(Received for publication, June 8, 1995)
From the
We have previously shown that a 222-kDa polypeptide
co-immunoprecipitates together with the type-I myoinositol
1,4,5-trisphosphate receptor (IPR) in WB rat liver
epithelial cell extracts, when the immunoprecipitation is carried out
with a type-I isoform specific antibody (Joseph, S. K.(1994) J.
Biol. Chem. 269, 5673-5679). Utilizing isoform-specific
antibodies raised to unique sequences within the COOH-terminal region
of IP
receptors, we now report that the
co-immunoprecipitating 222-kDa polypeptide is the type-III
IP
R isoform and that type-III IP
R antibodies
(Abs) can co-immunoprecipitate the type-I IP
R isoform.
Co-immunoprecipitation of IP
R isoforms was not due to
cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react
with type-I IP
R and vice versa; (b)
inclusion of the COOH-terminal type-III peptide had no effect on the
ability of type-I IP
R Ab to co-immunoprecipitate the
type-III IP
R but blocked the ability of type-III
IP
R Ab to co-immunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of
type-III IP
R, and immunoprecipitation with type-III
IP
R Ab does not co-immunoprecipitate any other isoforms.
However, type-I and type-II IP
R isoforms were
co-immunoprecipitated by their respective antibodies in hepatocyte
lysates. Sucrose density gradient analysis of WB cell lysates indicated
that the co-immunoprecipitating fraction is exclusively located at the
density expected for tetrameric receptors, suggesting that
co-immunoprecipitation was not a reflection of the nonspecific
aggregation of IP
R isoforms. Phosphorylation of either
type-I or type-III immunoprecipitates by protein kinase A indicated
that only the type-I IP
R could be phosphorylated in
vitro. Fractionation of WB cell membranes and immunofluorescence
studies showed that the type-I and type-III isoforms have very similar
sub-cellular localizations. We conclude that the WB cell contains both
type-I and type-III IP
R isoforms and that a proportion of
these receptors exist as heterotetramers.
The discharge of Ca from intracellular stores
in response to the activation of cell surface receptors is mediated by
the interaction of inositol 1,4,5-trisphosphate with a specific
receptor that functions as a ligand-gated Ca
channel(1, 2, 3) . Molecular cloning
studies have revealed the presence of three types of receptors encoded
by separate genes. The type-I IP
R (
)is
particularly enriched in the cerebellum region of the brain and has a
calculated molecular mass based on cDNA sequence of 313
kDa(4, 5) . The purified receptor has been shown to be
a homotetramer(6) . The type-I receptor mRNA undergoes
alternative splicing in two distinct regions of the receptor designated
S1 and S2. Several studies have shown that the type-I receptor with the
S2 region deleted is the predominant form in peripheral
tissues(7, 8) . The type-II IP
R is 69%
homologous to the type-I IP
R and was originally described
as being expressed in brain(9) . Subsequent studies have since
shown the presence of type-II IP
R mRNA in several
peripheral tissues(10) . The type-III IP
R has 62%
homology to the type-I IP
R and is expressed in several
epithelial cells including those of the kidney, pancreas, and
intestinal tract(11, 12) . Only partial sequences of a
type-IV (13) and type-V receptor (10) have been
reported, and their distribution and properties have not been
characterized.
In a previous study, we investigated the biosynthesis
and turnover of IPR in cultured WB rat liver epithelial
cells(14) . We found that immunoprecipitation of
S-labeled WB cell extracts with type-I IP
R
antibody resulted in the appearance of two
S-labeled
polypeptides with molecular masses of 235 and 222 kDa. Only the 235-kDa
band was immunoreactive with type-I-specific IP
R antibody.
The 222-kDa band, which was present in lower amounts relative to the
235-kDa band, was not identified but was shown not to be a proteolytic
clip of the type-I IP
R. Based on the cDNA the type-III
IP
R has a calculated molecular mass of 304-kDa and would
therefore be expected to run at a lower molecular mass than type-I
IP
R on SDS-PAGE. In the present study we have utilized
isoform-specific IP
R antibodies to show that the 222-kDa
band indeed corresponds to the type-III IP
R. Additional
data are shown demonstrating co-localization of both isoforms in WB
cells, and evidence is presented to indicate that a proportion of both
isoforms exist as heterotetrameric complexes.
Figure 1:
Co-immunoprecipitation of type-III
IPR with type-I IP
R in
S-labeled
WB extracts. Cells were labeled with Trans
S-label and
immunoprecipitated with either type-I Ab (lane 1) or type-III
Ab (lane 2) as described under ``Materials and
Methods.'' The immunoprecipitates were run out on a 5% SDS gel
together with an unlabeled sample of WB cell extract (lane 3).
The polypeptides were transferred to nitrocellulose, which was then
autoradiographed (Auto-rad, A). The same
nitrocellulose sheet was then sequentially immunoblotted with type-III
Ab (B), stripped, immunoblotted with type-I Ab (C),
stripped, and immunoblotted with both Abs (D).
From these data we conclude that the previously
unidentified S-labeled 222-kDa band present in type-I
immunoprecipitates corresponds to the type-III IP
R. Because
traces of type-I IP
R are also present in type-III
immunoprecipitates, the data would also suggest that a pool of both
receptors are associated and are co-immunoprecipitated. However, such a
conclusion relies on the antibodies being entirely specific for their
respective antigens. The recognition properties of the type-III
polyclonal antibody (GST-H3CT Ab; (12) ) is shown in Fig. 2A. Detergent extracts of cerebellum microsomes,
WB cells and hepatocytes were immunoblotted against type-I and type-III
antiserum. As expected, the type-I receptor that is enriched in
cerebellum produces a strong signal when probed against type-I Ab (Fig. 2A, upper panel). However, the
cerebellar type-I IP
R shows no cross-reactivity to the
type-III Ab (Fig. 2A, lower panel). Sufficient
amounts of type-I and type-III receptor are present in 50 µg of WB
cell extract proteins to enable a clear signal to be obtained from
immunoblotting without the necessity of immunoprecipitating the
extracts. This is not the case for hepatocytes where a weak signal for
type-I is seen only when 200 µg of extract protein is loaded into a
gel lane. The amount of type-III receptor in hepatocyte extracts was
below the detection limit. This is in agreement with a recent study
using reverse transcriptase-polymerase chain reaction that found only
3% of the total IP
R mRNA in rat liver encoded the type-III
isoform(10) . The co-immunoprecipitation of IP
R
isoforms seen in Fig. 1could be duplicated using monoclonal
antibodies to type-I and type-III IP
R (data not shown). The
immunoblot in Fig. 2B (lane 2) shows that the
type-III monoclonal Ab also selectively recognizes only the lower
222-kDa
S-labeled band in the type-I monoclonal Ab
immunoprecipitates.
Figure 2:
Specificity of IPR antibodies. A, protein from cerebellum microsomes (lane 1, 50
µg), WB cell membranes (lane 2, 50 µg), or hepatocytes (lane 3, 50 µg; lane 4, 100 µg; lane
5, 200 µg) were run out on a 5% SDS gel and subjected to
immunoblotting with CT-3 Ab (lower panel). The same blot was
then stripped and reprobed with CT-1 Ab (upper panel). B, WB cells were labeled with Trans
S-label and
immunoprecipitated with a type-I monoclonal antibody. The
autoradiograph of the immunoprecipitate is shown in lane 1,
and the immunoblot with type-III monoclonal antibody is shown in lane 2.
A further criteria of specificity in antibody
recognition is that immunoprecipitation of the target antigen should be
suppressed by inclusion of the peptide epitope used in immunization. In
the case of type-I IPR antibody, we have previously shown
that the CT-1 peptide blocks the immunoprecipitation of both
S-labeled bands from WB extracts(14) . In Fig. 3unlabeled WB extracts were immunoprecipitated with
polyclonal type-I or type-III antibody in the presence or absence of
CT-3 peptide. The immunoprecipitates were immunoblotted with either
type-I- (Fig. 3A) or type-III-specific antibodies (Fig. 3B). The results show that the CT-3 peptide does
not influence the ability of type-I antibody to immunoprecipitate the
type-I IP
R or to co-immunoprecipitate the type-III
IP
R. However, the CT-3 peptide markedly inhibits the
ability of the type-III antibody to immunoprecipitate the type-III
IP
R and co-immunoprecipitate the type-I IP
R
isoform. This selectivity of peptide inhibition suggests that
co-immunoprecipitation of receptors is not due to a lack of specificity
in the recognition properties of the IP
R antibodies.
Figure 3:
The effect of COOH-terminal type-III
receptor peptide on immunoprecipitation of receptor isoforms. 1-ml
aliquots of WB extracts were precleared and immunoprecipitated
overnight with either type-I or type-III antibody as described under
``Materials and Methods.'' Where present, the COOH-terminal
peptide of the type-III IPR (CT-3 peptide) was added at a
concentration of 100 µg/ml. The washed immunoprecipitates were run
out on 5% SDS-PAGE, and the gel was immunoblotted with type-I Ab (A) or type-III Ab (B). Cerebellum extract (0.5
µg of protein) was loaded in lane 1 of A. The
migration position of the 214-kDa myosin marker is
indicated.
Figure 4:
Protein kinase A phosphorylation of type-I
and type-III receptors. WB cell extracts were immunoprecipitated
overnight with CT-1 Ab (lane 1) or CT-3 Ab (lane 2).
The immunoprecipitates were washed once in WB solubilization buffer and
twice in phosphorylation buffer, which contained 120 mM KCl,
20 mM Tris-HCl (pH 7.2), 0.3 mM MgCl, 0.1
mM sodium orthovanadate, and 1% Triton X-100. The
immunoprecipitates were then incubated for 5 min at 37 °C in 0.25
ml of the phosphorylation buffer containing 10 µCi
[
-
P]ATP and 100 units/ml catalytic subunit
of protein kinase A (Sigma). The immunoprecipitates were recovered by
centrifugation, washed three times in phosphorylation buffer containing
1 mM unlabeled ATP, and quenched in SDS-PAGE sample buffer.
The samples were electrophoresed, transferred to nitrocellulose, and
autoradiographed (lanes 1 and 2). The location of the
type-I and type-III IP
R in lane 2 was determined
by consecutive immunoblotting of the same sheet of nitrocellulose with
CT-3 Ab (lane 3) and CT-1 Ab (lane 4). The identity
of the other phosphorylated bands seen below the myosin marker in lane 1 are not known.
Figure 5:
Comparison of the distribution of type-I
and type-III IPR receptors in WB cell membranes. WB cell
homogenates were fractionated on 50-80% sucrose density gradients
as described under ``Materials and Methods.'' 70 µl of
each fraction was treated with SDS sample buffer and electrophoresed on
a 7% gel. After transfer, the nitrocellulose sheet was cut below the
prestained myosin marker (214 kDa), and the upper part of the sheet was
consecutively immunoblotted with type-I and type-III IP
R
Ab. Similarly, the lower part of the nitrocellose sheet was probed
consecutively with antibodies against ribophorin-I, lamin-B, and
Na
/K
-ATPase.
Figure 6:
Indirect immunofluorescence of
IPR in WB cells. WB cells were grown on glass coverslips
and processed for immunofluorescence as described under
``Materials and Methods.'' All panels were photographed at
the same magnification through a
40 lens. A, staining
with type-I antibody; B, staining with type-III antibody; C, staining with anti-ribophorin antibody; D,
staining with type-III antibody preblocked with 100 µg/ml of
type-III COOH-terminal peptide.
Figure 7:
Analysis of WB cell lysates on sucrose
density gradients. 0.5-ml WB cell lysates (4-5 mg protein/ml)
were loaded on to 11-ml 5-20% sucrose density gradients prepared
in a buffer containing 150 mM NaCl, 50 mM Tris (pH
7.8), 1 mM EDTA, and 0.25% Triton X-100. The tubes were
centrifuged in a SW 41 Ti rotor at 100,000 g for 16 h.
34 fractions were collected from the top of each tube. Aliquots of
alternate fractions were run out directly on SDS-PAGE for
immunoblotting or were immunoprecipitated with the indicated antibody.
Immunoblotting of gradients containing 0.5 ml of cerebellum microsome
extract (1 mg protein/ml) or denatured cerebellum extract (treated for
15 min at 4 °C with 0.2% SDS) were used to localize the position of
type-I heterotetramer and monomer, respectively. Separate gradients
containing 1 mg of
-amylase (200 kDa) or thyroglobulin (669 kDa)
as size markers were processed, fractionated, and assayed for protein.
Only the data from WB cell extract fractionation is shown in A. B and C show the densitometric
quantitation of the data in A and includes the peak
localization of the size standards used in the experiment.
,
type-I;
, type-III.
Figure 8:
Co-immunoprecipitation of IPR
isoforms in hepatocyte lysates. Isolated hepatocytes were solubilized
as described previously(37) . 1-ml aliquots of hepatocyte
lysate (14 mg of protein/ml) were immunoprecipitated for 4 h at 4
°C with 5 µg of monoclonal antibodies to type-I, -II, or -III
IP
R. Where indicated, the COOH-terminal peptides of the
type-I and type-III IP
R were present at 50 µg/ml. The
immunoprecipitates were collected on protein A-Sepharose, run out on
SDS-PAGE, and immunoblotted with either type-I (A) or type-II (B) monoclonal antibodies.
The results of the present study establish that the lower
component of the S-labeled doublet of polypeptides
immunoprecipitated by the type-I IP
R Ab from WB cell
lysates is the type-III IP
R isoform. Although the presence
of multiple IP
R isoforms in specific cell types has been
inferred from mRNA analysis(10, 22, 23) , the
quantitation of relative levels of immunoreactive protein and the
subcellular localization of multiple isoforms has been analyzed in very
few cell types. In PC12 cells, an IP
R antibody that does
not discriminate between isoforms has been shown to detect a doublet of
polypeptides of which only the upper band is the type-I
IP
R(22) . The Jurkat T-cell has been shown to
contain all three isoforms, mast cells contain both type-I and type-II,
and only the type-II isoform can be detected in
macrophages(17) . WB rat liver epithelial cells contain high
levels of both type-I and type-III IP
R isoforms with no
detectable type-II isoform (data not shown). Isolated hepatocytes are
different in that they have a much lower total expression of receptors
and that they contain the type-I and type-II IP
R as the
predominant isoforms. A quantitation of the relative abundance of the
three isoforms in several cell lines and tissues has recently been
published(24) . In a megakaryocytic cell line, fluorescence
confocal microscopy was used to show that the type-I and type-II
receptors are localized differently (17) . This is clearly
different from our results in WB cells where no gross differences in
localization of type-I and type-III isoforms were noted from
subcellular fractionation or immunofluorescence measurements. Both
approaches also showed that the localization of the IP
R
isoforms was distinct from the distribution of ribophorin-I, a rough ER
marker. This supports the conclusion that in some cell types the
IP
R may be localized in a subcompartment of the
ER(25, 26, 27) .
The present study
demonstrates that antibodies specific to one IPR isoform
can co-immunoprecipitate additional isoforms. The same finding was also
noted by Wojcikiewicz in a recent study(24) .
Co-immunoprecipitation of different voltage-gated K
channels (28, 29) has been used as evidence for
the formation of heteromultimeric complexes in vivo. However,
the validity of these conclusions relies on the absolute specificity of
the antibodies that are used in this experimental approach. The isoform
specificity of the IP
R anti-peptide antibodies used in the
present studies are based on the following observations: (a)
The antibodies are raised against the nonconserved COOH-terminal
regions and do not cross-react with multiple isoforms on immunoblots.
Thus, the type-III polyclonal antibody does not react with the type-I
IP
R (from cerebellum or WB cells), and the type-I
polyclonal does not recognize the type-III IP
R. (b) The ability of an isoform-specific antibody to
immunoprecipitate that isoform and co-immunoprecipitate other isoforms
is blocked by the COOH-terminal cognate peptide of that antibody but
not by any other peptides. These results imply that
co-immunoprecipitation occurs as a result of the association of
receptor isoforms and not as a result of cross-reactivity. (c)
The type-III IP
R is absent from hepatocyte lysates.
Immunoprecipitation with the type-III antibody does not bring down any
type-I or type-II IP
R, as would be expected if the observed
antibody interactions are specific. (d) Finally, the
experimental observations on co-immunoprecipitation and subcellular
localization have been validated with two different antibodies for each
isoform (data not shown).
We have interpreted our results to
indicate that a pool of IP receptors in WB cells exists as
heterotetramers. The conclusion that the heterocomplexes are
heterotetramers rather than large aggregates of receptor is based on
our demonstration that the co-immunoprecipitating fractions of receptor
have a migration position on sucrose density gradients that is
consistent with that expected of a heterotetramer. Such experiments
also revealed that freshly prepared WB cell lysates contained low
amounts of disassembled receptors, including a pool of free monomers.
The same observation was made in our previous studies when the lysates
were fractionated by gel filtration(14) . It is therefore
possible that the heterotetramers assembled after lysis of the cells
and do not occur in vivo. However, the proportion of
S-labeled type-I and type-III receptor immunoprecipitated
by type-I antibody was not altered by lysing the cells in radioimmune
precipitation buffer (which contained 0.1% SDS, 1% sodium deoxycholate,
and 1% Triton X-100) or by pretreating the intact cells for 1 h with
100 µg/ml of the cleavable cross-linking agent
dithiobis(succinimidylpropionate) (data not shown). Because the amount
of co-immunoprecipitating receptor cannot be changed by these
experimental manipulations, we conclude that heteroligomerization of
IP
R pre-exists in the native WB cell membranes.
A random
association of two different monomers can generate five possible
combinations of tetramers. The proportion of homotetrameric and
heterotetrameric receptor would be dependent on the relative amounts of
the type-I and type-III isoforms. The ratio of type-I and type-III
isoforms in WB cells cannot be easily estimated by comparing
immunoblots because of the difficulties of standardizing and comparing
the reactivities of two different antibodies. An alternative is to
quantitate the S-labeled type-I and type-III isoforms
immunoprecipitated by their respective antibodies from the lysate of
S-labeled cells (cf. Fig. 1A). A
type-I/type-III ratio of 2.7 ± 0.2 (n = 3) is
obtained by this method. Assuming an entirely random association, the
relative proportions of the five tetrameric forms can be calculated
from the expansion of the polynomial (a + b)
= 1, where a and b are
the relative concentrations of the two different monomers(30) .
Taking a as the type-I and b as the type-III isoform
and the relative concentrations of a = 0.73 and b = 0.27, the calculated proportions of homotetrameric type-I
and type-III receptors would be 28 and 5% of the total receptor
concentration with the remaining 67% as heterotetramers.
Experimentally, the exact proportion of heterotetrameric type-I or
type-III is difficult to estimate, in part due to the presence of
nontetrameric forms in the lysate. However, it is clear from our data
that heterotetramers do not represent 67% of either isoform (cf. Fig. 1and Fig. 3). This suggests that they do not arise
by random mixing of monomers. Lower amounts of heterotetramers may
arise if not all combinations of monomers are equally favored or if the
ability to form heterotetramers is restricted to a localized pool of
receptors.
There are precedents for the combinatorial mixing of
subunits of channel proteins derived from separate gene products. The
formation of heteromultimeric members of the Shaker subfamily
of K channels has been viewed as allowing considerable
heterogeneity in channel gating and regulatory
properties(28, 29, 31) . The same could be
true for the IP
receptor channel, because each monomer is
believed to contribute transmembrane segments to the pore-forming
domain of the tetrameric channel(2) . A limiting concentration
of IP
has been shown to open only a population of
IP
R ion channels with further increases resulting in
activation of additional channels(32, 33) . One
interpretation of this ``quantal'' or ``increment
detection'' behavior of the channel is that it arises from
heterogeneity in sensitivity to
IP
(34, 35, 36, 37) .
Recently, it has been demonstrated that bacterially expressed
ligand-binding domains of the type-I and type-III IP
R have
affinities for IP
that differ by an order of
magnitude(22) . A mathematical model incorporating the presence
of high and low affinity IP
binding sites randomly
associated as homo- and heterotetramers has been used to describe the
kinetics of Ca
release from rat basophilic leukemia
cells(30) . Heteroligomeric IP
R ion channels would
be expected to have a wide range of sensitivity to IP
and
potentially could have different channel gating characteristics. The
inability to phosphorylate the type-III IP
R by protein
kinase A also points to important differences in the regulation of
IP
R isoforms. Heteroligomerization of isoforms may thus
provide an additional mechanism that generates considerable diversity
in the regulation of the IP
R Ca
ion
channel.
Note Added in
Proof-After submission of this paper, evidence for
heterotetramer formation of IPR subunits was reported in
several cell types (Monkawa, T., Miyawaki, A., Sugiyama, T., Yoneshima,
H., Yamamoto-Hino, M., Furuichi, T., Saruta, T., Hasegawa, M., and
Mikoshiba, K.(1995) J. Biol. Chem.270, 14700-14704).