Cooperative Formation of the Ligand-binding Site of the Inositol 1,4,5-Trisphosphate Receptor by Two Separable Domains*

Fumio YoshikawaDagger §, Hirohide IwasakiDagger , Takayuki MichikawaDagger , Teiichi FuruichiDagger , and Katsuhiko MikoshibaDagger parallel

From the Dagger  Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 and the parallel  Developmental Neurobiology Laboratory, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Inositol 1,4,5-trisphosphate (IP3)1 is a second messenger that mediates Ca2+ release from intracellular stores by binding to the IP3 receptor (IP3R) which is a tetrameric IP3-gated Ca2+ release channel (1, 2). Cerebellum has an extraordinary density of specific IP3-binding sites (3) and purified cerebellar IP3R protein binds IP3 in a stoichiometric manner, namely one subunit for one IP3 (4-6). Ca2+ release experiments using various synthetic inositol phosphates have suggested that molecular recognition of IP3 is markedly stereospecific (7). Type 1 IP3R (IP3R1) is the neuronal type and predominates in cerebellar Purkinje cells (8-10). It is 2749 amino acids long (molecular mass about 313 kDa) and is structurally divided into three parts as follows: a large N-terminal cytoplasmic arm region (residues 1-2275), a putative six membrane-spanning region clustered near the C terminus (residues 2276-2589) which is thought to constitute an ion channel by forming a tetramer, and a short C-terminal cytoplasmic tail region (residues 2590-2749) (11). A series of deletion mutants showed that the IP3R1 binds IP3 within the N-terminal 650 amino acids, independently of tetramer formation (12, 13).

We previously demonstrated the structural basis for molecular recognition of IP3 by mouse IP3R1 (mIP3R1) (14). The minimum region for high affinity binding has been localized within the 353 residues, 226-578, so that it appears to be close to the binding "core." Within the core region, we have identified 10 important basic amino acid residues all of which are well conserved in all IP3R family proteins cloned to date: three (Arg-265, Lys-508, and Arg-511) are critical and the other seven are required for specific binding. Nahorski and Potter (7) predicted that ionic interactions of positive charges on a binding site with negative charges on the three phosphate groups of IP3 would make major contributions to specific recognition and binding. Thus, we have proposed that the IP3-binding core forms a pocket with a positively charged inner surface lining of these basic residues which recognizes and binds a negatively charged IP3 ligand. Interestingly, all members of the IP3R family share extensive homology in the core sequence, except that the IP3R1 has an alternative splicing SI region (15 residues, 318-332) (9, 15), adjacent to which ~30 residues form the longest stretch of characteristic diversity within the family. An internal deletion of residues 316-352 leads to loss of binding (13), although neither the presence nor the absence of the SI segment (mIP3R1SI+ nor mIP3R1SI- subtype, respectively) significantly affects the binding,2 suggesting that the diversed stretch is not a prerequisite for binding but that its boundary should be strictly defined.

Recently, we have shown that limited trypsin digestion of mouse cerebellar membrane fractions causes fragmentation of the mIP3R1 into five major trypsin-resistant polypeptides and that these five tryptic fragments I-V have tight structural-functional coupling because of the following: (i) co-sedimentation of all four cytoplasmic peripheral fragments I-IV (40/37, 64, 76, and 40/36 kDa) together with the membrane-spanning integral fragment V (91 kDa) by centrifugation to pellet membrane proteins and immunoprecipitation with C terminal-specific antibody, and (ii) retention of IP3-induced Ca2+ release channel activity in such fragmented mIP3R1. As a result of trypsinization, the IP3-binding region is cleaved at the carboxyl side of Arg-343 and/or Arg-345, around which is the alternative splicing SI region, into two polypeptides: fragments I and II. These results have demonstrated that the native IP3-binding region consists of two major tightly folded structural components connected by one exposed loop near the SI region; the latter loop is very susceptible to trypsin attack, whereas the former two components are not. Similarly, fragments III, IV, and V would be reflected by well folded conformation of the native mIP3R1 channel. Thus, determination of the structure-function relationships among these components might clarify the molecular mechanism of ligand binding and channel opening of the mIP3R1 channel.

In the present study, we describe the structure-function relationships of the split tryptic IP3-binding region. We have characterized the IP3 binding properties of trypsin-fragmented mIP3R1, and we have demonstrated that the tryptic mIP3R1, completely fragmented into five polypeptides, still retains specific IP3 binding activity comparable to that of the intact receptor. To analyze further the split IP3 binding components, we have separately synthesized two glutathione S-transferase (GST) fusion proteins with these two components, and we have shown that high affinity binding site can be reconstituted in vitro by complementation with both distinct fusion proteins, each of which alone has no (N-terminal component) or low affinity (C-terminal component) binding. From these data, we propose that the functional structure of the IP3-binding pocket consists of two well folded structural domains that are non-covalently but tightly associated and a diverse loop-like structure between these two domains.

    EXPERIMENTAL PROCEDURES

Materials-- N-Tosyl-L-phenylalanyl chloromethyl ketone-treated bovine pancreas trypsin, soybean trypsin inhibitor, and gamma -globulin were purchased from Sigma; [3H]IP3 was from NEN Life Science Products; D-myo-inositol 1,4,5-trisphosphate was from Dojindo; D-myo-inositol 2,4,5-trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate were from Calbiochem; polyethylene glycol (PEG) 6000 was from Wako Pure Chemical; Solvable and Atomlight were from Packard; reduced glutathione was from Boehringer Mannheim, and GST fusion vector pGEX-2T and glutathione-Sepharose 4B were from Amersham Pharmacia Biotech. Site-specific anti-mIP3R1 antibodies (polyclonal antibodies N1, N3, anti-(1718-31), and 1ML1; monoclonal antibodies 10A6 and 18A10) have been described (18).

Preparation of Membrane Fractions from Mouse Cerebellum-- ddY mice (8-10 weeks old; Nippon SLC, Japan) were anesthetized and then decapitated, and cerebella were quickly dissected. The cerebella were homogenized by 10 strokes (850 rpm) in an ice-chilled glass Teflon Potter homogenizer containing 9 volumes of 0.32 M sucrose, 1 mM EDTA, 100 µM phenylmethylsulfonyl fluoride (PMSF), 10 µM pepstatin A, 10 µM leupeptin, and 5 mM Tris-HCl, pH 7.4. The homogenate was centrifuged at 1,000 × g for 15 min at 4 °C. The supernatant was re-centrifuged under the same conditions to completely remove P1 fraction. The second supernatant was centrifuged at 105,000 × g for 60 min at 2 °C. The precipitate (crude microsome) was resuspended with binding buffer (1 mM EDTA, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0 (at 4 °C)) to give a final concentration of about 15 mg/ml protein, frozen in liquid nitrogen, and stored at -80 °C until use. Protein concentrations were determined by a Bio-Rad protein assay kit using bovine serum albumin as a reference.

Trypsin Digestion-- Microsomal fraction (1 mg/ml protein) was incubated with the desired concentration of trypsin at 35 °C in trypsinizing buffer (120 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 8.0). The reaction was quenched with 10-fold weight excess of soybean trypsin inhibitor and 0.1 mM PMSF.

IP3 Binding Assay-- IP3 binding was carried out by incubating sample protein with 9.6 nM (or 4.8 nM for Scatchard analysis of microsomal fraction) [3H]IP3 in 100 µl of binding buffer for 10 min at 4 °C. The IP3/protein mixture was added to 4 µl of gamma -globulin (50 mg/ml) and 100 µl of PEG precipitation buffer (30% (w/v) PEG6000, 1 mM EDTA, and 50 mM Tris-HCl, pH 8.0, at 4 °C) and incubated for 5 min at 4 °C. The protein-PEG complexes were pelleted by centrifugation at 18,000 × g for 5 min at 4 °C. The pellet was dissolved in 180 µl of Solvable and then neutralized by adding 18 µl of acetic acid. The radioactivity of the neutralized samples was measured by mixing 5 ml of Atomlight followed by counting in a liquid scintillation counter (Beckman). The specific binding was defined as total binding minus nonspecific binding that was measured in the presence of 2-100 µM cold IP3.

In Scatchard plots, lines were fit to the data by the least squares method using either the single site relationship B/F = -B/Kd + Bmax/Kd or the two-site relationship B/F = Bmax1/(Kd1 + F) + Bmax2/(Kd2 + F) as appropriate, where B is the specific IP3 binding in pmol/mg protein, F is the concentration of the free IP3, Kd is dissociation constant for each separate site, and Bmax represents the maximal number of binding sites for each site (16).

Construction, Expression, and Purification of GST Fusion Protein-- Standard methods (PCR, cloning, and DNA sequencing) for construction of the mIP3R1 cDNA-derived plasmids were as described previously (14). DNA clones derived from PCR products were all confirmed by sequencing. To generate fusion constructs between GST and the IP3-binding region of mIP3R1, foreign nucleotide fragments as described below were attached to the mIP3R1 cDNA by PCR using appropriately designed primers. A BamHI site (GGATCC) was attached at the 5' terminus of the initiation codon (ATG) of the mIP3R1 for the GST-fused residue 1-343 (GN and GN(R265Q)) and at the 5' terminus of Arg-341 codon for the GST-fused residues 341-604 (GC), and TAAGAATTC (stop codon + EcoRI site) was attached at the 3' terminus of Arg-343 codon for the GN and GN(R265Q) and at the 3' terminus of Arg-604 codon for the GC. A fragment including the R265Q mutation was amplified using the R265Q mutant (14) as a template. Each genetically worked fragment coding the residues 1-343 (for GN), 1-343(R265Q) (for GN(R265Q)) and 341-604 (for GC) was digested by BamHI and EcoRI, and the resultant restriction fragment was cloned into the BamHI and EcoRI site of the GST fusion vector pGEX-2T. Escherichia coli JM109 strain was transformed with these expression constructs, and recombinant fusion proteins were expressed by the low temperature method that was developed to increase solubility of expressed IP3-binding proteins as described previously (14). Cells were harvested, resuspended in phosphate-buffered saline, and disrupted by sonication on ice. After centrifugation at 30,000 × g for 60 min at 2 °C, the supernatants were collected and subjected to a GST purification step through glutathione-Sepharose 4B column chromatography according to the manufacturer's protocol. The purified GST fusion proteins were subjected to SDS-PAGE analysis and IP3 binding assay.

    RESULTS

Characterization of IP3 Binding Activity of the Trypsin-fragmented mIP3R1-- We previously showed that limited trypsin digestion of mouse cerebellar membrane fractions fragmented the mIP3R1 into five major polypeptides (fragments I-V), in which the IP3-binding region was split into two fragments I and II (18). Intriguingly, these completely fragmented mIP3R1 still retained strong activity for IP3-induced Ca2+ release comparable with that of the intact receptor, suggesting that even the split IP3-binding site was sufficient to couple with gating the fragmented channel.

To characterize further the structural and functional properties of the split IP3-binding site, we analyzed the mIP3R1 in trypsin-digested and undigested microsomal fractions of mouse cerebellum for specificity, affinity, and capacity of IP3 binding. Microsomal fractions were digested with 5 µg/ml trypsin for 4 min at 35 °C and then pelleted by centrifugation. Almost all of the tryptic fragments as well as the intact mIP3R1 collected into the pellet. The pellet was solubilized with 1% Triton X-100 followed by centrifugation at 20,000 × g for 60 min at 2 °C to obtain the supernatant. The Triton extracts were then subjected to Western blot analysis using six site-specific antibodies, which specifically recognize the major tryptic fragments Ia/b (40/37 kDa), II (64 kDa), III (76 kDa), IVa/b (40/36 kDa), and V (91 kDa) (18). As shown in Fig. 1A, trypsinization resulted in complete fragmentation of the mIP3R1 into five tryptic fragments that were detectable with the site-specific antibodies (Trypsin +), but when pre-mixed with 50 µg/ml trypsin inhibitor and 0.1 mM PMSF prior to the digestion, no apparent mobility change was observed in the intact mIP3R1 band (Trypsin -). The IVa/b fragments were only detected at very low levels, since the epitope for anti-(1718-31) located in the alternative splicing SII region (40 residues 1692-1731) of these fragments is most labile to trypsinolysis (18). The size difference between the Ia and Ib fragments has been thought to be due to alternative splicing at the SI region (15 residues 318-332), the former derived from the SI+ subtype and the latter from the SI- subtype (15, 18).


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Fig. 1.   Effects of trypsinization on IP3 binding of the mIP3R1. Mouse cerebellar microsomal fractions (1.5 mg of protein) in 1.5 ml of the trypsinizing buffer were digested with 5 µg/ml trypsin for 4 min at 35 °C in the presence (Trypsin -) and the absence (Trypsin +) of 50 µg/ml trypsin inhibitor and 0.1 mM PMSF. The digested samples were centrifuged at 105,000 ×g for 60 min at 2 °C to collect the insoluble membrane fractions. The pellet was resuspended in 0.5 ml of the binding buffer, solubilized by the addition of 10% (w/v) Triton X-100 to give a final detergent concentration of 1%, and rotated for 30 min at 4 °C. The Triton-treated mixtures were centrifuged at 20,000 × g for 60 min at 2 °C to obtain the supernatants (Triton extracts). We confirmed by Western blotting that almost all of the tryptic fragments and the intact receptor were collected in these extracts (data not shown). The Triton extracts were subjected to Western blotting and IP3 binding assay. A, the Triton X-100 extracts (2.5 µg of protein) were separated by 8% SDS-PAGE and probed with N1, N3, 10A6, anti-(1718-31), 1ML1 and 18A10 antibodies which specifically recognize the Ia/b, II, III, IVa/b and V major tryptic fragments, respectively. Intact IP3R and major tryptic fragments are indicated by an arrowhead and arrows, respectively. B, specific [3H]IP3 (9.6 nM) binding to the Triton extracts (45 µg of protein). Nonspecific binding was measured in the presence of 2 µM cold IP3. Values are the mean ± S.D. of four experiments.

By using the Triton extracts containing these tryptic fragments, the following [3H]IP3-binding experiments were carried out. Although the IP3-binding core was completely separated into two fragments I and II, which were recognized by the N1 and N3 antibodies, respectively (Fig. 1A), the fragmented mIP3R1 (Trypsin +) still retained specific IP3 binding activity equivalent to that of the intact receptor (Trypsin -) (Fig. 1B). In [3H]IP3 binding competition experiments with various inositol phosphates, the fragmented mIP3R1 (Trypsin +) showed comparable ligand binding specificity to the intact receptor (Trypsin -) in the order of (1,4,5)IP3 > (2,4,5)IP3 > (1,3,4,5)IP4 (Fig. 2A). Scatchard analysis showed that the capacity for IP3 was unaffected by trypsin digestion; the Bmax (pmol/mg protein) was 23 for the fragmented mIP3R1 (Trypsin +) and 22 for the intact receptor (Trypsin -) (Fig. 2B). In addition, the affinity for IP3 (Kd value) was also comparable between the fragmented mIP3R1 (13 nM) and the intact receptor (19 nM). These results suggested that a rigid folded conformation of the tetrameric mIP3R1 complex in the native membrane might preserve its inherent IP3-binding pocket from trypsin digestion, even though the IP3-binding core was split into two near the center.


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Fig. 2.   Binding characteristics of the fragmented mIP3R1 with limited trypsin digestion. All samples used (Trypsin (-) and Trypsin (+)) were the same as those in Fig. 1. A, competition of specific [3H]IP3 (9.6 nM) binding to the Triton extracts (45 µg of protein) by various inositol phosphates: (1,4,5)IP3 (filled circles), (2,4,5)IP3 (open triangles), and (1,3,4,5)IP4 (filled squares). Values are normalized to 100% of controls measured without competitor. Nonspecific binding was measured in the presence of 2 µM IP3. Each point is the mean of two experiments. B, Scatchard plots of inhibition of specific [3H]IP3 (4.8 nM) binding to the Triton extracts (45 µg of protein) by cold (1,4,5)IP3. Nonspecific binding was measured in the presence of 10 µM IP3. The results of a typical experiment are shown. Kd and Bmax of each sample shown are the means of two experiments.

Release of IP3 Binding Activity from the Insoluble to Soluble Fraction by Prolonged Trypsin Digestion-- Most IP3 binding activity and fragments I and II were collected in an insoluble membrane fraction upon mild trypsin digestion, whereas a proportion of these fragments became soluble upon more extensive digestion. We next examined the temporal profile of IP3 binding activity in relation to increasing time of digestion (Fig. 3). Microsomal fractions were treated with 10 µg/ml trypsin for 0, 5, 10, 20, and 40 min and were separated in insoluble and soluble fractions by centrifugation at 105,000 × g for 60 min. The pelleted insoluble fractions were solubilized with 1% Triton X-100 to obtain Triton extract. An equivalent volume of the soluble fraction (sup) and the Triton extract of insoluble fraction (ppt) were subjected to [3H]IP3 binding (Fig. 3A) and Western blotting assay (Fig. 3B). As the digestion time with trypsin was extended to 10 min, both the insoluble and soluble fraction showed a sharp difference in temporal changes of IP3 binding activity; the former exhibited gradual attenuation, whereas the latter showed a gradual increase. When the digestion time was extended to beyond 20 min, no more marked changes were observed. These temporal profiles appear to parallel the immunoblotting patterns (Fig. 3B); as the digestion time was extended to 10 min, the levels of the Ia/b and II fragments, containing the epitopes for the N1 and N3, respectively, were decreased in the insoluble fraction but increased in the soluble fraction, and thereafter (at 20 and 40 min) no apparent change was seen in both fractions. However, more extensive digestion caused loss of binding activity and of these immunoreactive fragments (data not shown). These data indicated that the two tryptic Ia/b and II fragments were tightly associated with the tryptic mIP3R1-membrane complex and that some portions released by the prolonged digestions appeared to retain conformation for specific binding by interaction between fragments.


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Fig. 3.   Temporal profiles of IP3 binding activity of fragments I and II in soluble and insoluble membrane fractions upon prolonged trypsin digestion. A, mouse cerebellar microsomal fractions (1 mg/ml protein) were incubated with 10 µg/ml trypsin for 0, 5, 10, 20, and 40 min at 35 °C in trypsinizing buffer (closed). The reactions were quenched with 100 µg/ml trypsin inhibitor and 0.1 mM PMSF. As a control (open symbols), microsomal fraction was supplemented with trypsin inhibitor and PMSF followed by the addition of trypsin and then incubated for 40 min at 35 °C. The insoluble and soluble fractions were separated by centrifugation at 105,000 × g for 60 min at 4 °C. The pellet was then solubilized in 1% Triton X-100 solution in the same way as described in Fig. 1. Equivalent volumes of soluble fractions (circles) and Triton extract of insoluble fraction (squares) were subjected to the [3H]IP3 binding assay. Nonspecific binding was measured in the presence of 2 µM IP3. B, equivalent volumes of soluble fraction (sup) and Triton extract of insoluble fraction (ppt) prepared as described above were subjected to immunoblotting with the N1 and N3 antibody. An asterisk shows a nonspecific band.

Reconstitution of IP3 Binding Activity by Complementation between Two Recombinant Binding Components-- To examine the functional interaction between the two tryptic fragments I and II containing the split IP3-binding core, we tried to express recombinant proteins corresponding to these fragments in an E. coli system. However, it was difficult to obtain sufficient amounts of soluble proteins, especially for the fragment II (data not shown). To solve this, we constructed GST fusion proteins as shown in Fig. 4. The GN was composed of GST fused to the N-terminal residues 1-343, corresponding to almost the entire fragment I. We previously showed that Arg-265 within the IP3-binding core is one of three basic amino acid residues for which Gln substitutions (R265Q) caused a complete loss of binding activity (14). The GN(R265Q) had this R265Q mutation in the GN construct. The GC consisted of a GST fusion protein with residues 341-604, corresponding to almost all of the N-terminal 29-kDa subfragment derived from fragment II, which took place upon extensive trypsinolysis (18). These GST fusion proteins were expressed in E. coli and were purified through a glutathione column (Fig. 5A). [3H]IP3 binding assay showed that neither GN nor GN(R265Q) alone had any significant binding activity, whereas the GC alone showed some traces of activity (Fig. 5B).


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Fig. 4.   Constructs of GST fusion proteins carrying the IP3-binding domains of mIP3R1. A schematic representation of the N-terminal region of the mIP3R1 is shown at the top, where the IP3-binding core (residues 226-578; solid line) and the alternative splicing SI segment (residues 318-332; open box) are indicated. Boxes represents GST fusion proteins. All constructs were made in pGEX-2T; GN, GST + residues 1-343; GN(R265Q), R265Q mutant of GN; GC, GST + residues 341-604; GST, pGEX-2T vector alone as a control.


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Fig. 5.   Complementation of the IP3 binding activity by two separately expressed GST-fused IP3-binding domains. A, SDS-PAGE analysis of recombinant GST-fused IP3-binding domains. Two µg of GST fusion proteins, GN, GN(R265Q) and GC, and GST (see Fig. 4) were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Sizes of molecular makers in kDa are shown in the right. B, specific [3H]IP3 (9.6 nM) binding to the GST fusion proteins. IP3 binding of GST fusion proteins (7 µg) were individually analyzed: GST, GN, GN(R265Q), and GC. Two GST fusion proteins were mixed (7 + 7 µg), and then the IP3 binding activity of the mixtures was analyzed: GN+GC, GN(R265Q)+GC, GST+GC, GN+GST, and GN(R265Q)+GST. Nonspecific binding was measured in the presence of 2 µM IP3. Values are the mean ± S.D. of three experiments.

To examine whether the two split components worked cooperatively to bind IP3, we performed a complementation test by which two of these separately synthesized proteins were simply mixed in vitro (right half of Fig. 5B). The combinations of GN + GST, GC + GST, GN(R265Q) + GST, and GN(R265Q) + GC had no significant change in [3H]IP3 binding. In marked contrast, the combination of GN + GC displayed a dramatic increase in the specific binding activity, suggesting that the GN and the GC complemented each other to retrieve IP3 binding activity, even though both have a large extra GST moiety at the N terminus.

To characterize further this structural and functional reconstitution of an IP3-binding site by mixing two separately synthesized GN and GC, we carried out Scatchard analysis as shown in Fig. 6. The GN alone exhibited no binding activity as the control GST. On the other hand, the GC alone could form a very low affinity site with Kd value of 4.7 µM. It was of particular interest that the mixture of GN + GC displayed a plot fitted by two binding sites; the low affinity site appeared to be consistent with that of the GC alone, and the high affinity site had a Kd value of 11 nM, about 400-fold lower than that of the low affinity site and comparable to that of the intact mIP3R1. Judging from the Bmax value of 40 pmol/mg protein, it was estimated that about 2% of the total GC (about 1800 pmol/mg of protein) was involved in reconstitution of this high affinity site by complementation with the GN.


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Fig. 6.   Scatchard analysis of the specific IP3 binding to the GST fusion proteins. Scatchard plots of inhibition of specific [3H]IP3 (9.6 nM) binding to the GST fusion proteins by cold (1,4,5)IP3. Each protein (7 µg) was analyzed individually: GC, GN, and GST. The mixture of GN (7 µg) and GC (7 µg) was analyzed: GN+GC. In the plot of GN+GC, the quantity of protein is tentatively expressed as that of the GC regardless of the GN, since the GN alone has no significant activity. Nonspecific binding was measured in the presence of 200 µM IP3. An inset in the GN + GC plot shows an enlargement of a plot for a high affinity site.


    DISCUSSION

Mild trypsin digestion of a cerebellar membrane fraction generates five major tryptic fragments, including the N-terminal two fragments Ia/b (40/37 kDa) and II (64 kDa), that share the entire IP3-binding site and are tightly associated with the insoluble membrane fraction (18). In the present study, we have shown that the trypsinized IP3-binding site of mIP3R1 retains significant affinity, specificity, and binding capacity for IP3, comparable to those of the intact one. These results indicate that the trypsinized IP3-binding site may retain functional tertiary structure. Prolonged trypsinization caused concomitant loss of IP3 binding activity and release of fragments I and II from the insoluble mIP3R1-membrane complex to the soluble fractions. These results demonstrate that the folded conformation of the IP3-binding site is stably retained in either the insoluble or soluble form by a possible inter-fragment interaction. Joseph et al. (17) previously reported that prolonged trypsin digestions of rat cerebellar microsomes caused release of a 68-kDa fragment of IP3R with concomitant appearance of higher IP3 binding activity in the soluble fraction, whereas with mild digestion the 68-kDa fragment was retained in the membrane-bound insoluble form with lower activity. In the present study, however, we have shown that the affinity of the insoluble fraction from the mildly trypsinized samples is slightly higher than that of un-trypsinized samples. We consider that the 68-kDa fragment is likely to correspond to the 64-kDa fragment II identified in the present study and that the binding activity observed by Joseph et al. (17) could be attributed to a native-like interaction between fragments I and II which are simultaneously released, although Joseph et al. (17) did not note fragment I in both the soluble and insoluble fractions. This discrepancy between our results and the previous study (17) may be due to differences in preparation of membrane fractions, trypsin digestions, and/or binding assays (filtration versus PEG precipitation).

Neither the SI plus (mIP3R1SI+) nor minus (mIP3R1SI-) subtype showed any significant alteration in IP3 binding activity,2 whereas deletions of any other region within the IP3-binding core tested so far completely abolished the activity (12-14), suggesting that the SI region is the only redundant part of the binding core. Arg-343 and Arg-345, which lie between fragments I and II and are close to the alternative splicing SI region (residues 318-332), are very susceptible to limited trypsinolysis. Within the IP3-binding core of all members of the IP3R family, the most divergent sequences are found around the SI region. Thus, we suggest that in the vicinity of the SI region, Arg-343 and Arg-345 may form a flexible loop-like structure (residues ~318 to ~345) that interconnects two non-covalently but tightly associated well folded structural components; "binding domains" I (residues 1 or N terminus to ~317) and II (residues ~346 to ~923) corresponding to the trypsin-resistant fragments I and II, respectively (Fig. 7).


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Fig. 7.   Schematic model of the domain structure of the IP3-binding site. Trypsin-resistant fragments I and II of the mIP3R1 are designated as the binding domains I (residues 1 or N terminus to ~317) and II (residues ~346 to ~923). The N terminus of domain I in the native mIP3R1 could not been determined by amino acid sequencing (18). Around the region including the SI segment (residues 318-332), Arg-343 and Arg-345 probably form a flexible loop between two domains, exposed to the outside, thereby being hypersensitive to limited trypsinolysis. The domain II consists of at least two subdomains of the N-terminal 29 kDa (probably residues ~346 to ~604) and the C-terminal 38 kDa (probably residues ~605 to ~923), which are cleaved off with a more extensive trypsinolysis (18). The IP3-binding core has been experimentally determined to reside within residues 226-578, and therefore there are at least two putative core domains, I (residues at least 226 to ~317) and II (residues ~346 to ~604). The inner surface of the core domains may be lined with 10 basic amino acid residues for which Gln substitutions caused significant reduction of the binding activity (14). Of them, Arg-265, Lys-508, and Arg-511 are critical residues for the specific IP3 binding, because their single amino acid substitutions to either Gln or Ala caused a loss of the activity (14). The functional roles for the ligand binding of the binding domain I, core domains I and II, and the region between residues 1 and ~225 are indicated in italics as described under "Discussion."

Residues 224-579 of the mIP3R1 expressed in E. coli which almost correspond to the IP3-binding core (residues 226-578) show specific and high affinity IP3 binding activity, indicating that the binding core itself forms a functional structure (14). We recently showed that more extensive trypsin digestion further split fragment II into two subfragments, an N-terminal 29-kDa and a C-terminal 38-kDa fragment, and that the carboxyl side of Arg-603 and/or Lys-604 would be the sites for this extensive trypsinization, which lie in the vicinity of the C-terminal boundary of the IP3-binding core (18). Therefore, we suggest that the IP3-binding core consists of two "core domains" I (at least residues ~226 to ~317) and II (residues ~346 to ~604) in the binding domains I and II, respectively (Fig. 7).

The present study showed that the GST-fused N-terminal binding domain I (GN) had no significant binding activity, whereas the GST-fused C-terminal core domain II (GC) exhibited low affinity. Strikingly, simple mixing of these regions led to increased binding activity. The Kd value of this reconstituted high affinity IP3-binding site was almost comparable to that of the intact mIP3R1. These results suggested that the two separate GST fusion proteins mutually recognized and cooperatively formed a virtually native high affinity IP3-binding pocket. However, we could only reconstitute about 2% of the total input of the GC by just mixing the GN and the GC. This low rate of functional complementation may be due to physical interference by the N-terminal GST moiety attached to both fusion proteins, and/or due to low efficiency in direct interactions between GN and GC or in proper folding of expressed GN and GC, under the present conditions.

The present study provides evidence for the modular construction of the IP3-binding region with discrete functional domains. We suggest that the low affinity core domain II is a prototype of the IP3-binding structure (Fig. 7). The binding domain I, although it has no binding capacity by itself, could contribute to form a high affinity site cooperatively with the core domain II. Thus, the domain I seems to act as a "modulator" to potentiate the affinity of the IP3 binding prototype, core domain II. The functional property of this N-terminal binding domain I, however, is a little complicated as described previously (14); a short deletion of the N-terminal 31 amino acids from the N-terminal 734 amino acid region (T734) resulted in a significant reduction in the binding activity, although the resultant mutant included the entire IP3-binding core sequence. Such contradictory mutational effects were also found in serial N-terminal deletions up to residue 215. However, the authentic binding activity was markedly recovered, when deleted up to the first N-terminal 220, 223, or 225 amino acids, thereby indicating that the N-terminal boundary of IP3-binding core is at most residue 226. Notably, the mutant lacking the first N-terminal 223 amino acids showed more than 10-fold higher affinity for IP3 than that of the parental T734. Furthermore, a substitution of GST (26 kDa) for the first 223 amino acids of the N-terminal 604 amino acids (T604) significantly increased the affinity, as compared with the parental T604.2 These lines of peculiar evidence led us to hypothesize that the N-terminal first ~225 amino acids are not directly responsible for the binding but are somewhat related to its suppression. Then, as shown in Fig. 7 we hypothesize that there are at least three functional modules, two modulators in the domain I (N-terminal suppressor and C-terminal enhancer (core domain I)) and one proto-IP3-binding site in the core domain II. Co-operative regulation among these modules for ligand binding may influence the channel gating.

Mignery et al. (12) reported that the N-terminal cytoplasmic region of rat IP3R1 expressed in COS cells displayed an altered mobility (apparent decrease in mass of >50 kDa) on gel chromatography in the presence of IP3 and suggested that any conformational change induced upon binding to IP3 might be involved in coupling the ligand binding to the channel gating. We suppose that relative movement of the core domains I and II non-covalently associated with each other would occur upon ligand binding as described below. We previously demonstrated the significance of the 10 basic amino acid residues (Arg or Lys) in specific IP3 binding, all of which are well conserved within the IP3R family (14). Of these, Arg-265, Lys-508, and Arg-511 are critical. We thus suggested that these 10 basic residues, especially Arg-265, Lys-508, and Arg-511, contribute to form a positively charged pocket for binding to the negative charges on the three phosphate groups of IP3. Four of them (Arg-241, Lys-249, Arg-265, and Arg-269) are positioned in the core domain I and the other six (Arg-504, Arg-506, Lys-508, Arg-511, Arg-568, and Lys-569) in the core domain II (Fig. 7). Thus, the IP3-binding pocket constituting two core domains may be relatively expanded due to repulsion among the positive charges on the inner surface in the nonligand-bound state (open) and become narrow due to neutralization of the positive charges by interactions with the negative charges on IP3 in the ligand-bound state (closed). Finally, studies on the higher order structures of the IP3-binding site and the possible relative movement upon IP3 binding will shed light on the molecular basis of the gating of the mIP3R1 channel as well as the ligand binding.

    ACKNOWLEDGEMENT

We thank K. Yoshikawa for preparation of manuscript.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Howard Hughes Medical Institute, Department of Physiology, University of California San Francisco, Box 0725, San Francisco, CA 94143-0725.

To whom correspondence should be addressed: Dept. of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5320; Fax: 81-3-5449-5420; E-mail: tfuruich{at}ims.u-tokyo.ac.jp.

2 F. Yoshikawa and T. Furuichi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; IP3R1, type 1 inositol 1,4,5-trisphosphate; mIP3R1, mouse type 1 inositol 1,4,5-trisphosphate; PEG, polyethylene glycol; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis..

    REFERENCES
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ABSTRACT
INTRODUCTION
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