From the
The inositol 1,4,5-trisphosphate receptor (IP
Many cellular responses to hormones, neurotransmitters, and
growth factors are mediated by the intracellular second messenger
inositol 1,4,5-trisphosphate
(IP
The question remains as to whether distinct types of
IP
In the present study, we sought a cell line in
which all three types are abundantly expressed. Western blot analysis
using type-specific monoclonal antibodies against IP
In denaturing conditions, the solubilized
membrane proteins were boiled for 5 min in buffer A containing 6 M urea, 20 mM dithiothreitol, 1% SDS, and 1% Triton X-100
and dialyzed against 0.15 M NaCl, 5 mM EDTA, 0.1
mM PMSF, 10 µM leupeptin, 10 µM
pepstatin A, and 10 mM sodium phosphate, pH 7.2. The
concentrations of SDS were decreased by 3.3-fold dilution of the sample
with 0.15 M NaCl, 5 mM EDTA, 0.1 mM PMSF, 10
µM leupeptin, 10 µM pepstatin A, and 10
mM sodium phosphate, pH 7.2. These samples were subjected to
the same immunoprecipitation experiments as the non-denaturing sample.
In order to determine which types
of IP
In the present study, we demonstrated by immunoprecipitation
experiments using type-specific antibodies that IP
Northern blotting(22) , reverse
transcriptase-polymerase chain reaction (32), and in situ hybridization studies (21, 22) showed that the
mRNAs of IP
Hepatocytes have been often used for studies of
IP
Recently, Mathias et al. showed that mAb 18A10
inhibited IICR mediated by phospholipase C-
We thank Drs. Takayuki Michikawa, Michio Niinobe, and
J. M. Matheson for useful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
R)
exists as a tetrameric complex to form a functional inositol
1,4,5-trisphosphate-gated Ca
channel. Molecular
cloning studies have shown that there are at least three types of
IP
R subunits, designated type 1, type 2, and type 3. The
levels of expression of IP
R subunits in various cell lines
were investigated by Western blot analysis using type-specific
antibodies against 15 C-terminal amino acids of each IP
R
subunit. We found that all the three types of IP
R subunits
were expressed in each cell line examined, but their levels of
expression varied. To determine whether IP
Rs form
heterotetramers, we employed immunoprecipitation experiments using
Chinese hamster ovary cells (CHO-K1 cells), in which all three types
are abundantly expressed. Each type-specific antibody
immunoprecipitated not only the respective cognate type but also the
other two types. This result suggests that distinct types of
IP
R subunits assemble to form heterotetramers in CHO-K1
cells. We also detected heterotetramers in rat liver, in which
IP
R type 1 and type 2 are expressed abundantly. Previous
studies have shown some functional differences among IP
R
types, suggesting the possibility that various compositions of subunits
show distinct channel properties. The diversity of IP
R
channels may be further increased by the co-assembly of different
IP
R subunits to form homo- or heterotetramers.
)
(
)(1) . IP
releases Ca
from intracellular stores by
binding to an IP
receptor (IP
R)(2) ,
which composes an IP
-gated Ca
channel(3, 4, 5) . Molecular cloning
studies showed that there are at least three types of IP
R
subunits derived from distinct genes, designated type 1
(IP
R-1)(6, 7, 8, 9) , type 2
(IP
R-2)(10, 11, 12) , and type 3
(IP
R-3)(12, 13, 14) . Electron
microscopic observations(15, 16) ,
cross-linking(5) , and sucrose gradient centrifugation
experiments (17) have demonstrated that IP
Rs form
tetramers. Structural and functional analyses revealed that the
structure of IP
R can be divided into three functionally
different domains: an N-terminal IP
-binding domain, a
regulatory domain, and a channel domain near the C
terminus(18, 19) . The channel domain is sufficient for
assembly of the subunits to yield the tetrameric organization of the
IP
R (18, 19) and is well conserved (amino
acid identity of 65-70%) among members of the IP
R
family (12).
R subunits assemble to form heterotetramers. Previous
studies described only homotetramers (5, 20) probably
because in the studies cerebella were used as the material, where
IP
R-1 is predominant. In addition, because of the absence
of an antibody that specifically recognizes IP
R-2 or
IP
R-3, it was hard to determine IP
R subunit
composition. In situ hybridization studies showed distinct but
overlapping expression patterns of
IP
Rs(21, 22) . Some cell types coexpress two
or three types of the IP
R subunits. The coexistence of
different types in individual cells suggests the possibility that the
IP
R complex is composed of heteromeric structures. On the
basis of similarities in transmembrane topology, we have proposed that
IP
R is a member of the superfamily that includes the
voltage- and second messenger-gated ion channels(23) . The
voltage-gated K
channels (24, 25) and
the cyclic nucleotide-gated ion channels (26) have been
demonstrated to form hetero-oligomers. By analogy, IP
R-1,
IP
R-2 and IP
R-3 subunits might also assemble to
form heterotetramers.
R-1,
IP
R-2, and IP
R-3 showed that all three types
are abundantly expressed in Chinese hamster ovary cells (CHO-K1 cells).
We also found that IP
R-1 and IP
R-2 are
expressed in rat liver. By immunoprecipitation studies with the
type-specific antibodies, we present here biochemical evidence that the
three IP
R subunits co-assemble in CHO-K1 cells and rat
liver.
Preparation of Membrane Proteins
CHO-K1 cells
(kindly provided by Dr. Masahiro Nishijima, National Institute of
Health, Japan) were cultured in Ham's F-12 medium supplemented
with 10% fetal calf serum. The cells were mixed with 9 volumes of
solution containing 0.25 M sucrose, 1 mM EDTA, 0.1
mM phenylmethylsulfonyl fluoride (PMSF), 10 µM leupeptin, 10 µM pepstatin A, 1 mM 2-mercaptoethanol, and 5 mM Tris-HCl, pH 7.4, and
homogenized in a glass-Teflon Potter homogenizer with 10 strokes at
1,000 rpm. The homogenates were centrifuged at 1,000 g for 5 min at 4 °C. The supernatants were centrifuged at
105,000
g for 60 min at 2 °C to sediment the
membrane proteins. The pellets were resuspended in 1 mM EDTA,
0.1 mM PMSF, 10 µM leupeptin, 10 µM pepstatin A, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 7.4 (buffer A). Protein concentrations were measured
using the Bio-Rad protein assay kit. In the preparation of liver
membrane proteins, adult Wistar rat (7-8 weeks old, male) were
anesthetized by pentobarbital and perfused transcardially with
phosphate-buffered saline. After the liver was removed and minced, the
membrane proteins were prepared according to the same method as CHO-K1
cells.
Immunoprecipitation
The membrane proteins were
solubilized by the addition of 10% Triton X-100 to give final protein
and detergent concentrations of 3.0 mg/ml and 1.0%, respectively. The
solution was stirred for 30 min at 4 °C and centrifuged at 20,000
g for 60 min at 2 °C, and the supernatant was used
for the immunoprecipitation experiment. In non-denaturing conditions,
the solubilized membrane proteins were added to immunoprecipitation
buffer containing 0.15 M NaCl, 0.3% Triton X-100, 0.1% bovine
serum albumin, 5 mM EDTA, 0.1 mM PMSF, 10 µM pepstatin A, and 10 mM sodium phosphate, pH 7.2, and
precleared by Pansorbin (Calbiochem-Novabiochem Corp.). The precleared
supernatants were incubated with 6 µg/ml mAb 18A10
(anti-IP
R-1 antibody), mAb KM1083 (anti-IP
R-2
antibody), or mAb KM1082 (anti-IP
R-3 antibody) for 1 h at 4
°C. Then 6 µg/ml anti-rat IgG (Fc region-specific) and
anti-mouse IgG (Fc region-specific) were added to the mAb 18A10 sample
and the mAb KM1082 sample, respectively. Immune complexes were
collected with Pansorbin. The Pansorbin particles were washed three
times with 0.01% bovine serum albumin, 0.5% Triton X-100, 0.15 M NaCl, and 10 mM sodium phosphate, pH 7.2. The Pansorbin
pellets were mixed with SDS-PAGE sampling buffer containing 4% SDS, 10%
2-mercaptoethanol, 20% glycerol, and 0.125 M Tris-HCl, pH 6.8,
and then boiled for 3 min. After centrifugation, the supernatants were
subjected to 5% SDS-PAGE in the buffer system of Laemmli(27) .
After transferring the proteins electrophoretically to nitrocellulose
filters, immunodetections probed with type-specific antibodies were
performed. Bound antibodies were visualized by an ECL Western blotting
system (Amersham Corp.).
Expression of IP
cDNAs coding the entire protein of mouse
IPR Proteins from
cDNA
R-1 (6) and human IP
R-3 (12) were inserted into expression vector pBactS, which contains
a
-actin promoter and a simian virus 40 polyadenylation
sequence(6) . These plasmid DNAs were introduced into
NG108-15 cells by a calcium phosphate precipitation
technique(28) . 20 µg of DNA were used for each 10-cm cell
culture dish. When two cDNAs were co-transfected, 10 µg of each was
used. Three days after transfection, the cells were collected. The
membrane proteins of these transfected cells were prepared as mentioned
above.
Expression of IP
Mouse mAbs KM1112, KM1083, and KM1082 were
raised against synthetic peptides corresponding to 15 C-terminal amino
acids of IPR Subunits in
Cell Lines
R-1 (human IP
R-1
2681-2695)(8) , IP
R-2 (human IP
R-2
2687-2701)(12) , and IP
R-3 (human
IP
R-3 2657-2671)(12) , respectively (). Detailed characterizations of these antibodies were
described previously(29) .
R subunits are expressed in various cell lines, we
performed Western blot analysis using mAb KM1112
(anti-IP
R-1), mAb KM1083 (anti-IP
R-2), and mAb
KM1082 (anti-IP
R-3) (Fig. 1). Almost all of the three
types were expressed in each cell line examined, but the levels of
expression were cell type-specific. IP
R-1 was detected in
LLC-PK1, OK, CHO-K1, RINm5F, PC12A, C6, NG108-15, Jurkat, Raji,
and K562 cells and could be detected in Madin-Darby canine kidney cells
with a longer exposure on immunodetection. The level of expression of
IP
R-2 was most variable. IP
R-2 was detected in
Madin-Darby canine kidney, LLC-PK1, CHO-K1, RINm5F, C6, NG108-15,
Jurkat, Raji, and K562 cells and could be detected in OK and PC12A
cells with a longer exposure on immunodetection. IP
R-3 was
detected in all cell lines examined. We found that all three types were
abundantly expressed in CHO-K1 cells, and the tetrameric structure of
IP
R in this cell line was investigated.
Figure 1:
Expression of IPR types in
cell lines. 30 µg of membrane proteins of various cell lines were
analyzed by Western blot using mAb KM1112 (anti-IP
R-1, A), mAb KM1083 (anti-IP
R-2, B), and mAb
KM1082 (anti-IP
R-3, C). Madin-Darby canine kidney (MDCK), LLC-PK1 pig kidney, OK American opossum kidney, CHO-K1
Chinese hamster ovary, RINm5F rat insulinoma, PC12A rat
pheochromocytoma, C6 rat glial, NG108-15 mouse neuroblastoma
rat glioma hybrid, Jurkat human T cell leukemia, Raji human
Burkitt lymphoma, and K562 human chronic myelogenous leukemia cells are
shown. The lower molecular weight bands seem to be degraded proteins of
IP
Rs, because these bands were increased and the bands of
IP
Rs were decreased by incubating the samples at 37 °C
(data not shown). The bands of high molecular weight are seen in
IP
R-3, and are thought to represent dimer
complexes.
Heterotetramers in CHO-K1 Cells
Membrane proteins
of CHO-K1 cells were solubilized in 1% Triton X-100. To determine
whether distinct IPR subunits assemble to form
heterotetramers in CHO-K1 cells, we employed immunoprecipitation
experiments using the type-specific antibodies. We used mAb 18A10 (30, 31) for immunoprecipitation of IP
R-1,
because this antibody immunoprecipitates IP
R-1 more
effectively than mAb KM1112. We examined to what extent the mAb
immunoprecipitated their respective antigens from the solubilized
membrane proteins in CHO-K1 cells. The immunoprecipitates with mAbs
18A10, KM1083, and KM1082 were immunoblotted with mAbs KM1112, KM1083,
and KM1082, respectively. Judging from the intensity of the bands of
the immunoblotting, we estimated that mAbs 18A10, KM1083, and KM1082
immunoprecipitated 62% of IP
R-1, 98% of IP
R-2,
and 29% of IP
R-3, respectively (). Then, we
examined the presence of the three types of IP
Rs in each of
the immunoprecipitates. The immunoprecipitate from mAb 18A10 was
subjected to Western blot analysis using mAbs KM1112, KM1083, and
KM1082. Monoclonal antibody 18A10 immunoprecipitated not only
IP
R-1 but also significant amounts of IP
R-2 and
IP
R-3 (Fig. 2, lane 1). In the same way, mAb
KM1083 and mAb KM1082 immunoprecipitated all three IP
Rs
including their respective cognate antigens (Fig. 2, lanes 3 and 5). Although the band intensity does not necessarily
reflect the total amount of IP
R, it is possible to
quantitate the immunoblot results. The immunoprecipitate of each mAb
gave rise to immunosignals for the three IP
R subunits with
similar intensity (Fig. 2, lanes 1, 3, and 5). Similar results were obtained from repetitive experiments (n = 6). Besides, equal amounts of all three
IP
Rs were detected in the supernatant of each mAb (data not
shown). These results indicate that IP
R-1,
IP
R-2, and IP
R-3 associate with each other in
CHO-K1 cells and that homomeric IP
Rs account for a
relatively small fraction of the tetramer complexes. Nakade et al.(20) found that boiling the membranes in the presence of 6 M urea, 20 mM dithiothreitol, and 1% SDS dissociates
tetramer complexes of IP
R into monomers that are still
capable of being immunoprecipitated. Co-immunoprecipitations of
distinct types by each antibody were abolished under these denaturing
conditions, whereas the respective cognate antigens were still
immunoprecipitated ( Fig. 2lanes 2, 4, and 6); mAb 18A10 was somewhat ineffective in precipitating the
denatured IP
R-1. These findings exclude the possibility
that co-immunoprecipitation of distinct types occurs through
cross-reactivity of the antibodies or through nonspecific interaction
with Pansorbin.
Figure 2:
Immunoprecipitation of IPR
subunits from CHO-K1 cells. CHO-K1 membrane proteins solubilized in 1%
Triton X-100 (N, non-denaturing conditions; lanes 1, 3, and 5) or boiled in the presence of 6 M urea, 20 mM dithiothreitol, and 1% SDS (D,
denaturing conditions; lanes 2, 4, and 6)
were immunoprecipitated by the type-specific mAbs, as indicated at the top of each column. The immunoprecipitates were
separated by 5% SDS-PAGE and transferred to nitrocellulose filters.
Filters were incubated with mAb KM1112 (A), mAb KM1083 (B), and mAb KM1082 (C).
Next, we performed the same immunoprecipitation
experiments using cDNA-transfected cells. When NG108-15 cells
were transfected with both IPR-1 and IP
R-3
cDNAs, mAb 18A10 immunoprecipitated not only IP
R-1 (Fig. 3A, lane 1) but also IP
R-3 (Fig. 3B, lane 1). In the same way, mAb KM1082
immunoprecipitated not only IP
R-3 (Fig. 3B, lane 2) but also IP
R-1 (Fig. 3A, lane 2). IP
R-1 and IP
R-3 subunits
formed heterotetramers in the cells co-transfected with both cDNAs. On
the other hand, when the cells were transfected separately with
IP
R-1 or IP
R-3 cDNAs and mixed together prior
to solubilization of membranes, the precipitation of IP
R-3
by mAb 18A10 or of IP
R-1 by mAb KM1082 was not detected (Fig. 3, A, lane 5, and B, lane
4). With longer exposure, very faint bands of IP
R-1
precipitated by mAb KM1082 and of IP
R-3 precipitated by mAb
18A10 appeared. These precipitates probably represent heteromeric
complexes containing endogenous receptors of NG108-15 cells.
These results demonstrated that IP
R-1 and IP
R-3
subunits assemble in cDNA-transfected cells and also rule out the
possibility of artifactual rearrangement between subunits during the
experimental procedures. It is therefore concluded that
IP
R-1, IP
R-2, and IP
R-3 subunits
actually form heteromeric complexes in CHO-K1 cells.
Figure 3:
Immunoprecipitation of IPR
subunits from cDNA-transfected cells. The mixture of cDNAs of
IP
R-1 and IP
R-3 was used to transfect
NG108-15 cells. The solubilized membrane proteins of these cells
were prepared and are labeled Co-transfected. The cells
transfected separately with IP
R-1 or IP
R-3 cDNA
were combined. The solubilized membrane proteins of these cells were
prepared and are labeled MIX. The solubilized membrane
proteins of the co-transfected or mixed cells were immunoprecipitated
by mAb 18A10 (lanes 1 and 4) or mAb KM1082 (lanes
2 and 5). The immunoprecipitates together with the
solubilized membrane proteins (lanes 3 and 6) were
separated by 5% SDS-PAGE and transferred to nitrocellulose filters.
Filters were incubated with mAb KM1112 (A) or mAb KM1082 (B). ppt, precipitate.
Heterotetramers in Liver
We investigated the
presence of heterotetramers in tissues. Cerebellum and liver are often
used to study IP-mediated Ca
signaling.
IP
R-1 is predominant in the cerebellum, where it is
difficult to detect heterotetramers. On the other hand, in liver, we
found that IP
R-1 and IP
R-2 were abundantly
expressed, whereas IP
R-3 was scarcely detected (Fig. 4A). The cells composing the liver are relatively
homogenous, and most of them are hepatocytes. We examined whether
IP
R-1 and IP
R-2 subunits assemble to form
heterotetramers in liver. Immunoprecipitation experiments using liver
membrane proteins showed that mAb 18A10 immunoprecipitated
IP
R-2 as well as IP
R-1 (Fig. 4B, lanes 1 and 4) and that mAb KM1083 immunoprecipitated
IP
R-1 as well as IP
R-2 (Fig. 4B, lanes 2 and 5). These results indicate that
IP
R-1 and IP
R-2 assemble in liver. With a
longer exposure on immunodetection, we observed that mAb KM1082
immunoprecipitated IP
R-1 and IP
R-2 (data not
shown), suggesting that IP
R-3 subunits also associate with
other IP
R subunits in liver. It is therefore concluded that
IP
Rs form heterotetrameric complexes in liver.
Figure 4:
Immunoprecipitation of IPR
subunits from rat liver. A, 20 µg of membrane proteins of
liver were analyzed by Western blot using mAb KM1112 (lane 1),
mAb KM1083 (lane 2), and mAb KM1082 (lane 3). B, liver membrane proteins solubilized in 1% Triton X-100 were
immunoprecipitated by type-specific antibodies, as indicated at the top of each column. The immunoprecipitates were
separated by 5% SDS-PAGE and transferred to nitrocellulose filters.
Filters were incubated with mAb KM1112 (lanes 1-3), mAb
KM1083 (lanes 4-6), and mAb KM1082 (lanes
7-9).
Rs form
heterotetramers in CHO-K1 cells and the liver. Heterotetramers were not
generated by mixing the cDNA-derived homotetramers. In addition,
immunoprecipitation experiments in denaturing conditions showed no
cross-reaction between the antibodies. The results of these experiments
exclude the possibility that the receptor complexes are artifactually
formed during the experimental procedures. We also detected
heterotetramers in RINm5F and Jurkat cells (data not shown), suggesting
that the heterotetrameric complex formation of IP
R subunits
is common to the cells expressing two or three types of the
IP
R subunits.
R types were differentially expressed among
mouse tissues. Recently, Sugiyama et al.(33) reported
that the levels of expression of IP
R subunits are quite
different among hematopoietic cell lines and change dramatically with
stimuli that induce differentiation. There seem to be functional
differences among IP
R subunits. IP
binding
measurements using the ligand binding domains of IP
R-1 and
IP
R-2 indicated that IP
R-2 has a higher
affinity than IP
R-1(10) . This suggests that
different receptor types may respond to different IP
levels
within a cell. Mouse IP
R-1 contains two phosphorylation
sites of cAMP-dependent protein kinase at positions 1589 and 1755 (34),
which were shown to be phosphorylated by cyclic AMP-dependent protein
kinase both in vitro(35) and in intact
cells(36) . Recently, we showed that phosphorylation by cyclic
AMP-dependent protein kinase of IP
R-1 that was
immunoaffinity-purified from cerebella increased IP
-induced
Ca
release (IICR) in reconstituted lipid
vesicles(20) . These two sites are not conserved in
IP
R-2 and IP
R-3(12) . We have also found
that calmodulin binds IP
R-1 in the presence of
Ca
and have determined its binding site in
IP
R-1(37) . The calmodulin binding site is conserved
in IP
R-2 but not in IP
R-3. In fact, we were
able to detect the Ca
-dependent calmodulin binding to
IP
R-2 but not to IP
R-3. Therefore, each member
of the IP
R family is possibly regulated in different
manners by cross-talk with other intracellular signaling molecules.
Thus far, the channel properties of IP
R have been studied
only with IP3R-1 homotetramers(5) . The channel properties of
IP
R-2 and IP
R-3 homotetramers are currently
unknown. In voltage-dependent K
channels and cyclic
nucleotide-gated cation channels, the hetero-oligomeric channels have
distinct properties from the homomeric channels of the parent
subunits(24, 25, 26) . By analogy, the
heterotetramers of IP
R might also exhibit distinct
properties. The various subunit compositions of IP
R
tetramers could increase further the diversity of IP
R
channels.
-mediated Ca
signaling. We found that
IP
R-1 and IP
R-2 proteins are abundantly
expressed in the liver. This agrees with a recent study reported by De
Smedt et al.(38) , who investigated the levels of
expression of IP
R mRNAs in rat liver by reverse
transcriptase-polymerase chain reaction. Messenger RNAs encoding
IP
R-1, IP
R-2, and IP
R-3 accounted
for 29.4%, 61.1%, and 3.0%, respectively. Interestingly, cerebellar
Purkinje cells were shown to exhibit a lower IP
sensitivity
for IICR than hepatocytes(39, 40) . This difference may
be explained by the presence of IP
R-2 in the liver, which
seems to have a higher affinity for IP
than
IP
R-1(10) . Furthermore, our present study
demonstrated that a large proportion of IP
R-1 and
IP
R-2 subunits form heterotetramers in the liver. How the
heterotetramers contribute to the liver-specific
IP
-mediated Ca
signaling remains to be
examined.
in CHO cells.
(
)This inhibitory effect was also seen with
microinjected heparin. Because heparin is a competitive antagonist of
IP
Rs, it seems to inhibit IICR through all of the
IP
R channels. mAb 18A10 recognizes only IP
R-1
and inhibits IICR only through IP
R-1 (20). In this study,
we have shown that all the three IP
R subunits are expressed
in CHO cells. Why did mAb 18A10 completely inhibit IICR in spite of the
presence of a considerable amount of IP
R-2 and
IP
R-3, which are insensitive to mAb 18A10? If all of the
IP
R tetramers are homotetrameric, mAb 18A10 cannot inhibit
IICR through the homomeric channels composed of IP
R-2 or
IP
R-3 subunits. Because our data suggest that
IP
Rs form heterotetramers in CHO cells, one explanation for
the findings of Mathias et al. that mAb 18A10 completely
inhibits channel activity of these IP
Rs could be that
virtually all heterotetramers contain at least one IP
R-1
subunit. Therefore, the findings reported by Mathias et al. provide additional support to our biochemical findings that
IP
Rs form heterotetrameric complexes.
Table: IPR type-specific monoclonal
antibodies
, inositol 1,4,5-trisphosphate;
IP
R, IP
receptor; IP
R-1,
IP
R type 1; IP
R-2, IP
R type 2;
IP
R-3, IP
R type 3; PMSF, phenylmethylsulfonyl
fluoride; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal
antibody; IICR, IP
-induced Ca
release;
CHO, Chinese hamster ovary.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.