Trypsinized Cerebellar Inositol 1,4,5-Trisphosphate Receptor

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 IP3binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.

The type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP 3 )-gated Ca 2؉ release channel (calculated molecular mass ‫؍‬ ϳ313 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 IP 3 R1. Mouse IP 3 R1 (mIP 3 R1) was trypsinized into five major fragments (I-V) that were positioned on the entire mIP 3 R1 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 Nterminal cytoplasmic region where the IP 3 -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 mIP 3 R1-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. Ca 2؉ release experiments further revealed that even the completely fragmented mIP 3 R1 retained significant IP 3 -induced Ca 2؉ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP 3 -induced Ca 2؉ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP 3 R1, that is neighboring coupling between components I and II for IP 3

binding and long-distant coupling between the IP 3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.
Extracellular stimuli can activate hydrolysis of phosphatidylinositol 4,5-bisphosphate, a component of the plasma mem-brane, the result being production of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP 3 ) 1 (1). IP 3 diffuses into the cytoplasm and mediates the release of Ca 2ϩ from intracellular Ca 2ϩ storage organella, chiefly the endoplasmic reticulum, by binding to its receptor (IP 3 R). IP 3 R is a tetrameric intracellular IP 3 -gated Ca 2ϩ release channel (2). Molecular cloning has revealed that there are at least three distinct types of IP 3 R in mammals (3).
The cloned mouse IP 3 R1 (mIP 3 R1) itself was found to encode a complete IP 3 -operated Ca 2ϩ release channel as well as an IP 3 -binding receptor (21,22). We proposed that the mIP 3 R1 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 mIP 3 R1 binds IP 3 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 IP 3 binding core (25). The C-terminal membrane-spanning region, the most conserved region within the IP 3 R family, would form an ion channel. It was also shown that at least the Cterminal 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 IP 3 R1 channel would gate its channel embedded in Ca 2ϩ store membrane, apart from ϳ1,700 amino acids in the primary sequence. However, little is known of mechanisms underlying structural and functional coupling between IP 3 binding and channel opening.
The cerebellar IP 3 R1 was degradated by Ca 2ϩ -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 IP 3 R1 with limited trypsinolysis of cerebellar microsomes. They identified two trypsin-resistant bands of 68 and 94 kDa which, respectively, contained IP 3 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 noncovalent interactions among these tryptic fragments would affect retention of structural and functional coupling between IP 3 binding and channel opening.
We have now examined structure-function relationships of mIP 3 R1 by limited trypsinization of mouse cerebellar membrane fractions. We found that native mIP 3 R1 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 mIP 3 R1 channels retained significant IP 3 -induced Ca 2ϩ release (IICR) activity. We suggest that the native mIP 3 R1 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.
Preparation of Anti-peptide Antibodies-Peptides corresponding to residues 40 -55, 257-274, 560 -576, and 590 -604 of the mIP 3 R1 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-Nhydroxysuccinimide 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 peptideconjugated 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 M pepstatin A, and 10 M leupeptin)) and homogenized in a glass Teflon Potter homogenizer with 10 strokes at 850 rpm on ice. The homogenate was centrifuged at 1,000 ϫ 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 ϫ 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 ϳ15 mg/ml protein. Protein concentra-tions 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 Ϫ80°C until use. For Ca 2ϩ release experiments, the homogenate was centrifuged at 4,000 ϫ g for 20 min at 4°C. The supernatant was recentrifuged, under the same conditions. The second supernatant was then centrifuged at 105,000 ϫ g for 30 min at 2°C. The pellet was resuspended in Ca 2ϩ releasing buffer (110 mM KCl, 10 mM NaCl, 5 mM KH 2 PO 4 , 1 mM MgCl 2 , 1 mM DTT, and 10 mM Hepes-KOH, pH 7.2, at 24°C) and recentrifuged at 105,000 ϫ g for 30 min at 2°C to exchange the buffer. The resultant pellet was resuspended with the Ca 2ϩ releasing buffer to give a final concentration of ϳ10 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 Ca 2ϩ release experiments, microsomes (1 mg/ml protein) were digested by trypsin in Ca 2ϩ 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.
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 ϫ 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 l 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 ϫ 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 l of 1 mM EDTA, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0. 2.5 l of total (not fractionated) and cfg-sup, and 0.83 l of cfg-ppt, Triton-sup, and Triton-ppt were subjected to Western blotting. 100 l 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 g/ml soybean trypsin inhibitor, and protease inhibitor mixture I, and then precleared with 6 g/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 g/ml of monoclonal antibody (mAb) 18A10 against the mIP 3 R1 or normal rat IgG and for 60 min more with 6 g/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 ϫ 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 l of 1ϫ SDS-PAGE sample buffer for 30 min at 55°C. After removing detached Pansorbin particles by centrifugation, 5 l of the SDS-PAGE sample (IP-18A10 and IP-IgG) were analyzed by Western blotting.
Immunoaffinity Purification of Tryptic Fragments-500 g 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 g/ml trypsin for 2 min at 35°C. The reaction was quenched by adding 50 g/ml trypsin inhibitor and 0.1 mM PMSF. The trypsinized sample was centrifuged at 105,000 ϫ 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 g/ml trypsin inhibitor plus protease inhibitor mixture II (0.1 mM PMSF, 2 g/ml aprotinin, 5 M pepstatin A, and 1 M 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 ϫ g for 60 min at 4°C. The supernatant (ϳ15 ml) was diluted with 30 ml of purification buffer supplemented with 50 g/ml soybean trypsin inhib-itor 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 l 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 l of 1ϫ 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 l) 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 mIP 3 R1.
Ca 2ϩ Release Experiment-Changes in Ca 2ϩ concentration ([Ca 2ϩ ]) 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 [Ca 2ϩ ] was calculated as described (32), assuming a dissociation constant of 224 nM for Fura-2-Ca 2ϩ . Maximum and minimum values of R were obtained in the presence of an excess amount of CaCl 2 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 Ca 2ϩ releasing buffer were digested with trypsin, as described in the legends. Out of this trypsinized microsomal suspension, 200 l (200 g of protein) was taken, diluted to 500 l in a quartz glass cuvette with Ca 2ϩ releasing buffer, supplemented with creatine phosphate, creatine kinase, oligomycin, and Fura-2 to give a final concentration of 10 mM, 20 units/ml, 1 g/ml and 2 M, 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 [Ca 2ϩ ] were being carried out. Ca 2ϩ loading to microsomes was initiated with activation of Ca 2ϩ -ATPase by adding 2 mM ATP, and Ca 2ϩ release was triggered with activation of mIP 3 R1 by the addition of various amounts of IP 3 . Before closing each set of experiments, intramicrosomal Ca 2ϩ content was estimated by measuring ionophore 4-bromo A23187-induced Ca 2ϩ release. In a series of experiments, IP 3 and A23187 were added, when the basal [Ca 2ϩ ] was settled at ϳ220 nM.
In the experiments demonstrated in Fig. 6, direct trypsinization (at 5 g/ml trypsin for 4, 20, 40, and 60 min at 30°C) within measuring cuvettes was carried out ϳ1 min after the addition of 2 mM ATP, and the digestion was quenched by direct addition of 50 g/ml soybean trypsin inhibitor. After adding 10 M IP 3 , 4 M A23187 was applied when the [Ca 2ϩ ] was set at the concentration almost the same as when IP 3 was applied.

RESULTS
The mIP 3 R1 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 mIP 3 R1, 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 IP 3 -binding core (residue 226 -578) (25). Three monoclonal antibodies (mAbs) 4C11, 10A6, and 18A10 were raised against the purified cerebellar mIP 3 R1 (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 subsegment 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 mIP 3 R1 with an apparent mass of ϳ250 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 (ϳ320 kDa) comparable to that calculated from the open reading frame (17).
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 g/ml). Trypsin-resistant fragments were immunodetected in Western blots, using sitespecific antibodies (Fig. 2). No degradation of the intact mIP 3 R1 band of ϳ250 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 2 T. Michikawa and T. Furuichi, unpublished data. 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 (SIϪ) and the plus type (SIϩ), respectively, made up about 85 and 15% of the mIP 3 R1 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 g/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 g/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 g/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 g/ml), whereas fragment IVb was produced with a higher concentration (1 and 5 g/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 IP 3 R family as well as the alternative splicing SII region (40 residues, 1692-1731) (12,13). In the adult mouse cerebellum, the mRNAs for the SIIBϪ (minus residue 1715), SIIBCϪ (minus 1715-1731), SIIϩ, and SIIϪ (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 SIIϩ subtypes, the difference is either absence (SIIBϪ) or presence (SIIϩ) 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 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 mIP 3 R1 is labile to trypsin attack.
Structural Coupling of All the Tryptic Fragments by Noncovalent 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 membranespanning 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: (Ϫ), treated with 1 g/ml trypsin plus trypsin inhibitors for 2 min as a control; (ϩ), with 1 g/ml trypsin for 2 min; (ϩϩ); with 5 g/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 (ϩϩ) (lanes 3) readily removed the C-terminal 18A10 epitope but not the luminal 1ML1 one from the membrane-spanning fragment V, thus the 1ML1immuno-positive band produced by the digestion (ϩϩ) (lanes 3) was slightly shorter than that by the digestion (ϩ) (lanes 2). Unexpectedly, in addition to the mIP 3 R1 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 g/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.
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 mIP 3 R1 immunoprecipitated with the 18A10 cross-reacted with the other five sitespecific 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 (ϩ) 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 interfragment interactions; there is a supportive piece of evidence that typsinized microsomes treated by either reducing or nonreducing 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.
Functional Coupling in IP 3 -induced Ca 2ϩ Release from Trypsin-fragmented mIP 3 R1-We next examined the relation between the structural fragmentation of mIP 3 R1 channel complex with trypsin and the functional property as an IP 3 -gated Ca 2ϩ release channel. IP 3 -induced Ca 2ϩ release (IICR) activity in trypsinized microsomes was measured by monitoring extramicrosomal [Ca 2ϩ ], using the Ca 2ϩ fluorescent dye Fura-2 and a spectrofluorometer. First, microsomes were digested for 4 min under three different conditions: Ϫ, with 5 g/ml trypsin plus trypsin inhibitors as controls; ϩ, 1 g/ml trypsin; and ϩϩ, 5 g/ml trypsin. Fragmentation of the mIP 3 R1 in the digested microsomes was confirmed by Western blotting (Fig. 5A). The mIP 3 R1 was almost completely split into five fragments upon digestion ϩ and ϩϩ. The C-terminal tip including the 18A10 epitope was readily removed off upon the digestion ϩϩ, and the tips of the fragments III and IVa were also deleted by digestion ϩϩ. We confirmed that all these fragments produced by diges- 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 l 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. tion ϩ and ϩϩ could be precipitated by centrifugation (data not shown).
The trypsinized microsomes were made to load Ca 2ϩ through the activation of sarcoplasmic endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) by adding 2 mM ATP. Fig. 5B shows representative findings of ATP-induced Ca 2ϩ uptake (AICU) followed by IICR, from the un-fragmented (Trypsin(Ϫ)) and completely fragmented (Trypsin(ϩϩ)) mIP 3 R1 channels. IP 3 (1 M) and Ca 2ϩ ionophore A23187 (4 M) were added when the [Ca 2ϩ ] was ϳ220 nM. Trypsin (ϩϩ) microsomes showed no significant reduction in A23187-releasable Ca 2ϩ store size (estimated from A23187-induced Ca 2ϩ release; AICR) as well as AICU activity, a finding consistent with data that the SERCA/ Ca 2ϩ 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 [Ca 2ϩ ] level to ϳ220 nM in trypsin(ϩϩ) microsomes than in trypsin(Ϫ) microsomes (Fig. 5B). It is noteworthy that IICR from completely fragmented mIP 3 R1-channels in trypsin(ϩϩ) was in no way inferior to that from the 938 GGGFL Gly-6.9, Gly-6.1, Gly-6.6, Phe-6.8, Leu-6.9 IVa (40 kDa) Arg-1581 1582 NAARR Asn-6.2, Ala-7.4, Ala-7.8, Arg-7.9, Arg-7. Since amino acid sequence derived from Ia and Ib could not be detected, we considered the N termini to be blocked. c Phenylthiohydantoin. d Amino acids derived from two different peptides which probably overlapped are underlined. In this case, recovery of phenylthiohydantoin amino acid shows the total amount of the mixture.
FIG. 5. IP 3 -induced Ca 2؉ release from trypsin-fragmented mIP 3 R1. IP 3 -induced Ca 2ϩ release activity from tryptic mIP 3 R1 was measured using Ca 2ϩ 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 g/ml) and PMSF (to 0.1 mM). A, microsomal fractions (C) (1 mg of protein/ml) were trypsinized in Ca 2ϩ releasing buffer for 4 min: Trypsin(Ϫ), added 50 g/ml of trypsin inhibitor and 0.1 mM PMSF prior to incubation with 5 g/ml of trypsin; Trypsin(ϩ), incubation with 1 g/ml of trypsin; Trypsin(ϩϩ), incubation with 5 g/ml of trypsin. 2.5 g 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 l of the trypsinized microsomes (200 g of protein) was added to 300 l of Ca 2ϩ releasing buffer and supplemented with creatine phosphate (to 10 mM), creatine kinase (20 units/ml), oligomycin (1 g/ml), and Fura 2 (2 M). Trypsin(Ϫ) and -(ϩϩ) represent the samples prepared in A. Then, the mixture was subjected to Ca 2ϩ release assay. Extramicrosomal [Ca 2ϩ ] was measured by monitoring Fura 2 fluorescence, using a spectrofluorometer. ATP (2 mM) was added to load Ca 2ϩ into microsomal vesicles. In all the experiments, IP 3 and ionophore 4-Bromo A23187 were added, when the basal [Ca 2ϩ ] settled down to ϳ220 nM, i.e. IP 3 (1 M) was added to induce Ca 2ϩ release, after settling down again to ϳ220 nM [Ca 2ϩ ], A23187 (4 M) was added to measure Ca 2ϩ content releasable from the vesicles. Additional application of A23187 (2 M) showed no effect (data not shown). C, dose-response relation between IP 3 concentration and IICR activity. IICR activities in response to 30 nM, 100 nM, 300 nM, 1 M, 10 M, and 30 M IP 3 were measured. Trypsin(Ϫ), -(ϩ), and -(ϩϩ) indicate the same samples as described in A, and changes of Fura 2-fluorescence by application of these amounts of IP 3 were measured as in B. IICR activity was expressed as the value (⌬[Ca 2ϩ ]) given by subtracting the basal [Ca 2ϩ ] (ϳ220 nM) from peak heights of [Ca 2ϩ ] released by IP 3 .
Dose-response relations between IP 3 concentrations and IICR activities showed that the fragmented mIP 3 R1-channel in both trypsin(ϩ) and trypsin(ϩϩ) microsomes responded in an IP 3 dose-dependent manner (Fig. 5C). There was a slight augmentation of maximum release in the trypsin(ϩ). The doseresponse curve in the trypsin-(ϩϩ) shifted slightly to the right, that is a slight reduction in the IP 3 sensitivity but no change in the maximum release.
Trypsin-insensitive mIP 3 R1 Is a Non-functional Channel-Even after extended trypsinolysis, a trace but definite amount of the mIP 3 R1 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 mIP 3 R1, we analyzed the temporal profile of IICR in relation to digestion time (Fig. 6). Microsomes in the complete Ca 2ϩ releasing buffer were directly exposed to trypsin digestion (at 5 g/ml) for various times, 1 min after initiating AICU. The trypsin digestion was quenched by directly adding trypsin inhibitor (at 50 g/ml) to the cuvette, and then measurements of IICR and AICR were made. 10 M IP 3 was added to obtain the maximum IICR activity. Fig. 6A shows a temporal profile of extra-microsomal [Ca 2ϩ ] 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 M IP 3 and to 30 M IP 3 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 Ca 2ϩ 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 mIP 3 R1 displayed this lack of susceptibility to trypsinolysis. From this constant level of the undi- ] changes, respectively. C, small aliquots (2.5 g 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 mIP 3 R1 band, although films for detection of the other immuno-positive bands were used to be exposed only for 3-15 s. gested band, changes in IICR activity with prolonged digestion could not to be explained. Thus, the undigested mIP 3 R1 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.
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 Cterminal 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 mIP 3 R1 channel as it lacked the 18A10 epitope (Figs. 5 and 6). 18A10 functioned as a specific inhibitor for mIP 3 R1-mediated IICR (19,35). Fig.  7 shows that the 18A10 inhibited the IICR activity in the intact mIP 3 R1 by 60% (Trypsin(Ϫ)). As expected, the inhibitory effect was reduced, depending on trypsin digestion, and was abolished by completely removing 18A10 immunoreactivity (Trypsin(ϩϩ)). DISCUSSION We provided evidence that (i) the native mIP 3 R1 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 membranespanning integral fragment V in a non-covalent manner; (iii) such completely fragmented mIP 3 R1 retains IICR activity comparable to that of the intact one, indicating the tight structuralfunctional 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 mIP 3 R1 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 mIP 3 R1 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 mIP 3 R1 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 IP 3 R 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 IP 3 -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, Nterminal 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 IP 3 -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 mIP 3 R1 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 mIP 3 R1 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 mIP 3 R1 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 3 F. Yoshikawa and T. Furuichi, unpublished data. I-V, thus the mIP 3 R1-channel complex forms a compact structure. This view is supported by electron microscopic studies that a single native IP 3 R1 complex forms a compact squareshaped conformation (41,42).
The IP 3 R channel functions like an IP 3 /Ca 2ϩ signal converter (2), i.e. IP 3 binds to the N-terminal ligand-binding region, the information of which transduced to opening of the C-terminal Ca 2ϩ channel gate, leading to release into the cytoplasm of another second messenger Ca 2ϩ from stores. Mignery and Sü dhof (24) reported that the N-terminal 1803 residues molecularly expressed displayed a mobility shift (apparent decrease in mass of Ͼ50 kDa) on gel filtration chromatography in the presence of IP 3 , suggesting a conformational change in the cytoplasmic region upon ligand binding. Joseph et al. (29), however, reported that the preincubation of cerebellar microsomes with IP 3 does not affect the proteolytic pattern of the IP 3 R1, determined using two different anti-IP 3 R1 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 trypsinfragmented mIP 3 R1 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 IP 3 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 IP 3 binding. However, either in the presence or the absence of 1 M IP 3 , all the fragments were precipitated by centrifugation and immunoprecipitated by the 18A10 (data not shown). The function of the mIP 3 R1 channel is well regulated by a variety of modulatory systems as follows: phosphorylation with various protein kinases, binding to various modulators such as Ca 2ϩ , 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 mIP 3 R1 channel and then in the structural-functional coupling level. The IICR activities measured in the present study reflect not only the mIP 3 R1 itself but also other molecules such as the SERCA/Ca 2ϩ pump, the IP 3 metabolic enzymes such as IP 3 5-phosphatase and IP 3 3-kinase, and IP 3 R1 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.
IP 3 R shares a few fragmentary homologies with another intracellular Ca 2ϩ release channel, ryanodine receptor (RyR) (9,43), that is involved in Ca 2ϩ -induced Ca 2ϩ release (CICR) from the sarcoplasmic reticulum (44,45). The structural similarity probably relates to functional similarities as intracellular Ca 2ϩ 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 Ca 2ϩ release channel. Whether there is any similarity in structural-functional coupling for the ligand-gating mechanism between the IP 3 R-IICR channels and the RyR-CICR channels remains to be determined, although specific ligands differ between them (IP 3 versus Ca 2ϩ ). In the case of IP 3 R the IP 3 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 Ca 2ϩ activation is close to the channel region (53)(54)(55), and both regions locate in the same proteolytic fragment (49,56). However, it is well known that the liganding IP 3 is essential but not sufficient for the gating of the IP 3 R channel, and the concomitant action of cytosolic Ca 2ϩ at stimulatory levels is requisite for it as "co-agonist." It should be noted that the position of the cytoplasmic Ca 2ϩ site for IP 3 R, determined by 45 Ca 2ϩ overlay experiment (57) (Fig. 1), is topographically equivalent to that of the Ca 2ϩ activation site for RyR.