (lanes 3 and
6) or GST (lanes 2 and 5). IRBIT
binding was analyzed as in A.
View larger version (45K):
[in a new window]
Fig. 4.
The N-terminal region of IRBIT was essential
for interaction with IP3R1. A, schematic
representation of the structure of IRBIT and its GFP-tagged truncated
mutants. B, GST pull-down assay from the lysates of COS-7
cells expressing GFP-IRBIT (lanes 1-3), GFP-IRBIT-(1-277)
(lanes 4-6), GFP-IRBIT-(1-104) (lanes 7-9),
GFP-IRBIT-(105-530) (lanes 10-12), and GFP (lanes
13-15). The lysates of COS-7 cells expressing each construct
(input; I) were incubated with GST-EL (E) or GST
(G). Bound proteins were pulled down with
glutathione-Sepharose, eluted with glutathione, and subjected to
immunoblot analysis with anti-GFP antibody.
View larger version (64K):
[in a new window]
Fig. 5.
IRBIT co-localized with IP3R1 in
transfected COS-7 cells. IP3R1 was transiently
transfected into COS-7 cells with IRBIT (A and
B), GFP-IRBIT (C and D), and
GFP-IRBIT-(105-530) (E and F). The localization
of the corresponding proteins was analyzed by indirect
immunofluorescence (IP3R1 and IRBIT) and fluorescence
(GFP-IRBIT and GFP-IRBIT-(105-530)) confocal microscopy.
B, D, and F, cells were permeabilized
in saponin and cytosolic proteins were washed out prior to fixation.
Left panels show IRBIT (B), GFP-IRBIT
(D), and GFP-IRBIT-(105-530) (F). Middle
panels show IP3R1. Right panels show merged
images of fluorescence from left and middle
panels. B, lower panels are higher
magnification images of upper panels. Scale bars,
10 µm.
View larger version (67K):
[in a new window]
Fig. 6.
IRBIT associated with IP3R
in vivo. A, cerebellar lysates were
immunoprecipitated with anti-IRBIT or control antibody. The
immunoprecipitates were subjected to SDS-PAGE followed by Western
blotting with anti-IP3R1, anti-IP3R2,
anti-IP3R3, or anti-IRBIT antibody. B,
cerebellar lysates were immunoprecipitated with anti-IP3R1
or control antibody. The immunoprecipitates were subjected to Western
blotting with anti-IRBIT or anti-IP3R1 antibody.
C, cerebellar lysates were immunoprecipitated with
anti-IP3R2 or control antibody. The immunoprecipitates were
subjected to Western blotting with anti-IRBIT or anti-IP3R2
antibody. Arrowheads indicate immunoglobulin heavy
chains.
View larger version (29K):
[in a new window]
Fig. 7.
Physiological concentration of
IP3 selectively dissociated IRBIT from IP3R1.
A, the high salt extract of crude mouse cerebellar
microsomes was incubated with GST-EL. Bound proteins were pulled down
with glutathione-Sepharose and eluted with glutathione (Glu)
(a) or 0.1-10 µM IP3
(a), IP2 (b), IP4
(c), IP6 (d), or ATP (e).
IRBIT in the eluates were analyzed with anti-IRBIT antibody and Alexa
680-conjugated secondary antibody (a, lower, and
b-e). GST-EL in the glutathione and 0.1-10
µM IP3 eluate was analyzed with anti-GST
antibody (a, upper). B, the intensity
of the immunoreactive bands of IRBIT was quantified by infrared imaging
system, and relative intensity was plotted against concentration.
Results are shown as the mean ± S.D. from at least three
independent experiments.
View larger version (37K):
[in a new window]
Fig. 8.
IRBIT interacted with the
IP3-binding region of IP3R1 and Lys-508 was
critical for this interaction. A, schematic representation
of the structure of mouse IP3R1 and the recombinant GST
fusion proteins used in this study. The IP3 binding core
region is indicated with a gray box. Putative
membrane-spanning regions are indicated by solid vertical
bars. Roman numbers below IP3R1 indicate
the domain structure determined by the limited trypsin digestion (54).
Numbers above the lines represent corresponding amino acid
numbers. B, determination of the IRBIT binding region of
IP3R1. The high salt extract of crude mouse cerebellar
microsomes was incubated with GST fusion proteins described in
A. Bound proteins were pulled down with
glutathione-Sepharose, eluted with glutathione, and analyzed by Western
blotting using anti-IRBIT antibody. C, site-directed
mutagenesis analysis. The high salt extract was processed for pull-down
assay with GST-IbIIa, R441Q, and K508A as described in B.
Bound proteins were analyzed by Western blotting using anti-IRBIT
antibody (upper panel). GST fusion proteins pulled down with
glutathione-Sepharose were visualized by staining with Coomassie
Brilliant Blue (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(62), are reportedly involved in protein-protein interactions. The
C-terminal region of IRBIT may be one such domain. However, the
possibility that IRBIT has enzyme activity with a different substrate
specificity cannot be excluded.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Iwai for Sf9 expression and DNA sequencing, Dr. T. Uchiyama for providing GST-IbIIa, K508A, and R441Q proteins, Y. Makino at Okazaki National Research Institutes for protein sequencing, and Drs. T. Michikawa and M. Hattori for critical comments on this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan and from RIKEN, the Brain Science Institute of Saitama, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB092504.
¶ To whom correspondence should be addressed. Tel.: 81-3-5449-5319; Fax: 81-3-5449-5420; E-mail: hando@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M210119200
2 H. Ando, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; IP3R1, type 1 inositol 1,4,5-trisphosphate receptor; mGluRs, metabotropic glutamate receptors; B2Rs, B2 bradykinin receptors; IRBIT, IP3R-binding protein released with inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; GFP, green fluorescent protein; IP2, inositol 4,5-bisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP6, inositol 1,2,3,4,5,6-hexakisphosphate; PBS, phosphate-buffered saline; FRET, fluorescence resonance energy transfer; PIPES, 1,4-piperazinediethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve] |
2. | Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 11-21[CrossRef][Medline] [Order article via Infotrieve] |
3. | Furuichi, T., and Mikoshiba, K. (1995) J. Neurochem. 64, 953-960[Medline] [Order article via Infotrieve] |
4. | Patel, S., Joseph, S. K., and Thomas, A. P. (1999) Cell Calcium 25, 247-264[CrossRef][Medline] [Order article via Infotrieve] |
5. | Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Nature 342, 32-38[CrossRef][Medline] [Order article via Infotrieve] |
6. | Südhof, T. C., Newton, C. L., Archer, B. T., III, Ushkaryov, U. A., and Mignery, G. A. (1991) EMBO J. 10, 3199-3206[Abstract] |
7. |
Blondel, O.,
Takeda, J.,
Janssen, H.,
Seino, S.,
and Bell, G. I.
(1993)
J. Biol. Chem.
268,
11356-11363 |
8. | Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161[CrossRef][Medline] [Order article via Infotrieve] |
9. | Furuichi, T., Simon-Chazottes, D., Fujino, I., Yamada, N., Hasegawa, M., Miyawaki, A., Yoshikawa, S., Guénet, J.-L., and Mikoshiba, K. (1993) Recept. Channels 1, 11-24[Medline] [Order article via Infotrieve] |
10. | Mignery, G. A., and Südhof, T. C. (1990) EMBO J. 9, 3893-3898[Abstract] |
11. | Miyawaki, A., Furuichi, T., Ryou, Y., Yoshikawa, S., Nakagawa, T., Saitoh, T., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4911-4915[Abstract] |
12. |
Yoshikawa, F.,
Morita, M.,
Monkawa, T.,
Michikawa, T.,
Furuichi, T.,
and Mikoshiba, K.
(1996)
J. Biol. Chem.
271,
18277-18284 |
13. |
Zhu, C.-C.,
Furuichi, T.,
Mikoshiba, K.,
and Wojcikiewicz, R. J. H.
(1999)
J. Biol. Chem.
274,
3476-3484 |
14. | Zhu, C.-C., and Wojcikiewicz, R. J. H. (2000) Biochem. J. 348, 551-556[CrossRef][Medline] [Order article via Infotrieve] |
15. | Berridge, M. J., Bootman, M. D., and Lipp, P. (1998) Nature 395, 645-648[CrossRef][Medline] [Order article via Infotrieve] |
16. | Thrower, E. C., Hagar, R. E., and Ehrlich, B. E. (2001) Trends Pharmacol. Sci. 22, 580-586[CrossRef][Medline] [Order article via Infotrieve] |
17. | Mackrill, J. J. (1999) Biochem. J. 337, 345-361[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Patel, S.,
Morris, S. A.,
Adkins, C. E.,
O'beirne, G.,
and Taylor, C. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11627-11632 |
19. | Michikawa, T., Hirota, J., Kawano, S., Hiraoka, M., Yamada, M., Furuichi, T., and Mikoshiba, K. (1999) Neuron 23, 799-808[CrossRef][Medline] [Order article via Infotrieve] |
20. | Cameron, A. M., Steiner, J. P., Sabatini, D. M., Kaplin, A. I., Walensky, L. D., and Snyder, S. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1784-1788[Abstract] |
21. |
Cameron, A. M.,
Nucifora, F. C., Jr.,
Fung, E. T.,
Livingston, D. J.,
Aldape, R. A.,
Ross, C. A.,
and Snyder, S. H.
(1997)
J. Biol. Chem.
272,
27582-27588 |
22. | Dargan, S. L., Lea, E. J. A., and Dawson, A. P. (2002) Biochem. J. 361, 401-407[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Bultynck, G.,
De Smet, P.,
Weidema, A. F.,
Ver Heyen, M.,
Maes, K.,
Callewaert, G.,
Missiaen, L.,
Parys, J. B.,
and De Smedt, H.
(2000)
J. Physiol.
525,
681-693 |
24. | Bultynck, G., De Smet, P., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., De Smedt, H., and Parys, J. B. (2001) Biochem. J. 354, 413-422[CrossRef][Medline] [Order article via Infotrieve] |
25. | Cameron, A. M., Steiner, J. P., Roskams, A. J., Ali, S. M., Ronnett, G. V., and Snyder, S. H. (1995) Cell 83, 463-472[Medline] [Order article via Infotrieve] |
26. |
Joseph, S. K.,
and Samanta, S.
(1993)
J. Biol. Chem.
268,
6477-6486 |
27. |
Bourguignon, L. Y. W.,
Jin, H.,
Iida, N.,
Brandt, N. R.,
and Zhang, S. H.
(1993)
J. Biol. Chem.
268,
7290-7297 |
28. |
Hayashi, T.,
and Su, T.-P.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
491-496 |
29. |
Yoo, S. H.,
So, S. H.,
Kweon, H. S.,
Lee, J. S.,
Kang, M. K.,
and Jeon, C. J.
(2000)
J. Biol. Chem.
275,
12553-12559 |
30. |
Yoo, S. H.,
and Jeon, C. J.
(2000)
J. Biol. Chem.
275,
15067-15073 |
31. |
Thrower, E. C.,
Park, H. Y.,
So, S. H.,
Yoo, S. H.,
and Ehrlich, B. E.
(2002)
J. Biol. Chem.
277,
15801-15806 |
32. | Schlossmann, J., Ammendola, A., Ashman, K., Zong, X., Huber, A., Neubauer, G., Wang, G.-X., Allescher, H.-D., Korth, M., Wilm, M., Hofmann, F., and Ruth, P. (2000) Nature 404, 197-201[CrossRef][Medline] [Order article via Infotrieve] |
33. | Jayaraman, T., Ondrias, K., Ondiasova, E., and Marks, A. R. (1996) Science 272, 1492-1494[Abstract] |
34. |
Yokoyama, K.,
Su, I.,
Tezuka, T.,
Yasuda, T.,
Mikoshiba, K.,
Tarakhovsky, A.,
and Yamamoto, T.
(2002)
EMBO J.
21,
83-92 |
35. |
Yang, J.,
McBride, S.,
Mak, D.-O. D.,
Vardi, N.,
Palczewski, K.,
Haeseleer, F.,
and Foskett, J. K.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
7711-7716 |
36. | Tu, J. C., Xiao, B., Yuan, J. P., Lanahan, A. A., Leoffert, K., Li, M., Linden, D. J., and Worley, P. F. (1998) Neuron 21, 717-726[Medline] [Order article via Infotrieve] |
37. | Delmas, P., Wanaverbecq, N., Abogadie, F. C., Mistry, M., and Brown, D. A. (2002) Neuron 14, 209-220 |
38. | Kiselyov, K., Mignery, G. A., Zhu, M. X., and Muallem, S. (1999) Mol. Cell 4, 423-429[Medline] [Order article via Infotrieve] |
39. |
Boulay, G.,
Brown, D. M.,
Qin, N.,
Jiang, M.,
Dietrich, A.,
Zhu, M. X.,
Chen, Z.,
Birnbaumer, M.,
Mikoshiba, K.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14955-14960 |
40. | Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267[Medline] [Order article via Infotrieve] |
41. | Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179[Medline] [Order article via Infotrieve] |
42. |
Uchiyama, T.,
Yoshikawa, F.,
Hishida, A.,
Furuichi, T.,
and Mikoshiba, K.
(2002)
J. Biol. Chem.
277,
8106-8113 |
43. | Miyawaki, A., Furuichi, T., Maeda, N., and Mikoshiba, K. (1990) Neuron 5, 11-18[CrossRef][Medline] [Order article via Infotrieve] |
44. | Maeda, N., Niinobe, M., Nakahira, K., and Mikoshiba, K. (1988) J. Neurochem. 51, 1724-1730[Medline] [Order article via Infotrieve] |
45. |
Shen, L.,
Liang, F.,
Walensky, L. D.,
and Huganir, R. L.
(2000)
J. Neurosci.
20,
7932-7940 |
46. | Maeda, N., Niinobe, M., and Mikoshiba, K. (1990) EMBO J. 9, 61-67[Abstract] |
47. | Sugiyama, T., Furuya, A., Monkawa, T., Yamamoto-Hino, M., Satoh, S., Ohmori, K., Miyawaki, A., Hanai, N., Mikoshiba, K., and Hasegawa, M. (1994) FEBS Lett. 354, 149-154[CrossRef][Medline] [Order article via Infotrieve] |
48. | Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723[Abstract] |
49. | Dekker, J. W., Budhia, S., Angel, N. Z., Cooper, B. J., Clark, G. J., Hart, D. N. J., and Kato, M. (2002) Immunogenetics 53, 993-1001[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Wojcikiewicz, R. J. H.
(1995)
J. Biol. Chem.
270,
11678-11683 |
51. |
Joseph, S. K.,
Bokkala, S.,
Boehning, D.,
and Zeigler, S.
(2000)
J. Biol. Chem.
275,
16084-16090 |
52. | Laflamme, K., Domingue, O., Guillemette, B. I., and Guillemette, G. (2002) J. Cell. Biochem. 85, 219-228[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Luzzi, V.,
Sims, C. E.,
Soughayer, J. S.,
and Allbritton, N. L.
(1998)
J. Biol. Chem.
273,
28657-28662 |
54. |
Yoshikawa, F.,
Iwasaki, H.,
Michikawa, T.,
Furuichi, T.,
and Mikoshiba, K.
(1999)
J. Biol. Chem.
274,
316-327 |
55. | Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376[Medline] [Order article via Infotrieve] |
56. | Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Yuan, C.-S.,
Ault-Riché, D. B.,
and Borchardt, R. T.
(1996)
J. Biol. Chem.
271,
28009-28016 |
58. |
Gomi, T.,
Takata, Y.,
Date, T.,
Fujioka, M.,
Aksamit, R. R.,
Backlund, P. S., Jr.,
and Cantoni, G. L.
(1990)
J. Biol. Chem.
265,
16102-16107 |
59. |
Aksamit, R. R.,
Backlund, P. S., Jr.,
Moos, M., Jr.,
Caryk, T.,
Gomi, T.,
Ogawa, H.,
Fujioka, M.,
and Cantoni, G. L.
(1994)
J. Biol. Chem.
269,
4084-4091 |
60. |
Ault-Riché, D. B.,
Yuan, C.-S.,
and Borchardt, R. T.
(1994)
J. Biol. Chem.
269,
31472-31478 |
61. | Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C., and Südhof, T. C. (1995) Cell 81, 435-443[Medline] [Order article via Infotrieve] |
62. | Peles, E., Nativ, M., Campbell, P. L., Sakurai, T., Martinez, R., Lev, S., Clary, D. O., Schilling, J., Barnea, G., Plowman, G. D., Grumet, M., and Schlessinger, J. (1995) Cell 82, 251-260[Medline] [Order article via Infotrieve] |
63. | Bosanac, I., Alattia, J.-R., Mal, T. K., Chan, J., Talarico, S., Tong, F. K., Tong, K. I., Yoshikawa, F., Furuichi, T., Iwai, M., Michikawa, T., Mikoshiba, K., and Ikura, M. (2002) Nature 420, 696-700[CrossRef][Medline] [Order article via Infotrieve] |
64. | Peifer, M., Berg, S., and Reynolds, A. B. (1994) Cell 76, 789-791[Medline] [Order article via Infotrieve] |
65. |
Supattapone, S.,
Worley, P. F.,
Baraban, J. M.,
and Snyder, S. H.
(1988)
J. Biol. Chem.
263,
1530-1534 |
66. |
Worley, P. F.,
Baraban, J. M.,
Supattapone, S.,
Wilson, V. S.,
and Snyder, S. H.
(1987)
J. Biol. Chem.
262,
12132-12136 |
67. |
Hannaert-Merah, Z.,
Coquil, J. F.,
Combettes, L.,
Claret, M.,
Mauger, J. P.,
and Champeil, P.
(1994)
J. Biol. Chem.
269,
29642-29649 |
68. | Natsume, T., Hirota, J., Yoshikawa, F., Furuichi, T., and Mikoshiba, K. (1999) Biochem. Biophys. Res. Commun. 260, 527-533[CrossRef][Medline] [Order article via Infotrieve] |
69. | Yoshikawa, F., Uchiyama, T., Iwasaki, H., Tomomori-Satoh, C., Tanaka, T., Furuichi, T., and Mikoshiba, K. (1999) Biochem. Biophys. Res. Commun. 257, 792-797[CrossRef][Medline] [Order article via Infotrieve] |
70. |
Wojcikiewicz, R. J. H.,
Furuichi, T.,
Nakade, S.,
Mikoshiba, K.,
and Nahorski, S. R.
(1994)
J. Biol. Chem.
269,
7963-7969 |
71. |
Bokkala, S.,
and Joseph, S. K.
(1997)
J. Biol. Chem.
272,
12454-12461 |
72. | Oberdorf, J., Webster, J. M., Zhu, C. C., Luo, S. G., and Wojcikiewicz, R. J. H. (1999) Biochem. J. 339, 453-461[CrossRef][Medline] [Order article via Infotrieve] |
73. | Pollok, B. A., and Heim, R. (1999) Trends Cell Biol. 9, 57-60[CrossRef][Medline] [Order article via Infotrieve] |
74. | Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S., and Tsien, R. Y. (1991) Nature 349, 694-697[CrossRef][Medline] [Order article via Infotrieve] |
75. | Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Nature 388, 882-887[CrossRef][Medline] [Order article via Infotrieve] |
76. |
Hirose, K.,
Kadowaki, S.,
Tanabe, M.,
Takeshima, H.,
and Iino, M.
(1999)
Science
284,
1527-1530 |