Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3 Homotetramers by Controlled Proteolysis and Photoaffinity Labeling*

Karlien MaesDagger §, Ludwig MissiaenDagger , Jan B. ParysDagger , Patrick De SmetDagger ||, Ilse SienaertDagger ||, Etienne Waelkens**, Geert CallewaertDagger , and Humbert De SmedtDagger

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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-[alpha -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

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 [alpha -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-[alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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-[alpha -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).

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-[alpha -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-[alpha -32P]ATP was performed exactly as described in Maes et al. (24).

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-alpha -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-alpha -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.

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.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.



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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.

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).


                              
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Table I
Overview of antibodies

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.



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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.


                              
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Table II
Identification of proteolytic fragments of IP3R1 by site-specific antibodies and 8-azido-[alpha -32P]ATP labeling
IP3R1 was purified from Sf9 insect cells and subjected to a controlled proteolysis. The proteolytic fragments were identified with site-specific antibodies. The proteolytic fragments (represented in kDa), obtained after 2-30 min of incubation with chymotrypsin and recognized by a particular antibody are indicated by an "x." "xo" indicates that the used antibody detected the particular fragment with a very weak intensity, or that the fragment was very weakly labeled with 8-azido-[alpha -32P]ATP. The same method was used for IP3R1, which was labeled with 8-azido-[alpha -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."

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.


                              
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Table III
Identification of proteolytic fragments of IP3R3 by site-specific antibodies and 8-azido-[alpha -32P]ATP labeling
IP3R3 was purified from Sf9 insect cells and subjected to a controlled proteolysis. The proteolytic fragments were identified with site-specific antibodies. Proteolytic fragments (represented in kDa) recognized by a particular antibody are indicated by an "x". "xo" indicates that the used antibody detected the particular fragment with a very weak intensity. The same method was used for IP3R3, which was labeled with 8-azido-[alpha -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."

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-[alpha -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-[alpha -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."

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-[alpha -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.

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).



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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.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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