From the Laboratorium voor Fysiologie and the
** Laboratorium voor Biochemie, K.U.Leuven Campus Gasthuisberg,
Herestraat 49, B-3000 Leuven, Belgium
Received for publication, July 11, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Submillimolar ATP concentrations strongly enhance
the inositol 1,4,5-trisphosphate (IP3)-induced
Ca2+ release, by binding specifically to ATP-binding sites
on the IP3 receptor (IP3R). To locate those
ATP-binding sites on IP3R1 and IP3R3, both
proteins were expressed in Sf9 insect cells and covalently
labeled with 8-azido-[ Inositol 1,4,5-trisphosphate
(IP3)1 is an
intracellular second messenger that mediates the release of
Ca2+ from internal stores by binding to the IP3
receptor (IP3R), an intracellular Ca2+-release
channel (1). The IP3R is composed of three functionally different domains: an N-terminal IP3-binding region, a
large transducing domain, and a C-terminal channel region (2). The
transducing domain contains interaction sites for several modulators of
IP3-induced Ca2+ release such as
Ca2+, calmodulin, kinases, phosphatases, ATP, and FKBP12
(reviewed in Refs. 1 and 3). IP3Rs are encoded by three
different genes, resulting in the existence of IP3R1,
IP3R2, and IP3R3, and the various
IP3R isoforms are distributed in a tissue-specific manner
(4-7). Nearly all cell types coexpress at least two IP3R isoforms (4, 5, 8), which are mostly co-organized in heterotetrameric
structures (9-12). The different IP3R isoforms show
functional differences in their regulation by IP3 and by several modulators of IP3-induced Ca2+ release
(13-15).
ATP regulates the IP3R in a
concentration-dependent manner: Submillimolar
concentrations enhance IP3-induced Ca2+ release
(16-20), whereas millimolar levels of ATP inhibit
IP3-induced Ca2+ release by competing with
IP3 for the IP3-binding site (18-23). The
stimulatory effect of ATP is likely to occur via binding to one or more
sites on the IP3R, because purified IP3Rs bind
[ Materials--
CHAPS was obtained from Pierce (Rockford, IL).
Chymotrypsin, N-tosyl-L-phenylalanine
chloromethyl ketone, heparin-agarose, and
N-acetyl-D-glucosamine were from Sigma Chemical
Co. (St. Louis, MO). 8-Azido-[ Expression of IP3R1 and IP3R3 in Insect
Sf9 cells--
The full-length mouse IP3R1 and the
full-length rat IP3R3 were expressed in insect Sf9
cells as described by Sipma et al. (30) and by Maes et
al. (24), respectively.
Photoaffinity Labeling with 8-Azido-[ Purification of Recombinant IP3R1 and
IP3R3 from Sf9 Microsomes--
The purification of
IP3Rs was based on the method described by Parys et
al. (32). Microsomes of Sf9 cells expressing
IP3R1 and IP3R3 in a concentration of 10 mg of
protein/ml were centrifuged, and the pellet was solubilized (at 5 mg/ml) for 1.5 h at 4 °C in buffer A (50 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.83 mM benzamidine, and 10 mM 2-mercaptoethanol) with addition of 200 mM
NaCl, 77 nM aprotinin, 1.1 µM leupeptin, 0.7 µM pepstatin A, 2.5% CHAPS, and 1%
L- Controlled Proteolysis--
Purified IP3R was
partially digested with chymotrypsin (0.05 µg/ml) for 2, 5, 10, or 30 min on ice as described previously (32). The digestion was stopped by
the addition of 100 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone and
by boiling the samples for 5 min in sample buffer for SDS-PAGE.
Antibodies and Western Blotting--
The polyclonal antibody
against the C terminus of mouse IP3R1 (Rbt03), the mouse
monoclonal antibody against an N-terminal epitope of human
IP3R3 (MMAtype3) (Transduction Laboratories, Lexington, KY)
and the polyclonal antibody against the Ca2+-binding domain
cytI3b (amino acids 378-450) in the IP3-binding domain of
mouse IP3R1 (33) were characterized earlier (4, 30, 34). A
novel antibody was raised against the luminal Ca2+-binding
fragment LoopI17a of mouse IP3R1 (aa 2463-2528) (35). Two
rabbits were injected subcutaneously and intramuscularly with Freund's
complete adjuvant containing 0.5 mg of LoopI17a fused to GST. Animals
were boosted 2 weeks later with the same antigen in Freund's
incomplete adjuvant and regularly thereafter. After three boost
injections, both rabbits produced high titers of antibody. Both
antibodies (named anti-loopI17a-1 and anti-loopI17a-2) reacted with
mouse, rat, human, hamster, and rabbit IP3R1. They also
recognized rat IP3R3, although with lower
sensitivity.2 A polyclonal
antibody directed against residues 1829-1848 of human
IP3R1 was purchased from Alexis Corp. (Läufelfingen,
Switzerland). A polyclonal antibody against the C terminus of human
IP3R3 was from Santa Cruz Biotechnology (Santa Cruz, CA).
The various microsomal preparations were analyzed on 3-12%
Laemmli-type gels and transferred to Immobilon-P (Millipore Corp.,
Bedford, MA). Immunodetection of the proteins on the transfers was
exactly as described previously (30, 36).
45Ca2+
Fluxes--
IP3-induced Ca2+ release from
permeabilized A7r5 monolayers was described elsewhere (16). The added
ATP concentrations are indicated in the legend to Fig. 4. The medium
for the challenge with IP3 contained 120 mM
KCl, 30 mM imidazole-HCl, pH 6.8, and 1 mM EGTA.
Purification of IP3Rs from Sf9 Insect
Cells--
To allow an accurate analysis involving controlled
proteolysis and immunostaining, purification of the IP3R is
needed. IP3R1 has been purified from cerebellum (37, 38),
smooth muscle (39, 40) and oocytes (32). Until now, no
IP3R3 has been purified due to the lack of a known cell
type that abundantly expresses this isoform. We therefore expressed
IP3R1 or IP3R3 in Sf9 insect cells,
resulting in a 2.5 times higher expression of IP3R1 and a
>50 times higher expression of IP3R3 as compared with
rabbit cerebellar and 16HBE14o-microsomes, respectively (24, 30). In
the purification procedures described by Chadwick et al.
(39) and Parys et al. (32), microsomes were first
solubilized by a detergent followed by chromatography on heparin- and
lectin-based matrices. This method was based on the ability of heparin
to bind to the IP3R with high affinity (41-43) and on the
presence of N-glycosylation sites on 2 asparagine residues
present in IP3R1 (44). It has been suggested that
IP3R3 is also a glycoprotein (45), although only one
N-glycosylation site is predicted based on the primary sequence (46).
In this study, we have used an identical approach to purify recombinant
IP3R1 and IP3R3 overexpressed in Sf9
insect cells. It had to be verified whether the glycosylation patterns
of these proteins were the same as in mammalian cells. Briefly,
microsomes from Sf9 cells overexpressing either
IP3R1 or IP3R3 were solubilized with 2.5%
CHAPS. Subsequently, the solubilized microsomes were incubated with
heparin-agarose. After elution of the bound fraction, the latter was
incubated with wheat germ agglutinin-Sepharose. Both receptors could be
purified with high efficiency and were recognized by isoform-specific
antibodies (Fig. 1, A and
B, first lane of each blot),
confirming that they are both glycoproteins and that the
post-translational glycosylation of the IP3Rs in insect
cells is similar to that in mammalian cells. The purified IP3R1 migrated on SDS-PAGE with a molecular mass of 273 kDa, which deviated from the molecular mass of 313 kDa predicted from
the primary structure (Fig. 1A, first lane of
each blot). The purified IP3R3 also migrated
with a lower apparent molecular mass (248 kDa) than predicted (304 kDa)
(Fig. 1B, first lane of each blot). Because a similar behavior is also found for endogenous
IP3Rs from, e.g. cerebellar or 16HBE14o-cells
(20, 47), this discrepancy is likely due to aberrant mobility of higher
molecular mass proteins on SDS-PAGE.
Controlled Proteolysis and Identification of Proteolytic
Fragments--
The purified IP3Rs were subjected to a
controlled proteolysis with chymotrypsin (0.05 µg/ml, up to 30 min on
ice), and the digestion fragments were detected by a panel of different
site-specific antibodies (Fig. 1, A and B, and
Table I). No degradation of the intact
IP3R was observed during incubation without chymotrypsin (Fig. 1, first lane of each blot).
For IP3R1, four site-specific antibodies were used, of
which the epitopes were spread over the whole sequence. The
anti-cytI3b-2 antibody (30) is directed against a
Ca2+-binding site in the IP3-binding domain
(33). The anti-(1829-1848) antibody (Alexis Corp.) recognized an amino
acid stretch (residues 1829-1848) located in the regulatory domain
between the two putative ATP-binding sites (residues 1773-1780 and
2016-2021) (2, 26-28). A third antibody, anti-loopI17a-2, was raised
against the luminal Ca2+-binding fragment (35). Finally,
Rbt03 (30, 34) recognized the C terminus of IP3R1. The
proteolytic pattern, resulting from up to 30 min of incubation with
chymotrypsin, and as detected by the four antibodies against
IP3R1, is shown in Fig. 1A. We determined the
length of the fragments using Rainbow molecular mass markers. Based on
these data, we were able to localize the chymotrypsin-sensitive sites
on IP3R1 (Fig.
2A). The sum of the molecular
mass of the five major proteolytic fragments (40, 65, 80, 40, and 90 kDa) was close to the molecular mass of the intact IP3R1
(313 kDa). This result was in complete agreement with the study
of Yoshikawa et al. (48), where trypsin was used to
digest cerebellum-purified IP3R1 and where five similar
major proteolysis-insensitive fragments were found. Although we were
able to recognize most of the intermediate digestion products with
site-specific antibodies, two proteolytic fragments, which were
predicted based on Fig. 2A, could not be detected when the
digestion was performed for 30 min. Particularly, a 145-kDa fragment,
precursor of the 65- and 80-kDa fragments should be recognized by the
anti-cytI3b antibody and a 185-kDa fragment, precursor of the
successive fragments of 65, 80, and 40 kDa should be recognized by the
anti-(1829-1848) antibody. Because it is possible that these
intermediate fragments have a short life time, we decreased the time of
proteolysis to 2, 5, and 10 min, respectively (Fig. 1A,
insets of blots 1 and 2). Upon
staining with the anti-cytI3b-2 antibody (blot 1 and inset), we detected a proteolytic band corresponding to a
molecular mass of 145 kDa, which is most intense at 2 and 5 min of
incubation with chymotrypsin. This fragment is rapidly degraded into
smaller fragments, because it is poorly or not visible in the
proteolytic patterns representing 10 and 30 min of incubation of
IP3R1 with chymotrypsin. Upon staining with the
anti-(1829-1848) antibody, no clear fragment with a mass of 185 kDa
was visible, even at shorter time points (blot 2 and
inset). Because the corresponding predicted fragment was
also not detected in the study of Yoshikawa et al. (48), it
is conceivable that the latter intermediate fragment is rapidly
degraded into smaller subfragments during proteolysis and has
therefore a steady-state level below the detection limit for the
antibodies. All identified fragments, with indication of their
molecular mass and the recognizing antibodies, are represented in Table
II.
The same type of experiment was performed for IP3R3. Only
two site-specific antibodies are available for this isoform: the MMAtype3 antibody (Transduction Laboratories) (4) directed against the
N terminus, and the anti-CIII antibody (Santa Cruz Biotechnologies)
against the C terminus. However, the anti-loopI17a-2 antibody could
also recognize IP3R3,2 although with lower
sensitivity. The proteolytic pattern as stained by the three antibodies
against IP3R3 is shown in Fig. 1B. The time
dependence of the occurrence of the proteolytic fragments was also
investigated for IP3R3, but incubation with chymotrypsin for shorter times (2-10 min) revealed the same pattern of proteolytic fragments (data not shown). All identified fragments, with their molecular mass and the recognizing antibodies, are represented in Table
III. In addition we have verified the
N-terminal boundaries of some of the major proteolytic fragments by
N-terminal amino acid microsequencing (data not shown). A schematic
presentation of IP3R3 with the major proteolytic fragments
(105, 70, 35, and 95 kDa) is shown in Fig. 2B. The sum of
the molecular mass of the fragments was close to the molecular mass of
the intact receptor (304 kDa) as calculated from the cloned rat
IP3R3. The general structure of IP3R3 resembled
that of IP3R1: Both receptor isoforms were sensitive to
proteolysis at similar sites. Only the chymotrypsin-sensitive site that
is present in the IP3-binding domain of IP3R1,
could not be detected in IP3R3. This could however be due
to the lack of an antibody that recognized the relevant part of the
IP3-binding domain. Alternatively, it is also possible that
IP3R3 lacks the chymotrypsin-sensitive site in the
IP3-binding domain. It is conceivable that the
proteolysis-sensitive sites represent regions that are exposed on the
surface of the protein and thereby accessible to the proteolytic
enzymes as well as to different modulators of IP3-induced
Ca2+ release. Because functional IP3Rs are
mostly organized in heterotetramers (9-12), it can be expected that
corresponding regions of the different IP3R isoforms are
exposed at the surface of the receptor protein so that they can be
properly regulated.
Identification of Photoaffinity-labeled Proteolytic
Fragments--
In a previous study, we showed that two GST fusion
proteins, each containing a putative ATP-binding domain of
IP3R1, could bind ATP (29). Both predicted ATP-binding
domains were situated near chymotrypsin-sensitive sites (Fig.
2A). It is therefore likely that they are both accessible to
ATP in the intact protein. To prove this, we incubated microsomes from
Sf9 cells expressing recombinant IP3R1 with the
photoaffinity label 8-azido-[
IP3R3 contained only one of these proposed ATP-binding
sites, which is conserved in all IP3R isoforms and which is
also located near a chymotrypsin-sensitive site (Fig. 2B).
To confirm this, we performed the same photoaffinity labeling
experiment for the IP3R3 isoform. The smallest labeled band
of 95 kDa (Fig. 3B) was recognized by the anti-CIII antibody
(Table III), indicating that this band represented the proteolytic
fragment containing the putative ATP-binding site of IP3R3
(Fig. 2B). In summary, covalent labeling with
8-azido-[
The unequal number of ATP-binding sites found in IP3R1 and
IP3R3 may explain the differential modulation of these
isoforms by ATP. IP3R1 showed a higher affinity for ATP
than IP3R3 (13, 14, 24), suggesting that the upstream
ATP-binding site, which is only present in IP3R1, is a
high-affinity binding site. Moreover, IP3R3 displayed a
broader nucleotide specificity than IP3R1 (24), because it
bound equally well ATP and GTP. The latter property can be assigned to
the ATP-binding site present in IP3R3 and conserved in all
IP3R isoforms. The ATP-binding site that is only present in
IP3R1 was more specific for adenine nucleotides like ATP
and ADP (24).
ATP Dependence of IP3-induced Ca2+
Release--
In permeabilized A7r5 cells, which express
IP3R1 and IP3R3 in a 3 to 1 ratio (5), ATP
dependence of IP3-induced Ca2+ release was
found over a very broad concentration range (Fig. 4). ATP stimulated
IP3-induced Ca2+ release from the low
micromolar range up to 1 mM. At still higher ATP
concentrations, the release was inhibited probably due to competition
of ATP for the IP3-binding site (18-23). The broad concentration dependence in A7r5 cells is in very good agreement with
the different ATP affinities described previously for recombinant IP3R1 and IP3R3 (EC50 values of 1.6 µM and 177 µM, respectively) (24). This
difference in ATP affinities between IP3R1 and
IP3R3 was also observed by other groups: recent findings of
Hagar and Ehrlich (49) demonstrated that IP3R3 incorporated
in lipid bilayers was activated by ATP with an EC50
of about 3 mM, whereas a much lower EC50 (40 µM) was observed for IP3R1 (19). Moreover,
IP3-induced Ca2+ release in genetically
engineered DT40 B cells that express a single IP3R subtype
was also found to respond differently to ATP. In
IP3R1-expressing cells, the rate of Ca2+
release was enhanced by ATP with an EC50 of 0.39 mM, whereas IP3R3-expressing cells were much
less sensitive to ATP (14).
Because it was not possible to resolve the ATP concentration dependence
in A7r5 cells by curve-fitting procedures, the present data do not
allow a determination of whether the presence of two IP3R
isoforms in A7r5 is reflected in two separate stimulatory ATP-binding
sites. However, for preparations from rat cerebellum containing nearly
exclusively IP3R1, the maximum stimulation by ATP was found
at 50 µM. At 1 mM ATP,
IP3-induced Ca2+ release in cerebellar
preparations was close to control values (50), whereas 1 mM
ATP was the maximum stimulatory concentration in A7r5 cells. The much
higher maximum for ATP stimulation found for A7r5 cells is therefore
very probably a reflection of the presence of IP3R3. It was
also not possible to decide whether these properties of
IP3R3 are inferred in A7r5 cells by homo- or
heterotetramers. Coimmunoprecipitation experiments indicated that a
significant fraction of IP3R1 and IP3R3
expressed in A7r5 cells is present as heterotetramers.2 Our
data clearly showed that the presence of different ATP-binding sites on
IP3R1 and IP3R3 resulted in a nucleotide
sensitivity of IP3-induced Ca2+ release that
extended over a broad concentration range. The ATP concentration that
yielded maximum stimulation seems very variable and to be dependent on
the IP3R isoform composition in the particular cell type.
-32P]ATP. IP3R1 and
IP3R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by
site-specific antibodies. Two fragments of IP3R1 were
labeled, each containing one of the previously proposed ATP-binding
sites with amino acid sequence GXGXXG (amino
acids 1773-1780 and 2016-2021, respectively). In IP3R3,
only one fragment was labeled. This fragment contained the
GXGXXG sequence (amino acids 1920-1925), which
is conserved in the three IP3R isoforms. The presence of
multiple interaction sites for ATP was also evident from the
IP3-induced Ca2+ release in permeabilized A7r5
cells, which depended on ATP over a very broad concentration range from
micromolar to millimolar.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP in a specific manner (17, 20, 24). The
number and the localization of these sites have, however, not yet been
determined. Based on the glycine-rich amino acid sequence
GXGXXG (25), two ATP-binding sites were
postulated on the neuronal form of IP3R1 (aa 1773-1780 and
2016-2021). The former is only present in IP3R1, whereas
the latter is common to the three IP3R isoforms (2, 26-28). In a previous study, we have expressed the cDNA domains of
IP3R1 containing these glycine-rich motifs as glutathione
S-transferase (GST) fusion proteins in bacteria and showed
that they both were able to bind ATP (29). The aim of the present study
was to determine the location and number of ATP-binding sites on the
intact IP3R. We did this by photoaffinity labeling with
8-azido-[
-32P]ATP of microsomes of Sf9 insect
cells expressing recombinant IP3R1 or IP3R3
homotetramers, followed by purification and controlled proteolysis with
chymotrypsin. We found that controlled proteolysis of IP3R1
and IP3R3 yielded roughly the same major fragments,
indicating that both isoforms have a similar general structure.
Moreover, the results indicated that the IP3R1 contained
two ATP-binding sites, because two separate fragments obtained by
proteolysis were labeled. These two fragments each contained one of the
presumed ATP-binding sites (aa 1773-1780 and 2016-2021,
respectively), as identified using site-specific antibodies. In
IP3R3, only one proteolytic fragment was labeled. This
fragment contained the proposed ATP-binding site that is conserved in
all IP3R isoforms (aa 1920-1925). The unequal number of
ATP-binding sites in IP3R1 and IP3R3 could have
implications for the modulation of these isoforms by ATP. Recently, we
found that IP3R1 and IP3R3 have a different ATP
affinity (EC50 values of 1.6 and 177 µM,
respectively) (24). In this study, the ATP dependence of
IP3-induced Ca2+ release measured in
permeabilized A7r5 cells, which express both IP3R1 and
IP3R3, extended over a very broad range from micromolar to
millimolar concentrations. This finding confirms the presence of
multiple nucleotide-binding sites with different affinities for ATP
in IP3R1 and IP3R3.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP (2 mCi/ml, 12 Ci/mmol) was purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA).
45CaCl2 (2.2 mCi/ml, 134 µg of
Ca2+/ml), wheat germ agglutinin-Sepharose 6MB, Rainbow
molecular mass markers, the anti-mouse and anti-rabbit alkaline
phosphatase-coupled secondary antibodies, and the Vistra ECF substrate
were from Amersham Pharmacia Biotech AB (Uppsala, Sweden).
-32P]ATP of
Recombinant IP3R--
Microsomes of Sf9 insect
cells were prepared as described (31). Photoaffinity labeling of
microsomes containing either IP3R1 or IP3R3 with
8-azido-[
-32P]ATP was performed exactly as described
in Maes et al. (24).
-phosphatidylcholine. After centrifugation, the
supernatant was diluted with an equal volume of buffer A with addition
of 400 mM NaCl. The diluted supernatant was incubated for
30 min with heparin-agarose beads (112.5 µl/mg of protein). The
eluate obtained in buffer A with 600 mM NaCl, 0.75% CHAPS,
and 0.3% L-
-phosphatidylcholine, was incubated for 2 h with wheat germ agglutinin-Sepharose (75 µl/mg of protein). After wash steps in high (600 mM) and low (100 mM) salt conditions, the specifically bound proteins were
eluted in low salt conditions with 300 mM
N-acetyl-D-glucosamine. All centrifugation steps
were for 17 min at 35,700 × g at 4 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Controlled proteolysis of purified
IP3R1 (A) and IP3R3
(B). IP3Rs were purified from 1 mg of
Sf9 microsomes as described under "Experimental Procedures."
The duration of the controlled proteolysis with chymotrypsin (0.05 µg/ml on ice) is indicated at the top of each
lane. Control IP3Rs were not treated with
chymotrypsin. The proteins were separated by SDS-PAGE and transferred
to Immobilon-P. The blots were probed with site-specific antibodies: In
A, blot 1, anti-cytI3b-2 (dilution 1/300);
blot 2, anti-(1829-1848) (dilution 1/700); blot
3, anti-loopI17a-2 (dilution 1/1000); and blot 4, Rbt03
(dilution 1/10000). In B, blot 1, MMAtype3
(dilution 1/1000); blot 2, anti-loopI17a-2 (dilution 1/200);
and blot 3, anti-CIII (dilution 1/250). Positions of the
molecular mass markers (in kDa) are indicated.
Overview of
antibodies
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Fig. 2.
Schematic representation of the proteolytic
fragments of IP3R1 (A) and
IP3R3 (B). The top line
indicates the molecular mass and the number of amino acids of the
IP3R. The horizontal bar represents a scheme of
the IP3R with the chymotrypsin-sensitive sites (indicated
by the scissors), the size of the proteolytic fragments (in
kDa), the proposed ATP-binding sites, and the epitopes of the
site-specific antibodies.
Identification of proteolytic fragments of IP3R1 by
site-specific antibodies and 8-azido-[-32P]ATP
labeling
-32P]ATP. The same method was used for
IP3R1, which was labeled with
8-azido-[
-32P]ATP prior to purification and controlled
proteolysis. The presence of two different labeled sites (on fragments
of 90 and 40 kDa, respectively) follows from their specific
identification with different antibodies (Fig. 2A). The
details of the photoaffinity labeling, purification, and controlled
proteolysis are described under "Experimental
Procedures."
Identification of proteolytic fragments of IP3R3 by
site-specific antibodies and 8-azido-[-32P]ATP
labeling
-32P]ATP prior to purification and controlled
proteolysis. As illustrated in Fig. 2B, the smallest
labeled fragment was the 95-kDa C-terminal fragment. The details
of the photoaffinity labeling, purification, and controlled proteolysis
are described under "Experimental
Procedures."
-32P]ATP. Covalent
labeling of the ATP-binding sites by UV irradiation was followed by
purification and controlled proteolysis of IP3R1 and
identification of the labeled proteolytic fragments by site-specific antibodies. The two smallest labeled proteolytic fragments of IP3R1 (90 and 40 kDa, Fig.
3A) were recognized by the
Rbt03 antibody and the anti-(1829-1848) antibody, respectively (Table
II), indicating that they represented the proteolytic fragments
containing the previously proposed ATP-binding sites (Fig.
2A).
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Fig. 3.
Photoaffinity labeling of IP3R1
(A) and IP3R3 (B)
followed by controlled proteolysis. Microsomes from Sf9
insect cells were incubated with 20 µM
8-azido-[ -32P]ATP and subsequently irradiated with UV
light for 2.5 min. IP3R1 and IP3R3 were
purified from those microsomes after solubilization of the microsomes
with CHAPS and binding to heparin-agarose and subsequently wheat germ
agglutinin-Sepharose. IP3Rs were digested with 0.05 µg/ml
chymotrypsin for 30 min on ice (lanes 2) or were not treated
with chymotrypsin (lanes 1). After SDS-PAGE and blotting,
labeled IP3Rs were visualized using the Storm 840 PhosphorImager (Molecular Dynamics). Positions of the molecular mass
markers (in kDa) are indicated. The details of the photoaffinity
labeling and the proteolysis are described under "Experimental
Procedures."
-32P]ATP occurred at two different
proteolytic fragments of IP3R1 and only at one proteolytic
fragment of IP3R3. The labeled fragments contained the two
previously proposed ATP-binding sites, one of which is conserved in all
IP3R isoforms.
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Fig. 4.
ATP dependence of IP3-induced
Ca2+ release. Nonmitochondrial Ca2+ stores
in permeabilized A7r5 cells, loaded to steady state with
45Ca2+, were incubated in efflux medium for 10 min, at which time 1 µM IP3 plus the
indicated ATP concentration was added for 2 min. The stimulation of the
Ca2+ release by ATP is expressed as the percentage increase
in the Ca2+ release above the control value, obtained in
the absence of ATP. Data are the means of four independent experiments.
The error bars smaller than the data symbol are not
indicated.
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ACKNOWLEDGEMENTS |
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We thank Lea Bauwens, Jerry Renders, Luce Heremans, Anja Florizoone, Marina Crabbé, Hilde Van Weijenbergh, Irène Willems, Yves Parijs, and Raphael Verbist for their skillful technical assistance. We acknowledge the generous gifts of the p400C1 plasmid containing the IP3R1 cDNA by Drs. K. Mikoshiba and A. Miyawaki (University of Tokyo, Japan) and the pCB6+ plasmid containing the IP3R3 cDNA by Dr. G. I. Bell (Howard Hughes Medical Institute, University of Chicago, IL). We thank Drs. S. Joseph (Thomas Jefferson University School of Medicine, Philadelphia, PA) and G. Guillemette (University of Sherbrooke, Quebec, Canada) for the kind gift of anti-IP3R3 antibodies used in some control experiments.
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FOOTNOTES |
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* This work was supported in part by Grant 99/08 of the Concerted Actions, by Grant P4/23 of the Interuniversity Poles of Attraction Program of the Belgian State, and by Grants 3.0207.99 and G.0322.97 of the Foundation for Scientific Research-Flanders (FWO).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.
¶ A Research Associate of the FWO.
Senior Research Assistants of the FWO.
§ To whom correspondence should be addressed: Tel.: 32-16-345-736; Fax: 32-16-345-991; E-mail: Karlien.Maes@med.kuleuven.ac.be.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006082200
2 J. B. Parys, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; aa, amino acids; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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