Trypsinized Cerebellar Inositol 1,4,5-Trisphosphate Receptor
STRUCTURAL AND FUNCTIONAL COUPLING OF CLEAVED LIGAND BINDING AND CHANNEL 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
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel (calculated molecular mass = ~313 kDa/subunit). We studied structural and functional coupling in this protein complex by limited (controlled) trypsinization of membrane fractions from mouse cerebellum, the predominant site for IP3R1. Mouse IP3R1 (mIP3R1) was trypsinized into five major fragments (I-V) that were positioned on the entire mIP3R1 sequence by immuno-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I-IV were derived from the N-terminal cytoplasmic region where the IP3-binding region extended over two fragments I (40/37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the membrane-spanning channel region. All five fragments were pelleted by centrifugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggested that the native mIP3R1-channel is an assembly of four subunits, each of which is constituted by non-covalent interactions of five major, well folded structural components I-V that are not susceptible to attack by mild trypsinolysis. Ca2+ release experiments further revealed that even the completely fragmented mIP3R1 retained significant IP3-induced Ca2+ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP3-induced Ca2+ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP3R1, that is neighboring coupling between components I and II for IP3 binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Extracellular stimuli can activate hydrolysis of phosphatidylinositol 4,5-bisphosphate, a component of the plasma membrane, the result being production of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP3)1 (1). IP3 diffuses into the cytoplasm and mediates the release of Ca2+ from intracellular Ca2+ storage organella, chiefly the endoplasmic reticulum, by binding to its receptor (IP3R). IP3R is a tetrameric intracellular IP3-gated Ca2+ release channel (2). Molecular cloning has revealed that there are at least three distinct types of IP3R in mammals (3).

The IP3R activity as well as protein are extremely enriched in cerebella (4-8). The cloned cerebellar IP3R, now called type 1 IP3R (IP3R1), encodes an extraordinarily huge protein of 2749 amino acids (~313 kDa) (9-11) and has three regions (SI, SII, and SIII) that undergo alternative splicing (10, 12-14). The majority of IP3Rs expressed in the mouse cerebellum was IP3R1 (15, 16). The native IP3R1 forms largely a homo-tetramer in rodent cerebella (6, 17). Purified IP3R1 proteins showed a stoichiometric IP3 binding, namely one protomer binds to one IP3, with the affinity of Kd = 83-100 nM (6, 8), which is fairly comparable to the EC50 value (40-200 nM) in IP3-induced Ca2+ flux via the reconstituted purified IP3R1 liposomes (18-20), suggesting tight functional coupling of ligand binding and channel opening.

The cloned mouse IP3R1 (mIP3R1) itself was found to encode a complete IP3-operated Ca2+ release channel as well as an IP3-binding receptor (21, 22). We proposed that the mIP3R1 traverses the store membrane six times at the C-terminal membrane-spanning region (residues 2276-2589) (23). Thus both ends, the large N-terminal arm region (residues 1-2275) and the short C-terminal tail region (residues 2590-2749), face the cytoplasmic side. Deletion mutageneses showed that the mIP3R1 binds IP3 within the N-terminal 650 amino acids, independently of tetramer formation (21, 24). Furthermore, residues 226-578 were found to be close to the minimum for specific and high affinity ligand binding, thus assigned to the IP3 binding core (25). The C-terminal membrane-spanning region, the most conserved region within the IP3R family, would form an ion channel. It was also shown that at least the C-terminal part, including the membrane-spanning region, was sufficient for subunit assembly (26, 27). Based on these data, we asked how ligand binding to four individual binding pockets cytoplasmically extruding from a tetrameric IP3R1 channel would gate its channel embedded in Ca2+ store membrane, apart from ~1,700 amino acids in the primary sequence. However, little is known of mechanisms underlying structural and functional coupling between IP3 binding and channel opening.

The cerebellar IP3R1 was degradated by Ca2+-activated neutral protease calpain to two major fragments of 130 and 95 kDa immunoreacted with anti-C-terminal antibody (28). This finding suggested that the C-terminal channel region forms a folded structure. Joseph et al. (29) reported findings in a study on structural features of IP3R1 with limited trypsinolysis of cerebellar microsomes. They identified two trypsin-resistant bands of 68 and 94 kDa which, respectively, contained IP3 binding activity and membrane-spanning segments. A notable finding is that of a large portion of the 68-kDa peripheral fragment associated with the 94-kDa integral fragment. These results revealed a close association between both N-terminal and C-terminal tryptic fragments despite the disconnection of peptide bonds. Thus, it had to be determined if such non-covalent interactions among these tryptic fragments would affect retention of structural and functional coupling between IP3 binding and channel opening.

We have now examined structure-function relationships of mIP3R1 by limited trypsinization of mouse cerebellar membrane fractions. We found that native mIP3R1 consists of five major trypsin-resistant fragments (I-V), which were lined up on the entire sequence. All cytoplasmic fragments I-IV were tightly associated with the membrane-spanning fragment V, despite cleaving their peptide bonds, and such fragmented mIP3R1 channels retained significant IP3-induced Ca2+ release (IICR) activity. We suggest that the native mIP3R1 channel is an assembly of four subunits each of which is constituted by five major structural components, non-covalently but tightly associated, and that this structural coupling rather than peptide linkages connecting them would be a prerequisite for functional coupling in ligand binding and channel gating.

    EXPERIMENTAL PROCEDURES

Materials-- N-Tosyl-L-phenylalanyl chloromethyl ketone-treated bovine pancreas trypsin, soybean trypsin inhibitor, phosphocreatine, and oligomycin were purchased from Sigma; D-myo-inositol 1,4,5-trisphosphate and Fura-2 were from Dojindo (Japan); Pansorbin was from Calbiochem; rabbit anti-rat IgG (Fc-specific) was from Jackson ImmunoResearch Laboratories; normal rat IgG was from Cappel; CNBr-activated Sepharose 4B was from Amersham Pharmacia Biotech, creatine kinase was from Boehringer Mannheim, and 4-bromo A23187 was from Molecular Probes.

Preparation of Anti-peptide Antibodies-- Peptides corresponding to residues 40-55, 257-274, 560-576, and 590-604 of the mIP3R1 with an additional Cys residue on their N terminus were synthesized for preparation of polyclonal antibodies (pAbs) N1, N2, N4, and N5, respectively. The peptides were coupled to keyhole limpet hemocyanin via the N-terminal Cys, using a cross-linking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester. pAbs were raised in rabbits (New Zealand White; Nippon SLC, Japan) against the peptide-keyhole limpet hemocyanin conjugates. IgG fractions were purified from antisera using a protein A affinity column, Ampure PA kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol. For pAbs N1, N2, and N3, IgG fractions were further purified with the antigenic peptide-conjugated beads according to a standard protocol (30).

Preparation of Cerebellar Membrane Fractions-- ddY mice (8-10 weeks old; Nippon SLC) were anesthetized and decapitated. Cerebella were mixed with 9 volumes of homogenizing buffer (0.32 M sucrose, 1 mM EDTA, 5 mM Tris-HCl, pH 7.4, plus protease inhibitor mixture I (0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µM pepstatin A, and 10 µM leupeptin)) and homogenized in a glass Teflon Potter homogenizer with 10 strokes at 850 rpm on ice. The homogenate was centrifuged at 1,000 × g for 15 min at 4 °C, and then the supernatant was recentrifuged under the same conditions to remove the pellet. For crude microsomal fractions, the second supernatant was then centrifuged at 105,000 × g for 60 min at 2 °C. The pellet was resuspended with 1 mM EDTA, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0 (at 4 °C), to give a final concentration of ~15 mg/ml protein. Protein concentrations were measured using Bio-Rad protein assay kit with bovine serum albumin as a reference. The microsomal suspensions were frozen in liquid nitrogen and then were stored at -80 °C until use. For Ca2+ release experiments, the homogenate was centrifuged at 4,000 × g for 20 min at 4 °C. The supernatant was recentrifuged, under the same conditions. The second supernatant was then centrifuged at 105,000 × g for 30 min at 2 °C. The pellet was resuspended in Ca2+ releasing buffer (110 mM KCl, 10 mM NaCl, 5 mM KH2PO4, 1 mM MgCl2, 1 mM DTT, and 10 mM Hepes-KOH, pH 7.2, at 24 °C) and recentrifuged at 105,000 × g for 30 min at 2 °C to exchange the buffer. The resultant pellet was resuspended with the Ca2+ releasing buffer to give a final concentration of ~10 mg/ml protein.

Trypsin Digestion-- Microsomal fractions (1 mg/ml protein) were incubated with trypsin at 35 °C in trypsinizing buffer (120 mM KCl, 1 mM EDTA, 1 mM DTT, 20 mM Tris-HCl, pH 8.0, at 25 °C). For Ca2+ release experiments, microsomes (1 mg/ml protein) were digested by trypsin in Ca2+ releasing buffer at 35 °C, except for direct trypsinization in cuvettes while measuring fluorescence of Fura-2, as described in the legend of Fig. 6. The reaction was quenched with a 10-50-fold weight excess of soybean trypsin inhibitor and 0.1 mM PMSF or 10-fold weight excess of soybean trypsin inhibitor. We confirmed by Western blot analysis that each of these treatments completely blocked the activity of trypsin.

Western Blotting-- Samples were added to an equal volume of 2× SDS-PAGE sample buffer (4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (w/v) glycerol, and 125 mM Tris-HCl, pH 6.8) and incubated at 55 °C for 30 min. Then the mixtures were separated on 8% SDS-PAGE (25). Protein bands were analyzed by immunoblotting, using 11 site-specific antibodies (listed in Table I) and an ECL detection system (Amersham Pharmacia Biotech) (25).

Immunoprecipitation-- Immunoprecipitation was performed as described previously (31). 1.5 mg of microsomal fractions was resuspended with 1.5 ml of the trypsinizing buffer (1 mg/ml protein) and treated with trypsin on three different conditions, as described in Fig. 3. The trypsinized samples were centrifuged at 105,000 × g for 60 min at 2 °C to separate into soluble (cfg-sup) and insoluble membrane fractions. The pellet was resuspended with 0.5 ml of 1 mM EDTA, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0 (cfg-ppt). Then, the proteins were solubilized by adding 56 µl of 10% (w/v) Triton X-100 to give a final detergent concentration of 1% then rotated for 30 min at 4 °C. The Triton X-100-treated materials were centrifuged at 20,000 × g for 60 min at 2 °C to separate the supernatant (Triton-sup or Triton X-100 extract) and non-solubilized precipitate (Triton-ppt) that was resuspended with 556 µl of 1 mM EDTA, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0. 2.5 µl of total (not fractionated) and cfg-sup, and 0.83 µl of cfg-ppt, Triton-sup, and Triton-ppt were subjected to Western blotting. 100 µl of the Triton X-100 extract was diluted with 0.9 ml of phosphate-buffered saline supplemented with 5 mM EDTA, 0.2% Triton X-100, 50 µg/ml soybean trypsin inhibitor, and protease inhibitor mixture I, and then precleared with 6 µg/ml rabbit anti-rat IgG (Fc specific) and 0.2% (w/v) Pansorbin. The precleared supernatant (0.9 ml) was incubated at 4 °C for 60 min with 6 µg/ml of monoclonal antibody (mAb) 18A10 against the mIP3R1 or normal rat IgG and for 60 min more with 6 µg/ml of rabbit anti-rat IgG (Fc-specific). The mixtures were then incubated with 0.2% (w/v) Pansorbin for 60 min at 4 °C, and the Pansorbin-bound immune complexes were collected by centrifugation at 18,000 × g for 3 min at 4 °C. The pellets were washed three times with 1 ml of phosphate-buffered saline supplemented with 5 mM EDTA and 0.5% Triton X-100 and solubilized with 200 µl of 1× SDS-PAGE sample buffer for 30 min at 55 °C. After removing detached Pansorbin particles by centrifugation, 5 µl of the SDS-PAGE sample (IP-18A10 and IP-IgG) were analyzed by Western blotting.

Immunoaffinity Purification of Tryptic Fragments-- 500 µg of mAb 18A10 or normal rat IgG were coupled to 0.5 ml of CNBr-activated Sepharose 4B, according to the manufacturer's protocol. 45 ml of microsomes suspended in trypsinization buffer (1 mg/ml of protein) was digested with 1 µg/ml trypsin for 2 min at 35 °C. The reaction was quenched by adding 50 µg/ml trypsin inhibitor and 0.1 mM PMSF. The trypsinized sample was centrifuged at 105,000 × g for 60 min at 4 °C, and the pellet was resuspended in 15 ml of purification buffer (1 mM EDTA, 1 mM 2-mercaptoethanol, 50 mM Tris-HCl, pH 7.4) supplemented with 50 µg/ml trypsin inhibitor plus protease inhibitor mixture II (0.1 mM PMSF, 2 µg/ml aprotinin, 5 µM pepstatin A, and 1 µM leupeptin). The pellet suspension was solubilized by adding 10% (w/v) Triton X-100 to give a final detergent concentration of 1%, followed by rotation for 30 min at 4 °C. The mixture was centrifuged at 20,000 × g for 60 min at 4 °C. The supernatant (~15 ml) was diluted with 30 ml of purification buffer supplemented with 50 µg/ml soybean trypsin inhibitor plus protease inhibitor mixture II and precleared by use of 0.5 ml of normal rat IgG-Sepharose 4B beads. The precleared sample was then incubated with 0.5 ml of mAb 18A10-Sepharose 4B beads for 8 h at 4 °C with gentle rotation. The beads were then transferred into a column and washed with 10 ml of the purification buffer supplemented with 1% (w/v) Triton X-100. Absorbed proteins were eluted four times with 0.5 ml of 0.1 M glycine HCl, pH 2.5, and 0.1% (w/v) Triton X-100 and immediately neutralized by adding 25 µl of 1 M Tris. The eluates were combined, desalted on a PD10 column (Amersham Pharmacia Biotech), and lyophilized. The lyophilized pellet was solubilized with 130 µl of 1× SDS-PAGE sample buffer and incubated for 30 min at 55 °C.

N-terminal Sequencing of Tryptic Fragments-- Amino acid sequencing was done by APRO Life Science Institute (Naruto, Japan). The SDS-PAGE sample (20 µl) of the immunoaffinity purified proteins was applied to a 10% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membrane. The transferred protein bands were visualized by staining with Coomassie Brilliant Blue R-250, and membrane pieces of blotted bands of 91, 76, 64, 40, and 37 kDa were cut out of the blots. The protein-blotted membrane pieces were applied to a gas-phase protein sequencer (Hewlett-Packard). When two or three kinds of peptides were included in individual membrane pieces, sequences were determined on the basis of recovery of phenylthiohydantoin-amino acid upon the N-terminal sequencing and the sequence of the mIP3R1.

Ca2+ Release Experiment-- Changes in Ca2+ concentration ([Ca2+]) were monitored by measuring fluorescence of Fura-2 using a CAF110 spectrofluorometer (Japan Spectroscopic Co.). Fluorescence was recorded at 500 nm with alternate excitation wavelength of 340 and 380 nm. Ratio of fluorescence intensities (R, 340/380) was obtained every 0.5 s. The [Ca2+] was calculated as described (32), assuming a dissociation constant of 224 nM for Fura-2-Ca2+. Maximum and minimum values of R were obtained in the presence of an excess amount of CaCl2 and EGTA, respectively. In the experiments indicated in Figs. 5 and 7, 1.8 ml of microsomal fractions (1 mg of protein/ml) suspended in the Ca2+ releasing buffer were digested with trypsin, as described in the legends. Out of this trypsinized microsomal suspension, 200 µl (200 µg of protein) was taken, diluted to 500 µl in a quartz glass cuvette with Ca2+ releasing buffer, supplemented with creatine phosphate, creatine kinase, oligomycin, and Fura-2 to give a final concentration of 10 mM, 20 units/ml, 1 µg/ml and 2 µM, respectively, and then transferred into the spectrofluorometer. The suspension was continually stirred and maintained at 30 °C, while Fura-2 fluorescent measurements of extra-microsomal [Ca2+] were being carried out. Ca2+ loading to microsomes was initiated with activation of Ca2+-ATPase by adding 2 mM ATP, and Ca2+ release was triggered with activation of mIP3R1 by the addition of various amounts of IP3. Before closing each set of experiments, intra-microsomal Ca2+ content was estimated by measuring ionophore 4-bromo A23187-induced Ca2+ release. In a series of experiments, IP3 and A23187 were added, when the basal [Ca2+] was settled at ~220 nM. In the experiments demonstrated in Fig. 6, direct trypsinization (at 5 µg/ml trypsin for 4, 20, 40, and 60 min at 30 °C) within measuring cuvettes was carried out ~1 min after the addition of 2 mM ATP, and the digestion was quenched by direct addition of 50 µg/ml soybean trypsin inhibitor. After adding 10 µM IP3, 4 µM A23187 was applied when the [Ca2+] was set at the concentration almost the same as when IP3 was applied.

    RESULTS

The mIP3R1 Has Five Major Tryptic Fragments-- To detect and map fragments generated with limited trypsin digestion, we used a series of site-specific antibodies against the mIP3R1, a total of 11 which recognize epitopes widespread over the entire sequence (see Table I and Fig. 1). Four polyclonal antibodies (pAbs) N1, N2, N4, and N5 were newly developed for this study. Epitopes for the N2, N3, and N4 were designed within the IP3-binding core (residue 226-578) (25). Three monoclonal antibodies (mAbs) 4C11, 10A6, and 18A10 were raised against the purified cerebellar mIP3R1 (5, 7). pAb anti-(1564-85) recognizes the calmodulin (CaM) binding site (residues 1564-1585) (33). Sub-segments A, B, and C are further spliced in combination within the alternative splicing segment SII region (12, 13). pAb anti-(1718-31) recognizes the sub-segment C (residues 1716-1731).2 pAb 1ML1 recognizes a luminal loop between the membrane-spanning segments M5 and M6 (23), in contrast to all other antibodies that react with cytoplasmic portions. All antibodies specifically recognized cerebellar mIP3R1 with an apparent mass of ~250 kDa (lanes 1 and 3 in Fig. 2), except that the N1 and N2 also cross-reacted with nonspecific bands of 30 kDa and of 72/70 kDa, respectively (with asterisks in Control of Fig. 2). The discrepancy between the molecular mass of 250 kDa, estimated from SDS-PAGE and that of 313 kDa predicted from the primary structure, is likely due to aberrant mobilization of higher molecular weight proteins on SDS-PAGE, because the agarose-PAGE gave a value (~320 kDa) comparable to that calculated from the open reading frame (17).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Site-specific antibodies of the mIP3R1


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic representation of the trypsin-resistant fragments of the mIP3R1. Top line indicates amino acid number of the mouse IP3R1 (mIP3R1) of 2749 amino acids (SI+/SII+/SIII- splicing subtype). Middle line represents structure of the mIP3R1. The following regions are shown: IP3 binding core (residues 225-578) (25) with a solid horizontal bar; six putative membrane-spanning domain (MSD; 2276-2589) with six solid vertical bars; splicing segments SI (318-332) (10, 12) and SII (1692-1731) (12, 13) with open boxes; and the position (between 917 and 918) where splicing segment SIII (9 amino acids) (14) is inserted with vertical line. The sites for CaM-(1564-1585) (33), FKBP12-(1400-1401) (39), and putative ATP binding (1773-1778, 1775-1780, and 2016-2021), and cAMP-dependent protein kinase-(1588, 1755) (13) and cGMP-dependent protein kinase phosphorylation-(1755) (38) are indicated with arrows. The Ca2+-binding sites (2124-2146 and 2463-2528) (57) are marked with dotted horizontal lines. The epitopes for antibodies used in this experiment are indicated as short solid bars above the line representing structure of the mIP3R1. The bottom row shows major tryptic fragments that are positioned relative to the linear sequence. The molecular mass of each fragment is indicated in kDa.


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 2.   Fragmentation of cerebellar mIP3R1 by limited trypsin digestion. Crude microsomal fractions (1 mg protein/ml) from mouse cerebellum were treated with trypsin, under various conditions. The reactions were quenched by addition of a 10-fold excess weight of trypsin inhibitor and 0.1 mM PMSF. 2.5 µg of the protein was separated on 7.5% SDS-PAGE, followed by analysis with Western blotting, using 11 site-specific antibodies shown on the left shoulder of each blot (N1, N2, N3, N4, N5, 4C11, 10A6, anti-(1564-85), anti-(1718-31), 1ML1, and 18A10). All incubation was done at 35 °C. In the control column: control, lane 1-4; untreated, lanes 1 and 3; incubation for 20 min without trypsin, lane 2; added 50 µg/ml trypsin inhibitor and 0.1 mM PMSF prior to incubation with 5 µg/ml of trypsin for 20 min, lane 4. In the other columns (lanes 5-32), undigested (lanes 5, 12, 19, and 26), incubation with trypsin at the concentration of 0.2 (lanes 6-11), 1 (lanes 13-18), 5 (lanes 20-25), and 20 (lanes 27-32) µg/ml for 1 (lane 6, 13, 19, and 26), 2 (lanes 7, 14, 21, and 28), 4 (lanes 8, 15, 22, and 29), 6 (lanes 9, 16, 23, and 30), 10 (lanes 10, 17, 24, and 31), and 20 (lanes 11, 18, 25, and 32) min. Tryptic fragments are indicated with arrows. Nonspecific bands are shown with asterisks. Molecular size markers in kDa are on the left.

Microsomal fractions from mouse cerebellum were digested at 35 °C for 0, 1, 2, 4, 6, 10, and 20 min with graded concentrations of trypsin (0.2, 1, 5, and 20 µg/ml). Trypsin-resistant fragments were immunodetected in Western blots, using site-specific antibodies (Fig. 2). No degradation of the intact mIP3R1 band of ~250 kDa was observed by incubating without trypsin (lane 2) and with trypsin after pretreatment with protease inhibitors (lane 4), indicating no traces of endogenous protease activity in the reaction mixtures, and that pretreatment with protease inhibitors could completely block exogenous trypsin activity. In contrast, digestion even with the lowest concentration of trypsin used led to a rapid disappearance of the intact band and concomitant appearance of tryptic products, which were probed with each antibody (lanes 5-32).

The fragmentation patterns probed by N1 and N2 were almost the same except for nonspecific bands: 32 kDa with the N1, 72/70 kDa and its derivative 64/62 kDa with the N2 (with asterisks in Fig. 2). A 37-kDa band was immunodetected as the major and the smallest product with both pAbs. A 40-kDa band was faint with a similar digestion profile as the 37-kDa band. We assumed that the difference in size and intensity between these two bands resulted from a splicing variation in the SI region (15 residues, 318-332), since the minus type (SI-) and the plus type (SI+), respectively, made up about 85 and 15% of the mIP3R1 mRNAs from adult mouse cerebella (12) which appeared to correspond to the 37- and 40-kDa bands, respectively. We designated the 40- and 37-kDa bands as fragments Ia and Ib, respectively.

The immunoblotting patterns probed with N3, N4, and N5 and 4C11 were much the same and were characteristic of a 64-kDa major band, designated as fragment II, which predominantly avoided attack by highest concentrations of trypsin.

A doublet of 105/100-kDa band was detected using antibodies N1, N2, N3, N4, N5, and 4C11. We assumed that the 105/100-kDa doublet was further digested into two fragments, at a site flanked by the N2 and N3 epitopes. Fragment Ia/b (40/37 kDa) was recognized with N1 and N2 and fragment II (64 kDa) was recognized with N3, N4, N5, and 4C11. Fragment Ia/b and II were thus lined up close to each other, as depicted in Fig. 1.

Intensive digestion at 20 µg/ml trypsin gave rise to a 29-kDa sub-band immunoreactive to the N5 (Fig. 2, lanes 29-32), which was also detected with the N3 and N4 with longer exposure to an ECL film (data not shown). With the same digestion, a 38-kDa sub-band reacted with the 4C11. The digestion profile of both bands was much the same; the longer the digestion time at 20 µg/ml trypsin was extended, the more the immunoreactivity became obvious. From these data, we assumed that fragment II contained a tryptic site susceptible to attack by such extensive trypsin digestion, between the N5 and 4C11 epitope, the result being generation of two sub-fragments of 29 and 38 kDa.

The banding patterns detected with the 10A6 and anti-(1564-85) were much the same except for the susceptibility to trypsin, and a band of 76 kDa, designated as fragment III, was the major tryptic product (Fig. 2). The anti-(1564-85) epitope in fragment III was considerably less stable than that of the 10A6 with prolonged digestion to even 1 µg/ml trypsin. We thus assumed that the C terminus of the fragment III was located near residue 1585, the C terminus of the anti-(1564-85) epitope.

There was a close resemblance in the immuno-intensity and trypsin-sensitivity profile in that there was a faint band of 140 kDa among blots probed with antibodies N3, N4, N5, 4C11, 10A6, and anti-(1564-85) (Fig. 2). We assumed that the 140-kDa band further split into two, and fragment II reacted with the N3, N4, N5, and 4C11 and fragment III with the 10A6 and anti-(1564-85). Thus, fragments II and III were likely contiguous (Fig. 1).

The anti-(1718-31) reacted weakly to two bands of 40 and 36 kDa, designated as fragments IVa and IVb, respectively. Fragment IVa was produced by digestion at a lower trypsin concentration (0.2 and 1 µg/ml), whereas fragment IVb was produced with a higher concentration (1 and 5 µg/ml). Anti-(1718-31) immunoreactivities to both fragments were digested with more extensive trypsinolysis. It is notable that these two fragments likely contain largely diverse stretches in the IP3R family as well as the alternative splicing SII region (40 residues, 1692-1731) (12, 13). In the adult mouse cerebellum, the mRNAs for the SIIB- (minus residue 1715), SIIBC- (minus 1715-1731), SII+, and SII- (minus 1692-1731) subtype were found to be produced in a relative ratio of 50, 26, 20, and 4%, respectively (12). Considering size and immuno-intensity, it was unlikely that both fragments were splicing variants because the anti-(1718-31), specific to the sub-segment C, reacted only with the SIIB- and SII+ subtypes, the difference is either absence (SIIB-) or presence (SII+) being only single Gln-1715 residue. We thus assumed that the difference between fragments IVa and IVb was related to proteolysis; however, the possibility of a difference in trypsin sensitivity among splicing variants would need to be ruled out.

A 116-kDa band was faintly observed using three distinct antibodies, the anti-(1564-85), anti-(1718-31) (Fig. 2), and with 10A6 in case of a longer exposure for immunodetection (data not shown). We assumed that this faint 116-kDa band was composed of the fragments III (76 kDa) and IVa/b (40/36 kDa) which were in close proximity, as shown in Fig. 1.

The blotting patterns probed by 1ML1 and 18A10 were much the same, and a 91-kDa band, designated as fragment V, was a major tryptic band (Fig. 2). Immunoreactivity of fragment V to the 18A10 was more susceptible to trypsinolysis than that to the 1ML1, indicating that the C-terminal tip, including the 18A10 epitope of the mIP3R1 is labile to trypsin attack.

The anti-(1718-31), 1ML1, and 18A10 (Fig. 2) displayed a similar blotting pattern of a 130-kDa band. We assumed that fragments IVa/b (40/36 kDa) and V (91 kDa) located within the vicinity and were composed of this 130-kDa band (Fig. 1).

Thus, we detected five major tryptic fragments: 40/37 (Ia/b), 64 (II), 76 (III), 40/36 (IVa/b), and 91 kDa (V); these seemed to be lined up on and to cover the entire cerebellar mIP3R1 (Fig. 1). The molecular mass of these major fragments was 305-314 kDa, a value close to the mass (313 kDa) deduced from the cloned mIP3R1.

Structural Coupling of All the Tryptic Fragments by Non-covalent Interactions-- Our assumption was that tryptic fragments I-IV contained most of the N-terminal cytoplasmic arm region, whereas fragment V consists of all the membrane-spanning segments and the C-terminal tail region. To determine if cytoplasmic peripheral fragments were released from the integral fragment-membrane complex upon trypsinolysis, we first carried out precipitation tests by centrifugation (cfg in Fig. 3) of microsomal fractions subjected to three different digestions: (-), treated with 1 µg/ml trypsin plus trypsin inhibitors for 2 min as a control; (+), with 1 µg/ml trypsin for 2 min; (++); with 5 µg/ml trypsin for 6 min. The resultant supernatants (cfg sup, lanes 4-6) and membrane pellets (cfg ppt, lanes 7-9) were probed by Western blotting using various antibodies (Fig. 3). The digestion (++) (lanes 3) readily removed the C-terminal 18A10 epitope but not the luminal 1ML1 one from the membrane-spanning fragment V, thus the 1ML1-immuno-positive band produced by the digestion (++) (lanes 3) was slightly shorter than that by the digestion (+) (lanes 2). Unexpectedly, in addition to the mIP3R1 which remained intact and the membrane-spanning fragment V, all cytoplasmic peripheral fragments I-IV produced were collected in the pellet fraction (lane 7-9). More extensive digestion (at 10 µg/ml trypsin for 40 min) released only about half the number of these peripheral fragments from the membrane (data not shown). These data suggest that fragments I-IV were somehow attached to or were associated with the membrane or formed an aggregate or complex that could be pelleted by the centrifugation.


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 3.   All tryptic fragments were co-immunoprecipitated with the C terminal-specific mAb 18A10. Tryptic microsomal fractions were analyzed by immunoprecipitation using the mAb 18A10. All trypsinizations were carried out at 35 °C and quenched by adding 50 µg/ml soybean trypsin inhibitor and 0.1 mM PMSF. Microsomal fractions were trypsinized under three conditions (total, lanes 1-3): -, added 50 µg/ml of trypsin inhibitor and 0.1 mM PMSF prior to incubation with 1 µg/ml trypsin for 2 min as a control; +, incubation with 1 µg/ml trypsin for 2 min; ++, incubation with 5 µg/ml trypsin for 6 min. The trypsinized microsomes were centrifuged at 105,000 × g for 60 min (cfg., lanes 4-9) to separate into soluble (sup, lanes 4-6) and insoluble membrane (ppt, lane 7-9) fractions. The insoluble fractions (cfg.-ppt) were solubilized with 1% Triton X-100 (Triton, lanes 10-15) and re-centrifuged to separate into Triton X-100 extract (sup, lanes 10-12) and insoluble (ppt, lanes 13-15) fractions. The Triton extracts (Triton-sup) were subjected to the immunoprecipitation experiments (IP). The immunoprecipitates with mAb 18A10 and normal rat IgG are shown in lane 16-18 (18A10) and 19-21 (IgG), respectively. Aliquots of each step were separated on 8% SDS-PAGE and subjected to Western blotting using the antibodies shown on the left shoulders (N1, N3, 10A6, anti-(1718-31), 1ML1, and 18A10). Asterisks indicate bands of rat and rabbit IgG used as primary and secondary antibodies for immunoprecipitation, respectively. The intact mIP3R1 and major tryptic fragments are marked by arrows. Molecular size markers in kDa are on the right.

All the tryptic fragments as well as the intact mIP3R1 precipitated were solubilized with 1% Triton X-100 (Triton in Fig. 3) and were largely separated into soluble fractions (sup, lanes 10-12) from insoluble (ppt, lanes 13-15) materials upon re-centrifugation.

To determine if each peripheral fragment I-IV was associated with the membrane directly or through the integral fragment V, we next performed immunoprecipitation experiments (IP) of the Triton extract with either the mAb 18A10 specific to the fragment V (18A10, lanes 16-18) or the normal rat IgG as controls (IgG, lanes 19-21). The intact mIP3R1 immunoprecipitated with the 18A10 cross-reacted with the other five site-specific antibodies (lanes 16). No significant immunoreactivity to these antibodies was precipitated with the normal IgG (lanes 19-21), except for bands of IgG used (asterisks in Fig. 3). In marked contrast, all five tryptic fragments produced by digestion (+) co-immunoprecipitated with the 18A10, together with fragment V, the only one containing its epitope (lane 17). These data ruled out the possibility that the cytoplasmic fragments formed aggregates or complexes to be pelleted by centrifugation; all five tryptic fragments seemed to be held together, or fragments I-IV were directly attached to or indirectly associated with the fragment V. These inter-fragment interactions were stable even after cleaving the peptide bonds off by trypsinization and stripping membranes off by Triton solubilization. No disulfide bond seemed to be involved in the inter-fragment interactions; there is a supportive piece of evidence that typsinized microsomes treated by either reducing or non-reducing SDS-PAGE sample buffer prior to SDS-PAGE application gave almost the same immunoblotting pattern (data not shown).

N-terminal Sequencing of Tryptic Fragments-- To determine tryptic cleavage sites, all the major tryptic fragments were purified at once from Triton extracts of trypsinized microsomal fractions, using immunoaffinity beads coupled to the 18A10. An SDS-PAGE pattern of the affinity purified proteins is shown in Fig. 4. We confirmed that the 91-, 76-, and 64-kDa bands thus purified were reactive with the 18A10 (specific to fragment V), 10A6 (fragment III), and N3 (fragment II), respectively (data not shown). The 40- and 37-kDa proteins purified reacted with both N1 and anti-(1718-31), indicating that fragments Ia/b and IVa/b overlapped (data not shown). The N-terminal pentapeptide sequences of these purified fragments were then determined (Table II), as described under "Experimental Procedures." With the 37-kDa doublet, including fragments Ib and IVb, only the sequence of the fragment IVb and not that of fragment Ib was readable. In addition, recovery of the phenylthiohydantoin-amino acids upon sequencing of the 37-kDa band was much lower than expected from the amount estimated by SDS-PAGE which meant a possible block of the N terminus of fragment Ib. With the purified 40-kDa doublet, including fragments Ia and IVa, three different kinds of sequences attributed to the N terminus of fragment IVa (not to that of fragment Ia) were determined. Similarly, the N terminus of fragment Ia also seemed to be blocked. SDS-PAGE analysis of purified tryptic fragments also showed that the darkness of stained bands corresponding to fragments II and III (and probably Ib too) was fairly comparable with that of the 18A10-reactive fragment V (Fig. 4), hence their interactions may be stoichiometric.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   Simultaneous purification of all the tryptic fragments by the C terminal-specific mAb 18A10-immunoaffinity chromatography. All five tryptic fragments were co-purified using the mAb 18A10-immobilized Sepharose column. Triton X-100 extracts of trypsinized microsomal fractions were applied on an affinity column. 20 µl of concentrated eluates was separated on 7.5% SDS-PAGE and stained by Coomassie Brilliant Blue R-250. The major five tryptic fragments are indicated on the right. Molecular size markers in kDa are on the left.

                              
View this table:
[in this window]
[in a new window]
 
Table II
N-terminal sequences of major tryptic fragments
N-terminal 5 amino acids of the major tryptic fragments of the mIP3R1 were determined as described under "Experimental Procedures."

Functional Coupling in IP3-induced Ca2+ Release from Trypsin-fragmented mIP3R1-- We next examined the relation between the structural fragmentation of mIP3R1 channel complex with trypsin and the functional property as an IP3-gated Ca2+ release channel. IP3-induced Ca2+ release (IICR) activity in trypsinized microsomes was measured by monitoring extramicrosomal [Ca2+], using the Ca2+ fluorescent dye Fura-2 and a spectrofluorometer. First, microsomes were digested for 4 min under three different conditions: -, with 5 µg/ml trypsin plus trypsin inhibitors as controls; +, 1 µg/ml trypsin; and ++, 5 µg/ml trypsin. Fragmentation of the mIP3R1 in the digested microsomes was confirmed by Western blotting (Fig. 5A). The mIP3R1 was almost completely split into five fragments upon digestion + and ++. The C-terminal tip including the 18A10 epitope was readily removed off upon the digestion ++, and the tips of the fragments III and IVa were also deleted by digestion ++. We confirmed that all these fragments produced by digestion + and ++ could be precipitated by centrifugation (data not shown).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   IP3-induced Ca2+ release from trypsin-fragmented mIP3R1. IP3-induced Ca2+ release activity from tryptic mIP3R1 was measured using Ca2+ fluorescent indicator dye Fura 2 and a spectrofluorometer. All the trypsin digestions were carried out at 35 °C and stopped by adding soybean trypsin inhibitor (to 50 µg/ml) and PMSF (to 0.1 mM). A, microsomal fractions (C) (1 mg of protein/ml) were trypsinized in Ca2+ releasing buffer for 4 min: Trypsin(-), added 50 µg/ml of trypsin inhibitor and 0.1 mM PMSF prior to incubation with 5 µg/ml of trypsin; Trypsin(+), incubation with 1 µg/ml of trypsin; Trypsin(++), incubation with 5 µg/ml of trypsin. 2.5 µg of proteins were separated on 8% SDS-PAGE and immunodetected with the antibodies shown on the top (N1, N3, 10A6, anti-(1718-31), 1ML1 and 18A10). The intact receptor and tryptic fragments are indicated by arrows. B, 200 µl of the trypsinized microsomes (200 µg of protein) was added to 300 µl of Ca2+ releasing buffer and supplemented with creatine phosphate (to 10 mM), creatine kinase (20 units/ml), oligomycin (1 µg/ml), and Fura 2 (2 µM). Trypsin(-) and -(++) represent the samples prepared in A. Then, the mixture was subjected to Ca2+ release assay. Extramicrosomal [Ca2+] was measured by monitoring Fura 2 fluorescence, using a spectrofluorometer. ATP (2 mM) was added to load Ca2+ into microsomal vesicles. In all the experiments, IP3 and ionophore 4-Bromo A23187 were added, when the basal [Ca2+] settled down to ~220 nM, i.e. IP3 (1 µM) was added to induce Ca2+ release, after settling down again to ~220 nM [Ca2+], A23187 (4 µM) was added to measure Ca2+ content releasable from the vesicles. Additional application of A23187 (2 µM) showed no effect (data not shown). C, dose-response relation between IP3 concentration and IICR activity. IICR activities in response to 30 nM, 100 nM, 300 nM, 1 µM, 10 µM, and 30 µM IP3 were measured. Trypsin(-), -(+), and -(++) indicate the same samples as described in A, and changes of Fura 2-fluorescence by application of these amounts of IP3 were measured as in B. IICR activity was expressed as the value (Delta [Ca2+]) given by subtracting the basal [Ca2+] (~220 nM) from peak heights of [Ca2+] released by IP3.

The trypsinized microsomes were made to load Ca2+ through the activation of sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA) by adding 2 mM ATP. Fig. 5B shows representative findings of ATP-induced Ca2+ uptake (AICU) followed by IICR, from the un-fragmented (Trypsin(-)) and completely fragmented (Trypsin(++)) mIP3R1 channels. IP3 (1 µM) and Ca2+ ionophore A23187 (4 µM) were added when the [Ca2+] was ~220 nM. Trypsin (++) microsomes showed no significant reduction in A23187-releasable Ca2+ store size (estimated from A23187-induced Ca2+ release; AICR) as well as AICU activity, a finding consistent with data that the SERCA/Ca2+ pump in the SR is relatively resistant to limited trypsinolysis (34). However, as the digestion time was extended, the active AICU activity tended to be attenuated, since it took longer to settle the [Ca2+] level to ~220 nM in trypsin(++) microsomes than in trypsin(-) microsomes (Fig. 5B). It is noteworthy that IICR from completely fragmented mIP3R1-channels in trypsin(++) was in no way inferior to that from the unfragmented ones trypsin(-) (Fig. 5B).

Dose-response relations between IP3 concentrations and IICR activities showed that the fragmented mIP3R1-channel in both trypsin(+) and trypsin(++) microsomes responded in an IP3 dose-dependent manner (Fig. 5C). There was a slight augmentation of maximum release in the trypsin(+). The dose-response curve in the trypsin-(++) shifted slightly to the right, that is a slight reduction in the IP3 sensitivity but no change in the maximum release.

Trypsin-insensitive mIP3R1 Is a Non-functional Channel-- Even after extended trypsinolysis, a trace but definite amount of the mIP3R1 band that was almost of the same size as the intact one was detected in case of a longer exposure for immunodetection. To rule out the possibility that IICR activity measured in this study was related to undigested mIP3R1, we analyzed the temporal profile of IICR in relation to digestion time (Fig. 6). Microsomes in the complete Ca2+ releasing buffer were directly exposed to trypsin digestion (at 5 µg/ml) for various times, 1 min after initiating AICU. The trypsin digestion was quenched by directly adding trypsin inhibitor (at 50 µg/ml) to the cuvette, and then measurements of IICR and AICR were made. 10 µM IP3 was added to obtain the maximum IICR activity. Fig. 6A shows a temporal profile of extra-microsomal [Ca2+] change in one session with 40 min digestion. As shown in Fig. 6B, prolonged digestion led to a concomitant decline in IICR activity. Microsomes digested for 60 min exhibited no significant response to 10 µM IP3 and to 30 µM IP3 successively added (asterisk in 60 min of Fig. 6B). The attenuation of IICR was unlikely related to bleaching of Fura 2 due to prolonged exposure to excitation light, because significant IICR activity remained detectable in the control microsome (without fragmentation) exposed to the light for the same period (right upper trace of Fig. 6B). In contrast to the attenuation of IICR activity, AICU activity was retained even after prolonged digestion; rather, higher AICR activity was observed, because the SERCA seemed to be fairly resistant to this degree of trypsinolysis, thus more Ca2+ was sequestered by a longer incubation time up to the application of A23187 (Fig. 6B). Small aliquots of the releasing mixtures at various times were withdrawn and subjected to Western blotting analysis with the 4C11 and 18A10. Fig. 6C represents temporal profiles of digestion patterns after longer exposure for immunodetection of the undigested bands. A decline in the IICR activity was nearly parallel to the remaining amounts of the immunoreactive fragment II but not those of the C-terminal 18A10 epitope on the fragment V, because of differences in susceptibility to trypsinolysis. In contrast, immunoreactivity of the undigested bands was little changed in the course of digestion, suggesting that only a particular fraction of mIP3R1 displayed this lack of susceptibility to trypsinolysis. From this constant level of the undigested band, changes in IICR activity with prolonged digestion could not to be explained. Thus, the undigested mIP3R1 proteins might be not only trypsin-tolerant but non-functional due to malfolding or aberrant configuration (e.g. inside-out transmembrane topology), possibly related to sample preparations.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of prolonged trypsinization on IP3-induced Ca2+ release. Microsomal fractions (200 µg of protein) were suspended in 0.5 ml of the Ca2+ releasing buffer supplemented with creatine phosphate (10 mM), creatine kinase (20 units/ml), oligomycin (1 µg/ml), and Fura-2 (2 µM). The microsome suspension in a measuring cuvette was continually stirred and maintained at 30 °C. About 1 min after the addition of ATP (2 mM), trypsin (5 µg/ml) was directly added to the cuvette. After 4, 20, 40, or 60 min, the reaction was quenched by the addition of soybean trypsin inhibitor (50 µg/ml), and then,10 µM IP3 was added to measure the IP3-induced Ca2+ release. Finally, when basal [Ca2+] was recovered again at the same level as when IP3 was added, 4 µM A23187 was applied to determine the A23187-induced Ca2+ release. A, the trace represents change in extramicrosomal [Ca2+] upon 40-min digestion, 10 µM IP3 and 4 µM A23187 additions. B, 10 µM IP3 (upper trace) and 4 µM A23187 (lower trace) induced [Ca2+] change after 0-, 4-, 20-, 40-, or 60-min trypsin digestions. As controls (right traces), IP3- and A23187-induced Ca2+ release were measured after a 60-min incubation with both trypsin and trypsin inhibitor. Arrows indicate time points of addition of IP3 or A23187. In the 60-min digestion trace, a second application of IP3 (30 µM) carried out was shown (arrow with asterisk). The [Ca2+] at which IP3 and A23187 were applied was appropriately fit: ~207, ~224, ~185, ~146, ~137, and ~85.7 nM [Ca2+] in the 0-, 4-, 20-, 40-, 60-min trypsin digestion and the 60-min control with trypsin plus its inhibitor, respectively. The calibration bars are as follows: x axis, 60 s for all traces; y axis, 50 and 200 nM for IP3 and A23187-induced [Ca2+] changes, respectively. C, small aliquots (2.5 µg of protein) in the above measurements were removed and subjected to Western blotting probed with the 4C11 and 18A10. These ECL films were subjected to longer exposure (5-10 min) on immunodetection to detect the undigested full-length mIP3R1 band, although films for detection of the other immuno-positive bands were used to be exposed only for 3-15 s.

Inhibition of IICR by the C Terminus-specific mAb 18A10 Was Non-effective When the Epitope Was Removed by Mild Trypsin Digestion-- As shown in Figs. 3, 5A, and 6C, the C-terminal 18A10 epitope could be easily removed from fragment V by relatively mild trypsin digestion. We could measure the apparent IICR activity in such a fragmented mIP3R1 channel as it lacked the 18A10 epitope (Figs. 5 and 6). 18A10 functioned as a specific inhibitor for mIP3R1-mediated IICR (19, 35). Fig. 7 shows that the 18A10 inhibited the IICR activity in the intact mIP3R1 by 60% (Trypsin(-)). As expected, the inhibitory effect was reduced, depending on trypsin digestion, and was abolished by completely removing 18A10 immunoreactivity (Trypsin(++)).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Inhibition of IICR by the C terminal-specific mAb 18A10. Microsomal fractions were treated with trypsin under the same three conditions as described in Fig. 5A. Trypsinized microsomal fraction (200 µg of protein) was incubated with 50 µg/ml of the 18A10, normal rat IgG (NRG), or no IgG (-) in 0.5 ml of the Ca2+ releasing buffer for 30 min at 4 °C. Then the mixture, supplemented with creatine phosphate (10 mM), creatine kinase (20 units/ml), oligomycin (1 µg/ml), and Fura-2 (2 µM), was subjected the Ca2+ release assay. The IICR activities in response to 100 nM IP3 were measured. Procedures for the Fura-2-fluorescence measurement of extra-microsomal [Ca2+] changes were otherwise the same as described in Fig. 5B.


    DISCUSSION

We provided evidence that (i) the native mIP3R1 consists of five major fragments I-V resistant to limited trypsin digestion, which means that there are four most exposed or disordered regions of the polypeptide backbone, all highly susceptible to the trypsinolysis and that there are five well folded structural components; (ii) all the cytoplasmic peripheral fragments I-IV are directly and/or indirectly associated with the membrane-spanning integral fragment V in a non-covalent manner; (iii) such completely fragmented mIP3R1 retains IICR activity comparable to that of the intact one, indicating the tight structural-functional coupling of these five split fragments; (iv) the inhibition of IICR by binding of the mAb 18A10 to the C terminus is due to physical interference of mIP3R1 channel gating (or coupling) structure, since the C terminus itself is not indispensable to IICR.

Limited proteolysis provides direct evidence of protein folding; regions accessible to proteases occur in extended linker regions or loops often exposed on the surface of the protein between tightly folded domains and which would be expected to be almost entirely resistant to attack by low concentrations of proteases (36). Trypsin sensitivity of the mIP3R1 channel noted in the present study would reflect the overall conformation of the native one. Trypsin cleaves the carboxyl side of arginyl and lysyl bonds. Although the mIP3R1 has 321 residues of Arg and Lys that are scattered over the primary structure of 2749 amino acids, substantially only four sites/regions are highly sensitive to limited trypsinolysis, generating five major trypsin-resistant fragments. With prolonged incubation time or higher trypsin concentrations, fragment IV was readily proteolyzed, but the other fragments remained fairly stable, except that the C-terminal tips of the fragment III and V were labile. These results suggest that fragments I, II, III, and V would be tightly folded, whereas fragment IV would be relatively relaxed. Judging from size and position, the fragments II and V appear to be equivalent to the 68- and 94-kDa fragments, respectively, as defined by Joseph et al. (29).

All members of the IP3R family consist of long stretches with extensive homology separated by short stretches with a characteristic divergency (37). Interestingly, all four trypsin hypersensitive regions were found to be localized in these variable regions. These regions would be flexible loops between the trypsin-resistant and folded structures. It should be noted that two hypersensitive sites, Arg-343/Arg-345 and Arg-922/Arg-937, are close to the alternative splicing regions SI (10, 12) and SIII (9 residues, NNDVEKLKS, inserted between Gly-917 and Ser-918) (14), respectively. Near the SIII, accessibility of trypsin would also be suggestive, since there is a potential site for protein kinase C (KLKS) (14).

The IP3-binding core stretches over two split fragments I and II. Both fragments have a relatively tight association and would cooperate for ligand binding.3 With further extensive digestion, fragment II further cut into at least two parts, N-terminal 29-kDa and C-terminal 38-kDa subfragments. Based on size and surface probability (predicted using a computer program), the carboxyl side of Arg-603 and/or Lys-604 are the most plausible sites for this second trypsinization in fragment II, which are likely to be in the vicinity of the C-terminal boundary of the defined IP3-binding core (25).

Fragment IV contains Ser-1587 and Ser-1755 phosphorylated by protein kinase A and protein kinase G (13, 38), supporting our view it would be a relatively relaxed structure. CaM binds residues 1564-1585 (33). As trypsin cut nearby, the carboxyl side of Arg-1581, Arg-1585, or Arg-1586, these residues are probably positioned on the surface. The immunophilin FK506-binding protein 12 (FKBP12) binds to a Leu-Pro pair (residues 1400-1401) in fragment III (39). The region surrounding this site, however, was not so susceptible to trypsin digestion. It might be that the mIP3R1 prepared under the present experimental conditions attached FKBP12, which covered tryptic site(s) such as Lys-1395 and Arg-1407 near the binding site. Similarly, it is impossible to rule out that binding of unidentified auxiliary protein(s) to the mIP3R1 may occlude tryptic sites, resulting in the fragmentation patterns we observed.

All of four cytoplasmic fragments I-IV were directly and/or indirectly associated with the membrane-spanning fragment V in a non-covalent manner. It has been reported about many proteins that split complemental fragments of a protein, generated by limited proteolysis, can be associated with each other to form a functional structure as does the intact protein (40). These inter-fragment interactions probably contribute to folding, stability, and functions of native proteins in general. We propose that the native mIP3R1 is composed of four subunits each of which is a non-covalent and tight assembly of five well folded components I-V, corresponding to the tryptic fragments I-V, thus the mIP3R1-channel complex forms a compact structure. This view is supported by electron microscopic studies that a single native IP3R1 complex forms a compact square-shaped conformation (41, 42).

The IP3R channel functions like an IP3/Ca2+ signal converter (2), i.e. IP3 binds to the N-terminal ligand-binding region, the information of which transduced to opening of the C-terminal Ca2+ channel gate, leading to release into the cytoplasm of another second messenger Ca2+ from stores. Mignery and Südhof (24) reported that the N-terminal 1803 residues molecularly expressed displayed a mobility shift (apparent decrease in mass of >50 kDa) on gel filtration chromatography in the presence of IP3, suggesting a conformational change in the cytoplasmic region upon ligand binding. Joseph et al. (29), however, reported that the preincubation of cerebellar microsomes with IP3 does not affect the proteolytic pattern of the IP3R1, determined using two different anti-IP3R1 antibodies, the epitopes of which are the C terminus and residues 401-414. This means that a large conformational change probably does not occur at least within the components II and V (the 68- and 94-kDa fragments in their report). We found that the trypsin-fragmented mIP3R1 retains IICR activity and that functional coupling probably occurs through non-covalent interactions in these five structural components, independently of peptide linkages connecting them. Therefore, the gating signal triggered by IP3 binding may (i) change in relative position of each component including the binding components (I and II) and the channel component (V), and/or (ii) a series of propagation of conformational changes from one component to the next through interfaces, in which case the change would be delicate regarding II and V at least. We asked whether fragments I-IV were released into the soluble fraction by such putative conformational changes triggered by IP3 binding. However, either in the presence or the absence of 1 µM IP3, all the fragments were precipitated by centrifugation and immunoprecipitated by the 18A10 (data not shown). The function of the mIP3R1 channel is well regulated by a variety of modulatory systems as follows: phosphorylation with various protein kinases, binding to various modulators such as Ca2+, ATP, CaM, and FKBP12, etc. The functional sites for these modulatory systems are scattered chiefly in components III, IVa, and V (Fig. 1). We think that some aspects in these modulations would be related with subtle adjustments of the inter-component interactions, leading to alteration in configuration of the mIP3R1 channel and then in the structural-functional coupling level. The IICR activities measured in the present study reflect not only the mIP3R1 itself but also other molecules such as the SERCA/Ca2+ pump, the IP3 metabolic enzymes such as IP3 5-phosphatase and IP3 3-kinase, and IP3R1 modulatory proteins such as CaM and FKBP12, etc. Possible involvement(s) of changes of these molecules by trypsinolysis in IICR activity would need to be ruled out.

IP3R shares a few fragmentary homologies with another intracellular Ca2+ release channel, ryanodine receptor (RyR) (9, 43), that is involved in Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (44, 45). The structural similarity probably relates to functional similarities as intracellular Ca2+ release channels are common to the two families. A series of limited proteolyses of the skeletal muscle RyR have been carried out with trypsin (34, 46-48) and calpain (49-52). The proteolytic fragments were also tightly associated in a non-covalent manner, and the fragmented RyR channel retained CICR activity. These findings suggest that these interactions among the folding components resistant to proteolysis play an important role in formation of functional configuration, as the intracellular Ca2+ release channel. Whether there is any similarity in structural-functional coupling for the ligand-gating mechanism between the IP3R-IICR channels and the RyR-CICR channels remains to be determined, although specific ligands differ between them (IP3 versus Ca2+). In the case of IP3R the IP3 ligand-binding site is far from the channel region in the primary sequence (although the distance in the tertiary structure is yet unclear), whereas in the case of RyR the region for Ca2+ activation is close to the channel region (53-55), and both regions locate in the same proteolytic fragment (49, 56). However, it is well known that the liganding IP3 is essential but not sufficient for the gating of the IP3R channel, and the concomitant action of cytosolic Ca2+ at stimulatory levels is requisite for it as "co-agonist." It should be noted that the position of the cytoplasmic Ca2+ site for IP3R, determined by 45Ca2+ overlay experiment (57) (Fig. 1), is topographically equivalent to that of the Ca2+ activation site for RyR.

    ACKNOWLEDGEMENTS

We thank T. Iizuka for technical support in preparing rabbit antisera; A. Takahashi for preparing mAbs; E. Majima for amino acid sequencing analyses; H. Mizuno, R. Ando, and C. Fujiwara-Hirashima for Ca2+ release experiments; A. Mizutani for fruitful discussions; K. Yoshikawa for preparation of manuscript; and M. Ohara for proofreading.

    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, Dept. 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 T. Michikawa and T. Furuichi, unpublished data.

3 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; RyR, ryanodine receptor; IP3R1, type 1 inositol 1,4,5-trisphosphate receptor; mIP3R1, mouse type 1 inositol 1,4,5-trisphosphate receptor; IICR, IP3-induced Ca2+ release; pAb, polyclonal antibody; mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; [Ca2+], Ca2+ concentration; AICU, ATP-induced Ca2+ uptake; AICR, A23187-induced Ca2+ release; SERCA, sarcoplasmic endoplasmic reticulum Ca2+-ATPase; FKBP12, FK506-binding protein 12; CaM, calmodulin; CICR, Ca2+-induced Ca2+ release..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  2. Furuichi, T., and Mikoshiba, K. (1995) J. Neurochem. 64, 953-960[Medline] [Order article via Infotrieve]
  3. Furuichi, T., Khoda, K., Miyawaki, A., and Mikoshiba, K. (1994) Curr. Opin. Neurobiol. 4, 294-303[Medline] [Order article via Infotrieve]
  4. Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161[CrossRef][Medline] [Order article via Infotrieve]
  5. Maeda, N., Niinobe, M., Nakahira, K., and Mikoshiba, K. (1988) J. Neurochem. 51, 1724-1730[Medline] [Order article via Infotrieve]
  6. Supattapone, S., Worley, P. F., Baraban, J. M., and Snyder, S. H. (1988) J. Biol. Chem. 263, 1530-1534[Abstract/Free Full Text]
  7. Maeda, N., Niinobe, M., Inoue, Y., and Mikoshiba, K. (1989) Dev. Biol. 133, 67-76[Medline] [Order article via Infotrieve]
  8. Maeda, N., Niinobe, M., and Mikoshiba, K. (1990) EMBO J. 9, 61-67[Abstract]
  9. Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Nature 342, 32-38[CrossRef][Medline] [Order article via Infotrieve]
  10. Mignery, G. A., Newton, C. L., Archer, B. T., III, and Südhof, T. C. (1990) J. Biol. Chem. 265, 12679-12685[Abstract/Free Full Text]
  11. Yamada, N., Makino, Y., Clark, R. A., Pearson, D. W., Mattei, M.-G., Guénet, J.-L., Ohama, E., Fujino, I., Miyawaki, A., Furuichi, T., and Mikoshiba, K. (1994) Biochem. J. 302, 781-790[Medline] [Order article via Infotrieve]
  12. Nakagawa, T., Okano, H., Furuichi, T., Aruga, J., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6244-6248[Abstract]
  13. Danoff, S. K., Ferris, C. D., Donath, C., Fischer, G., Munemitsu, S., Ullrich, A., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2951-2955[Abstract]
  14. Nucifora, F. C., Jr., Li, S.-H., Danoff, S., Ullrich, A., and Ross, C. A. (1995) Mol. Brain Res. 32, 291-296[CrossRef][Medline] [Order article via Infotrieve]
  15. Furuichi, T., Simon-Chazottes, D., Fujino, I., Yamada, N., Hasegawa, M., Miyawaki, A., Yoshikawa, S., Guénet, J.-L., and Mikoshiba, K. (1993) Recept. Channels 1, 11-24[Medline] [Order article via Infotrieve]
  16. Yamamoto-Hino, M., Miyawaki, A., Kawano, H., Sugiyama, T., Furuichi, T., Hasegawa, M., and Mikoshiba, K. (1995) Neuroreport 6, 273-276[Medline] [Order article via Infotrieve]
  17. Maeda, N., Kawasaki, T., Nakade, S., Yokota, N., Taguchi, T., Kasai, M., and Mikoshiba, K. (1991) J. Biol. Chem. 266, 1109-1116[Abstract/Free Full Text]
  18. Ferris, C. D., Huganir, R. L., Supattapone, S., and Snyder, S. H. (1989) Nature 342, 87-89[CrossRef][Medline] [Order article via Infotrieve]
  19. Nakade, S., Rhee, S. K., Hamanaka, H., and Mikoshiba, K. (1994) J. Biol. Chem. 269, 6735-6742[Abstract/Free Full Text]
  20. Hirota, J., Michikawa, T., Miyawaki, A., Furuichi, T., Okura, I., and Mikoshiba, K. (1995) J. Biol. Chem. 270, 19046-19051[Abstract/Free Full Text]
  21. Miyawaki, A., Furuichi, T., Ryou, Y., Yoshikawa, S., Nakagawa, T., Saitoh, T., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4911-4915[Abstract]
  22. Miyawaki, A., Furuichi, T., Maeda, N., and Mikoshiba, K. (1990) Neuron 5, 11-18[CrossRef][Medline] [Order article via Infotrieve]
  23. Michikawa, T., Hamanaka, H., Otsu, H., Yamamoto, A., Miyawaki, A., Furuichi, T., Tashiro, Y., and Mikoshiba, K. (1994) J. Biol. Chem. 269, 9184-9189[Abstract/Free Full Text]
  24. Mignery, G. A., and Südhof, T. C. (1990) EMBO J. 9, 3893-3898[Abstract]
  25. Yoshikawa, F., Morita, M., Monkawa, T., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 18277-18284[Abstract/Free Full Text]
  26. Sayers, L. G., Miyawaki, A., Muto, A., Takeshima, H., Yamamoto, A., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1997) Biochem. J. 323, 273-280[Medline] [Order article via Infotrieve]
  27. Joseph, S. K., Boehning, D., Pierson, S., and Nicchitta, C. V. (1997) J. Biol. Chem. 272, 1579-1588[Abstract/Free Full Text]
  28. Magnusson, A., Haug, L. S., Walaas, I., and Østvold, A. C. (1993) FEBS Lett. 323, 229-232[CrossRef][Medline] [Order article via Infotrieve]
  29. Joseph, S. K., Pierson, S., and Samanta, S. (1995) Biochem. J. 307, 859-865[Medline] [Order article via Infotrieve]
  30. Ohmi, S., Tsujimura, K., and Inagaki, M. (eds) (1994) Cell Bio/Technology, Protocol for Preparation of Anti-peptide Antibody (in Japanese), Shujunsha, Tokyo
  31. Monkawa, T., Miyawaki, A., Sugiyama, T., Yoneshima, H., Yamamoto-Hino, M., Furuichi, T., Saruta, T., Hasegawa, M., and Mikoshiba, K. (1995) J. Biol. Chem. 270, 14700-14704[Abstract/Free Full Text]
  32. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract]
  33. Yamada, M., Miyawaki, A., Saito, K., Nakajima, T., Yamamoto-Hino, M., Ryo, Y., Furuichi, T., and Mikoshiba, K. (1995) Biochem. J. 308, 83-88[Medline] [Order article via Infotrieve]
  34. Shoshan-Barmatz, V., and Zarka, A. (1988) J. Biol. Chem. 263, 16772-16779[Abstract/Free Full Text]
  35. Nakade, S., Maeda, N., and Mikoshiba, K. (1991) Biochem. J. 277, 125-131[Medline] [Order article via Infotrieve]
  36. Price, N. C., and Johnson, C. M. (1989) in Proteolytic Enzymes: A Practical Approach (Beynon, R. J., and Bonds, J. S., eds), pp. 163-180, IRL Press at Oxford University Press, Oxford
  37. Maranto, A. R. (1994) J. Biol. Chem. 269, 1222-1230[Abstract/Free Full Text]
  38. Komalavilas, P., and Lincoln, T. M. (1994) J. Biol. Chem. 269, 8701-8707[Abstract/Free Full Text]
  39. Cameron, A. M., Nucifora, F. C., Jr., Fung, E. T., Livingston, D. J., Aldape, R. A., Ross, C. A., and Snyder, S. H. (1997) J. Biol. Chem. 272, 27582-27588[Abstract/Free Full Text]
  40. Taniuchi, H., Parr, G. R., and Juillerat, M. A. (1986) Methods Enzymol. 131, 185-217[Medline] [Order article via Infotrieve]
  41. Takei, K., Mignery, G. A., Mugnaini, E., Südhof, T. C., and De Camilli, P. (1994) Neuron 12, 327-342[Medline] [Order article via Infotrieve]
  42. Katayama, E., Funahashi, H., Michikawa, T., Shiraishi, T., Ikemoto, T., Iino, M., Hirosawa, K., and Mikoshiba, K. (1996) EMBO J. 15, 4844-4851[Abstract]
  43. Mignery, G. A., Südhof, T. C., Takei, K., and De Camilli, P. (1989) Nature 342, 192-195[CrossRef][Medline] [Order article via Infotrieve]
  44. McPherson, P. S., and Campbell, K. P. (1993) J. Biol. Chem. 268, 13765-13768[Free Full Text]
  45. Meissner, G. (1994) Annu. Rev. Physiol. 56, 485-508[CrossRef][Medline] [Order article via Infotrieve]
  46. Chu, A., Sumbilla, C., Scales, D., Piazza, A., and Inesi, G. (1988) Biochemistry 27, 2827-2833[Medline] [Order article via Infotrieve]
  47. Meissner, G., Rousseau, E., and Lai, F. A. (1989) J. Biol. Chem. 264, 1715-1722[Abstract/Free Full Text]
  48. Chen, S. R. W., Airey, J. A., and McLennan, D. H. (1993) J. Biol. Chem. 268, 22642-22649[Abstract/Free Full Text]
  49. Callway, C., Seryshev, A., Wang, J.-P., Slavik, K. J., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884[Abstract/Free Full Text]
  50. Gilchrist, J. S. C., Wang, K. K. W., Katz, S., and Belcastro, A. N. (1992) J. Biol. Chem. 267, 20857-20865[Abstract/Free Full Text]
  51. Brandt, N. R., Caswell, A. H., Brandt, T., Brew, K., and Mellgren, R. L. (1992) J. Membr. Biol. 127, 35-47[Medline] [Order article via Infotrieve]
  52. Rardon, D. P., Cefali, D. C., Mitchell, R. D., Seiler, S. M., and Jones, L. R. (1990) Circ. Res. 67, 84-96[Abstract]
  53. Chen, S. R. W., Zhang, L., and McLennan, D. H. (1993) J. Biol. Chem. 268, 13414-13421[Abstract/Free Full Text]
  54. Bhat, M. B., Zhao, J. Y., Takeshima, H., and Ma, J. (1997) Biophys. J. 73, 1329-1336[Abstract]
  55. Bhat, M. B., Zhao, J. Y., Zang, W., Balke, C. W., Takeshima, H., Wier, W. G., and Ma, J. (1997) J. Gen. Physiol. 110, 749-762[Abstract/Free Full Text]
  56. Marks, A. R., Fleisher, S., and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149[Abstract/Free Full Text]
  57. Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.