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synaptic density of Purkinje cells.In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that.
The EMBO Journal vol.9 no. 1 pp.61 - 67, 1990

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of lnsP3 receptor complex Nobuaki Maeda', Michio Niinobe' and Katsuhiko Mikoshiba1' 2 1Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Yamadaoka, Suita Osaka 565 and 2National Institute for Basic Biology, Myodaijicho, Okazaki 444, Japan Communicated by J.-P.Changeux

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the postsynaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P4W protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P4W protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bm. of 2.1 pmol/i4g and a KD of 83 nM. It reacted with three different monoclonal antibodies against P4N protein, indicating that P4W protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells. Key words: cerebellum/inositol 1,4,5-trisphosphate receptor/ monoclonal antibody/P4W protein -

spines which normally are formed on the dendritic tree. This causes defective synaptic contacts with the parallel fibers (Hirano and Dembitzer, 1975). In addition, the staggerer mutant lacks calcium spikes on the Purkinje cells, although sodium spikes are present (Crepel et al., 1984). Immunohistochemical studies at the electron microscopic level indicate that P4W protein is present in the Purkinje cells at the plasma membrane and at the endoplasmic reticulum (Maeda et al., 1989a), which are considered to be involved in calcium signalling. Worley et al. (1987a,b) reported that inositol 1,4,5-trisphosphate (InsP3) receptors are highly concentrated in the cerebellum. Supattapone et al. (1988a) purified InsP3 receptor from rat cerebellum and reported that it is a glycoprotein with a mol. wt of 260 000. InsP3 receptor is considered to be involved in the calcium release processes from the endoplasmic reticulum and the calcium influx through the plasma membrane (Berridge, 1987; Kuno and Gardner, 1987; Shah et al., 1987). The calcium spike phenomena are thought to be closely associated with InsP3 production (Murphy and Miller, 1988; Meyer and Stryer, 1988). Therefore, we considered the possibility that P4W protein is an InsP3 receptor protein. In the previous study, we reported the procedure for purifying P4W protein from mouse cerebellum (Maeda et al., 1988). Because P4W protein is highly insoluble, it was solubilized with a solution containing guanidinium chloride and Zwittergent 3-14 (Maeda et al., 1988), but the InsP3 binding activities were completely inactivated in this process. In this study, we reexamined the solubilization and purification procedure for P400 protein in a mild condition, and we found that P4W protein is indeed an InsP3 receptor. In addition, several important differences were observed in the characteristics of the InsP3 receptor protein from those reported by Supattapone et al. (1988a). These include the yield, the solubility in Triton X-100, and the size and shape of the receptor protein. The possible causes of these differences are discussed.

Introduction

P4W protein is a ubiquitous 250 kd glycoprotein, which is

present in the brain, spleen, thymus, liver and heart (Mikoshiba and Changeux, 1978; Mikoshiba et al., 1979; Maeda et al., 1988) and is especially abundant in cerebellar Purkinje cells (Mikoshiba and Changeux, 1978; Mikoshiba et al., 1979, 1985; Maeda et al., 1988). Developmental studies indicated that the expression of P400 protein in Purkinje cells is closely associated with their dendritic arborization growth (Maeda et al., 1988, 1989a). Analysis of the cerebella of various neurologically deficient mutant mice indicated that the Purkinje cells in the staggerer mutant cannot accumulate P400 protein (Mallet et al., 1976; Mikoshiba et al., 1979; Maeda et al., 1989a). The Purkinje cells in this mutant have poor dendritic arborization and lack © Oxford University Press

Results Solubilization of InsP3 receptor and P400 protein To determine whether P4W protein is an InsP3 receptor, we first tried to purify P4W protein from mouse cerebellum under mild conditions. As we have reported previously, most of the P400 protein was recovered in the precipitate when cerebellar microsome fractions were treated with a 1 % Triton X-100 solution and centrifuged at 105 000 g for 60 min (Maeda et al., 1988). InsP3 binding activities also precipitated under the same conditions. Supattapone et al. (1988a) reported that InsP3 binding sites were solubilized in the 1 % Triton X-100 solution from rat cerebellum at a critical tissue concentration and the receptors did not precipitate after centrifugation at 120 000 g for 2 h. 61

N.Maeda, M.Niinobe and K.Mikoshiba

However, most InsP3 binding activities in the microsome fraction of mouse cerebellum were recovered in the precipitate under such a condition. Using CHAPS, octylglucoside or Zwittergent 3-14 gave the same result. The most InsP3 binding activities and P400 protein were recovered in the supernatant when the microsomes were treated with % Triton X-l00 and centrifuged at 20 000 g for 60 min. The effects of CHAPS, octylglucoside and Zwittergent 3-14 were more limited than that of Triton X-100. Thus Triton X-100 and the above centrifugation were chosen to solubilize the InsP3 receptor. 0

4

05 ( C)

10

1 3 5 7191113

15

200

'

1

E

E z

0~~~~~.05 z

z I

w~~~~~~~

0.01 (d)

1 468

15t

10

5

9 10

0.2

0.1

0.1 ---

0.05

5

15

10

FRACTION NUMBER

Fig. 1. Purification of P400/InsP3 receptor protein. The Triton X-100 was applied to a column of DE52 and the proteins were eluted with a linear NaCl gradient (0.05-0.5 M NaCI indicated by dashed line) (a). The collected fractions indicated by a horizontal bar in (a) were applied to a column of heparin-agarose. After washing the column with a buffer containing 0.25 M NaCI, the receptors were eluted with 0.5 M NaCl (the position of the buffer change is indicated with a arrowhead) (b). The peak fractions of InsP3 binding (indicated by a horizontal bar in (b) were collected and were applied to a column of lentil lectin-Sepharose. After washing, the receptors were eluted with 0.8 M c-methyl-D-mannoside (the position of the application of ci-methyl-D-mannoside is indicated by a arrowhead) (c). The receptor fractions indicated by a horizontal bar in (c) were applied to a column of hydroxylapatite. The receptors were eluted with a linear sodium phosphate gradient (0.025-0.15 M sodium phosphate indicated by dashed line) (d). Aliquots of the each fraction were assayed for protein concentration (0) and for InsP3 binding activity (0). Insets show the results of the immunoblotting of the fractions indicated by the fraction number with a monoclonal antibody against P400 protein, 18A10.

extract

Purification of InsP3 receptor In a preliminary experiment, we observed that InsP3 binding sites are enriched in a crude mitochondrial fraction (P2 fraction) and in a microsomal fraction (P3 fraction). Therefore, we used the P2 + P3 fraction from mouse cerebella as a starting material. A sequence of four purification steps was used to purify InsP3 receptor. The P2 + P3 fraction was prepared from ddY mouse cerebella and treated with 1 % Triton X-100. The solubilized proteins were applied to a column of DE52. The InsP3 binding activity was absorbed by DE52 and was eluted as a single peak by increasing the NaCl concentration (Figure la). The InsP3 receptor was further purified on a heparin-agarose column. The InsP3 binding activity was adsorbed on heparin-agarose at 0.25 M NaCl, and was eluted by increasing the concentration of NaCl to 0.5 M (Figure lb). Next, the sample was applied to a column of lentil lectin-Sepharose. About 50% of the InsP3 binding activities was adsorbed on the lentil lectin-Sepharose and was eluted by 0.8 M a-methyl-D-mannoside (Figure lc). Concanavalin A - Sepharose absorbed all of the InsP3 binding activity, but only a small part was eluted by camethyl-D-mannoside. So lentil lectin-Sepharose was used to improve the yield. The carbohydrate-binding specificity of lentil lectin is different from that of concanavalin A. Lentil lectin requires a fucose residue attached to the asparaginelinked N-acetylglucosamine residue for tight binding, but concanavalin A does not (Kornfeld et al., 1981). Thus the heterogeneous behaviour of the receptor on the lentil lectin - Sepharose seems to be the result of the heterogeneity of the carbohydrate chains. Finally, the sample was applied to a column of hydroxylapatite, and the receptors were eluted with a gradient of sodium phosphate (Figure ld). SDS-PAGE analysis of the InsP3 binding fraction of this

Table I. Purification of InsP3 receptor

Step

Protein

InsP3 binding

Specific

Recovery

Purification

mg

pmol

activity

(%)

(fold)

(pmol/mg) P2 + P3 fraction Triton extract DE52 Heparin-agarose Lentil lectin-Sepharose

Hydroxylapatite

62

500

538

1.1

100

I

260

615

2.4

114

2.2

423 361 215 169

9.6 36 195 338

79 67 40 31

8.7 33 177 307

44 9.9 1.1 0.5

Characterization of (B)

Table II. Inhibition of specific [3H]Ins-1,4,5-P3 binding by various inositol phosphates, heparin and Ca2 + Binding of [3H]Ins-1,4,5-P3 (% of control)

Displacing agent CL

.3 I-> i2 0

X

00

A11K 1 2-97K

Bound(pmol/pg)

-

10-8

10-7

0

None

-66K

Ins-l-P1

-43K

Ins-1,4-P2

-31K

Ins-1,3,4-P3

ae

O

P400/lnsP3 receptor complex

105

IP3 (M)

Ins-1,4,5-P3 Ins-2,4,5-P3

Fig. 2. Characterization of the purified InsP3 receptor. (A) Binding assays contained 0.42 Mg of purified receptor, 11.2 nM [3H]InsP3 and various concentrations of cold InsP3 in 50 M1 of I mM EDTA, 10 zM pepstatin A, 10 MM leupeptin, 0.1 mM PMSF, 50 mM Tris-HCI, pH 8.0. The samples were incubated for 10 min at 4°C and then processed for PEG precipitation as described in Materials and methods. (B) The purified receptor (1 Mg) was analyzed by SDS-PAGE (5-20% polyacrylamide gradient gel). The positions of mol. wt markers are shown on the right.

hydroxylapatite chromatography revealed that the 250 kd protein is only one protein band (Figure 2B). When the receptor protein was applied to a column of Sepharose CL-4B, it was eluted as a very broad peak (data not shown). Because of this breadth, the exact molecular mass is unknown, but it is > 106 daltons. Table I summarizes the purification of InsP3 receptor protein. Approximately 0.5 mg of receptor protein can be obtained from 10 g of mouse cerebellum. Supattapone et al. (1988a) obtained 0.02 mg of receptor protein from 16 rat cerebella (-'-4 g of tissue). Thus, from the same amount of the tissue, 10 times more receptor protein can be obtained by our method than by the procedure of Supattapone et al. (1988a). Scatchard analysis of the InsP3 binding to the purified receptor indicated that the KD was 83 nM and the Bmax was 2.1 pmol/Atg of protein (Figure 2A). The KD value of the original P2 + P3 fraction was 34 nM, which is close to that of the purified receptor. The binding of [3H]InsP3 to the purified P4W/InsP3 receptor is highly specific (Table II). Ins-2,4,5-P3 was a relatively potent competitor for [3H]InsP3 binding and the Ins-1,3,4,5-P4 was less effective. Ins-1,4-P2 and Ins-l-PI were inactive at the concentration of 10 rIM. Such specificity is comparable to that of InsP3-sensitive calcium mobilization (Berridge and Irvine, 1984). Heparin and Ca2+ inhibited the [3H]InsP3-binding to the P2 + P3 fraction of cerebellum. Heparin also inhibited the [3H]InsP3 binding to the purified receptor but the sensitivity to Ca2+ was lost in the purified state (Table II). The P2 + P3 fraction of cerebellum has a higher affinity for [3H]InsP3 at alkaline pH. The binding of [3H]InsP3 to the purified P4W/InsP3 receptor retained the dependency on pH (data not shown). Electron microscopic observation of the purified receptor Electron microscopy of the purified receptor showed a very

large particle. Negative staining revealed positive (Figure 3A) and negative (Figure 3B-E) images of the

Ins-1,3,4,5-P4 Ins-P6

Heparin

Ca2+

A

1 AM 10 MM I FAM 10 MM 1 MM 10 AM 1 MM 10 AM 1 MM 10 MM 1 MM 10 AM 1 MM 10 AM 20 Mg/ml 1 mM

y.

B

.:

...}

P2 + P3

Purified receptor

100 105 93 102 87 104 90 14 0 47 0 98 22 92 25 0 0

100 101 85 95 97 92 92 3 0 27 4 59 12 95 46 0 99

C

E

Fig. 3. Electron microscopy of the purified receptor complex. (A) The positive image shows squares (25 nm/side, arrowheads). Occasionally, the squares are joined to each other at sides (arrow). (B) - (E) The negative images of the receptor complexes. The 7 x 25 nm rod-like images may be edge views of the receptor complexes (E). Bar, 50 nm.

receptor. The positive image shows the structures as squares (25 nm/side) (Figure 3A, indicated by arrowheads). The particles are occasionally joined together at their sides (Figure 3A and B, indicated by arrows). The substructures of the particles were not clear. There was a small proportion of 7 x 25 nm rod-like images (Figure 3E). These may be edge views of the receptor molecules.

Identification of P400 protein as an InsP3 receptor We prepared three monoclonal antibodies against P400 protein, 4C1 1, lOA6 and 18A10 (Maeda et al., 1988, 1989a). These antibodies were produced by injecting partially purified P4W protein from mouse cerebellum into a rat and fusing the spleen cells from the rat with Sp2 mouse myeloma cells. These antibodies react strongly with P4W protein from mouse and rat cerebellum. Figure 4(A) shows the reactions of monoclonal antibodies with P4w protein partially digested with Staphylococcus aureus V8 protease. The 4C1 1 and 10A6 reacted with the common peptide at the higher mol.

63

N.Maeda, M.Niinobe and K.Mikoshiba (B) b c

(A) b

a

c

~ . ~ .W

Table III. Selective absence of [3H]InsP3 binding in the cerebellum of ped mutant mouse

G

Binding of [3H]InsP3 (mean + SD, c.p.m.)

*

--qfiI

Cerebrum Cerebellum

w 43K

Fig. 4. Analysis with monoclonal antibodies. (A) Purified P4W protein was partially digested with Staphylococcus aureus V8 protease. The proteins were electrophoresed (5-12.5% SDS-PAGE) and immunodetected with 4Cl 1 (a), 1OA6 (b) and 18A10 (c) after the transfer of the proteins to a nitrocellulose sheet. The positions of mol. wt markers (in kd) are shown on the right. The triangle indicates the position of intact P4W protein. (B) Purified InsP3 receptor protein was electrophoresed (5% SDS-PAGE) and transferred to a nitrocellulose sheet. The blots were stained with amido black (a), or immunostained with 4C 11 (b), 1OA6 (c) and 18A 10 (d).

Normal

pcd

490 120 3867 + 400

432 + 5 64 + 11

The cerebrums and the cerebella of a 2 month old pcd/pcd mouse and its normal littermate were dissected and homogenized in 10 vol of 0.32 M sucrose, 1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mM Tris-HCI, pH 8.0. The homogenates were centrifuged at 400 000 g for 20 min at 2°C and the pellets were homogenized and centrifuged again as above. The resultant pellets were resuspended in the above buffer (protein concentration = 0.6 mg/ml). One hundred microliters of the samples were incubated with 10 nM [3H]InsP3 for 10 min at 4°C. The incubations were terminated with rapid filtration through Whatman GF/B filter and the radioactivities were measured by liquid scintillation counting.

A A

x 8--

E

>

>:6 4-

2

MML

WM ., I1 . 243 81

.

27

9

.

3

A

1

DILUTION (fold)

B

Fig. 5. Immunoprecipitation of InsP3 receptor with a monoclonal antibody against P4W protein. Triton X-100 extract of the cerebellar P2 + P3 fraction was prepared as described in Materials and methods. One hundred microliters of the extract was diluted with 900 il of

0.1% bovine serum albumin (BSA), 0.15 M NaCl, 5 mM EDTA, 0.1 mM PMSF, 10 jsM leupeptin, 10 liM pepstatin A, 10 mM Tris-HCI, pH 8.0. Five microliters of the original and the diluted solutions of 18A10 monoclonal antibody (the concentration of the antibody of the original solution is - 100 jig/ml) were added to the samples, which were then incubated for 60 min at 4°C. Five microliters of the rabbit anti-rat IgG (Fc region specific) was added and the solutions were incubated for 30 min at 4°C. One hundred microliters of the 10% (w/v) suspension of Pansorbin (Calbiochem) was added to the sample. The sample tubes were gently rotated for 30 min at 4°C and then the Pansorbin particles were washed three times with 1 ml of 0.15 M NaCl, 5 mM EDTA, 0.1 mM PMSF, 10 jM leupeptin, 10 lsM pepstatin A, 10 mM Tris-HCI, pH 8.0 (buffer A). The Pansorbin pellets were resuspended in 100 ld of buffer A and then incubated with 10 nM [3H]InsP3 for 10 min at 4°C. Incubations were terminated by filtration through Whatman GF/B filters, followed by a rapid washing with 2 ml of buffer A. The filters were dried and the radioactivities were measured with liquid scintillation counter. Only a low level of 3H binding was observed when normal rat IgG was used instead of the 18A10 monoclonal antibody (0).

wt portion, but their reaction patterns were quite different from each other at the lower mol. wt portion. 18A10 and the other two showed almost no common reaction product. It is apparent that these monoclonal antibodies recognize the three different epitopes on P4W protein. The subclasses

64

Fig. 6. Autoradiographs of [32P]InsP3 binding sites in the cerebella of 40-day-old normal and pcd mutant mice. In contrast to the normal littermate (A), the cerebellum of the pcd mutant showed almost no autoradiographic grains (B). ML, molecular layer; WM, white matter; CN, deep cerebellar nucleus.

of 4C 11, 1OA6 and 18A 10 are IgG2a, IgG2b and IgG2a respectively. Each fraction of the chromatographies shown in Figure 1 was analyzed by the immunoblotting method using a monoclonal antibody against P4W protein, 18A10. The immunoblot analysis revealed that P400 protein and InsP3 binding activity co-migrated completely in each purification step (Figure 1). The purified InsP3 receptor also reacted on immunoblot analysis with the other two kinds of monoclonal antibodies against P4W protein, 4C 11 and 1OA6 (Figure 4B).

Characterization of P400/lnsP3 receptor complex

Figure 5 indicates that InsP3 receptor activity was immunoprecipitated dose-dependently by 18A10 monoclonal antibody. In this condition, only 250 kd P40 protein was specifically immunoprecipitated (data not shown). P40 protein is rather ubiquitous, but it is especially abundant in cerebellar Purkinje cells (Maeda et al., 1988). In order to determine whether the InsP3 receptor is also abundant in Purkinje cells, we analyzed the brains of Purkinje cell degeneration (pcd) mutant mice. In this mouse, Purkinje cells accumulate the normal level of P400 protein before degeneration, but the Purkinje cells begin to degenerate at 15-18 days of age and almost no P4w protein is detected in the cerebella of 50-day-old mutants (Maeda et al., 1989a,b). In contrast to the high level of binding activity in the normal cerebellum, there is almost no 3H-binding in the cerebella of the pcd mutant mice (Table HI). There is a low level of binding activity in the normal cerebrum, and the pcd mutation had no effect on the [3H]InsP3 binding in the cerebrum (Table IE). Figure 6 shows the autoradiographs of [32P]InsP3 binding sites in the cerebella of normal and pcd mutant mice. In the normal cerebellum, the molecular layer had the greatest density of autoradiographic grains, and the white matter and the deep cerebellar nucleus showed moderate density (Figure 6A). In contrast, there were almost no autoradiographic grains in the pcd mouse cerebellum (Figure 6B). These results indicate that InsP3 receptors are concentrated exclusively in the Purkinje cells in the cerebellum, and suggest that InsP3 receptors are distributed in the dendrites, axons and axon terminals of Purkinje cells. This is consistent with the immunohistochemical study described below. Thus it is clear that P4W protein is an InsP3 receptor protein (P4W/InsP3 receptor protein).

'A

-

Immunohistochemical localization of P400/lnsP3 receptor protein Figure 7 shows the immunohistochemistry of P4W/InsP3 receptor protein. This protein is present in the dendrites, cell body and axon of the Purkinje cells, but the nuclei of Purkinje cells were not stained (Figure 7A and D). At the deep cerebellar nucleus, the synaptic boutons of Purkinje cells around the cell bodies of large neurons and in the neuropiles are densely stained (Figure 7C). Figure 7(B) shows the dendrite of a Purkinje cell which is cut longitudinally (indicated by arrowheads). The plasmalemma and its closely associated structures (these are smooth endoplasmic reticulum, as we have previously confirmed) are densely stained. The insides of the dendrites are relatively negative, but both densely stained sides of the dendrites are connected in places with immunopositive structures (Figure 7B, indicated by arrow). In the cell body of the Purkinje cell, the cytoplasm around the nucleus is positively stained and the marginal part of the cell soma is also stained strongly

(Figure 7D).

Discussion The results presented here demonstrate that the cerebellar Purkinje cell marker, P4w protein, is an InsP3 receptor protein. The purified receptor exhibited a single, high-affinity InsP3 binding site with a KD of 83 nM, which is similar to that reported by Supattapone et al. (1988a). Scatchard plot analysis yielded a Bmax value for the purified receptor of

D r."

Ia..

#1 Fig. 7. Immunohistochemistry with 4C1 1 monoclonal antibody. (A) The dendrites, cell bodies and the axons (arrowheads) of Purkinje cells are positively stained. (B) The plasmalemma and its closely associated structures of the dendrites are stained (arrowheads). The densely stained both sides of the dendrites are connected with immunopositive structure (arrow). (C) The neurons at the deep cerebellar nucleus (arrow) are surrounded with densely stained synaptic boutons of Purkinje cells. (D) The cytoplasm of the Purkinje cell is immunopositive, but the staining of the cell surface and the associated sites are much denser (arrowheads). x500.

2.1 pmol/,tg of protein. We recently cloned the cDNA of P4W protein and estimated that the mature P4W protein is a 2749 amino acid polypeptide with a mol. wt of 313 K (Furuichi et al., 1989). If we postulate that the receptor has one binding site per molecule, the Bmax is calculated to be 3.2 pmol/tg of protein. The measured Bmax is close to this value and, therefore, it seems that there is one high-affinity binding site for each receptor molecule. Although the mol. wt, KD and B,,,ax of the purified InsP3 receptor are similar to those reported by Supattapone et al. (1988a), the InsP3 receptor purified by us showed several characteristics different from those reported by them. In our preparation, the InsP3 receptor precipitated when the microsome fraction was treated with 1 % Triton X-100 and centrifuged at 105 000 g for 60 min, but it was recovered in the supernatant when centrifugation was at 20 000 g. On the other hand, Supattapone et al. (1988a) reported that the InsP3 receptor was solubilized from cerebellar membrane with 1 % Triton X-100 and it did not precipitate after centrifugation at 120 000 g for 120 min. The InsP3 receptor purified by Supattapone et al. (1988a) is a globular protein with a Stokes radius of 10 nm. Electron microscopic observation of the receptor purified by us indicated that it is a 25 x 25 nm square. Since Supattapone et al. (1988a) prepared cerebellar membrane by homogenization with

65

N.Maeda, M.Niinobe and K.Mikoshiba

Polytron, it may be that their relatively violent homogenization process destroyed the subunit structure of the receptor complex. The different behaviour of the two receptors in the centrifugation may also be explained by such a loss of the subunit structure in their purification steps. InsP3 is a well-known second messenger which initiates calcium release from the endoplasmic reticulum (Berridge, 1987; Shah et al., 1987). And in T lymphocytes, InsP3 opens the calcium channel at the plasma membrane (Kuno and Gardner, 1987). Immunohistochemical analysis at the electron microscopic level revealed that P4w/InsP3 receptor is present at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of the Purkinje cells (Maeda et al., 1989a). Such a localization of this protein is compatible with the hypothesis that P400/InsP3 receptor protein is directly involved in the calcium release processes from the endoplasmic reticulum and the calcium influx through the plasma membrane of Purkinje cells. Recently, Ross et al. (1989) reported that InsP3 receptor is located in the rough and smooth endoplasmic reticulum and the nuclear membrane of Purkinje cells, but not in the plasma membrane. Our monoclonal antibodies against P4W/InsP3 receptor only weakly stained the nuclear membrane of Purkinje cells. These discrepancies may be due to the differences in the epitopes recognized by the antibodies, the ages of animals or the condition of the fixation. Anderson et al. (1989) purified the ryanodine receptor - Ca2+ release channel complex from cardiac sarcoplasmic reticulum. Electron microscopic observation of this Ca2+ release channel revealed a 4-fold symmetric, four-leaf clover structure, which fills a 30 x 30 nm box (Anderson et al., 1989). The size and the shape of the InsP3 receptor complex are similar to those of the ryanodine receptor-Ca2 + release channel complex. The behaviour of the InsP3 receptor on the heparin - agarose and hydroxylapatite columns is also similar to those of the ryanodine receptor complex (Inui et al., 1987). Thus they may be closely related molecules, although the mol. wt of the subunit is different (the ryanodine receptor complex is composed of 360-400 kd subunits, and the cDNA cloning of the P4w/InsP3 receptor indicated that the mol. wt of the subunit is 313 kd). In our recent cross-linking experiment and in a reconstitution study, we observed that the native P400/InsP3 receptor has a tetrameric structure and constitutes a cation-permeable channel (manuscript in preparation). An important question is why the Purkinje cells have an exceptionally large amount of InsP3 receptor. Autoradiographic studies of rat brain with [3 P]InsP3 indicated that the highest density of InsP3 receptors occurs in the molecular layer of the cerebellum and the second highest density of the receptors is in the CAl region of the hippocampus (Worley et al., 1987a, 1989). The CA3 region of the hippocampus has a low density of the receptors (Worley et al., 1989). We also obtained similar results using mouse brain (M.Niinobe et al., unpublished observation). The P400/InsP3 receptor protein is a rather ubiquitous protein and is present in the hippocampus in a lesser amount than in the cerebellum, and it is also concentrated in the CAl region (S.Nakanishi et al., manuscript in preparation). In the Purkinje cells, the P400/InsP3 receptor is present in the dendrites, cell soma and axon and in the synaptic boutons at the deep cerebellar nucleus. Activation of several types of glutamate receptors, such as the quisqualate-selective

66

sub-type, stimulate the inositol phosphate/ diacylglycerol second messenger pathway (Murphy and Miller, 1988; Sladeczek et al., 1988). Purkinje cells receive two kinds of excitatory inputs: inputs from parallel fibres and from the climbing fibres. When these two inputs are activated together, the synaptic transmission between parallel fibres and Purkinje cells undergoes long-term depression, which is thought to be the physiological basis for motor learning in the cerebellum (Ito and Kano, 1982; Ito et al., 1982). Quisqualate receptors have been reported to be selectively involved in long-term depression (Kano and Kato, 1987). And the increase in the concentration of Ca2 + in the dendrites of Purkinje cells is necessary for long-term depression to occur (Kano and Kato, 1987). The P4w/InsP3 receptors in the dendrites and the spines of Purkinje cells may be involved in such a process. On the other hand, the InsP3-dependent release of Ca2+ from the intracellular storage sites induces the release of acetylcholine from the synaptic terminals of NG108-15 cells (Yano et al., 1984; Higashida, 1988). So P400/InsP3 receptors in the synaptic terminals of Purkinje cells may also be involved in the neurotransmitter release processes. In the previous study, we demonstrated that the expression of P400 protein in the Purkinje cells occurs closely associated with the growth of dendritic arborization and that the Purkinje cells in the staggerer mutant mouse cannot accumulate P400 protein (Maeda et al., 1989a). The Purkinje cells of the staggerer mutant have poor dendritic arborization, and the spines that are normally formed on the dendrites are absent (Hirano and Dembitzer, 1975). Ca2+ is an important factor in neurite growth (Mattson, 1988), so the P4w/InsP3 receptor protein may play essential roles in the morphogenesis of Purkinje cells. In the post-nuclear fraction from mouse cerebellum, the P400/InsP3 receptor protein is efficiently phosphorylated by cyclic AMP-dependent protein kinase (A kinase) (Mikoshiba et al., 1985; Supattapone et al., 1988b; Yamamoto et al., 1989), and also by the calcium calmodulin-dependent protein kinase II but with slower kinetics than that of A kinase (Yamamoto et al., 1989). This protein is also phosphorylated in the cultured Purkinje cells, but the stimulation of the phosphorylation of P4w/InsP3 receptor protein by dibutyryl cyclic AMP has not been observed (Yamamoto et al., 1989). Thus it is uncertain at present whether the cyclic AMPdependent phosphorylation of the P400/InsP3 receptor protein has physiological significance.

Materials and methods Materials

[3H]InsP3 and [32P]InsP3 were purchased from NEN. InsP3 and heparin agarose were obtained from Sigma. Bio Gel HTP was obtained from Bio Rad, lentil lectin-Sepharose from Pharmacia, and DE52 from Whatman. Vectastain ABC kit was purchased from Vector Laboratories, and anti-rat IgG (Fc region specific) from Jackson Immunoresearch Laboratories. Ins-l-PI and Ins-P6 were obtained from Sigma. Ins-2,4,5-P3, Ins-14-P2 and Ins-1 ,3,4,5-P4 were purchased from Boehringer Mannheim. Ins-1,3,4-P3 was kindly given by Dr Masato Hirata. All of the other chemicals were the highest purity of commercially available reagents. Preparation of membranes Adult ddY mice were anaesthetized and then killed by decapitation and the cerebella were dissected. Ten grams of fresh cerebella were mixed with 9 vol of the solution containing 0.32 M sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulphonyl fluoride (PMSF), 10 ltM leupeptin, 10 /M pepstatin

Characterization of P400/lnsP3 receptor complex A. 1 mM 2-mercaptoethanol and 5 mM Tris-HCI, pH 7.4, and were homogenized in a glass-Teflon Potter homogenizer with 10 strokes at 850 r.p.m. The homogenate was centrifuged at 1000 g for 5 min at 2°C, and washed again under the same conditions. The combined supernatants were centrifuged at 105 000 g for 60 min at 2°C to precipitate P2 + P3 fraction.

Solubilization and purification of P400/InsP3 receptor protein The P2 + P3 fraction was resuspended in the solution containing 1 mM EDTA, 0.1 mM PMSF, 10 yM leupeptin, 10 jtM pepstatin A, 1 mM 2-mercaptoethanol and 50 mM Tris-HCI, pH 8.0. Membranes were solubilized by addition of 10% (w/v) Triton X-100 to give final protein and detergent concentrations of 3.0 mg/ml and 1.0% respectively. The sample was stirred for 30 min at 0°C and then centrifuged at 20 000 g for 60 min at 2°C. The supernatant was applied to a column of DE52 (2.6 x 10 cm) equilibrated in 1 % Triton X- 100, 1 mM EDTA, 10 ,tM leupeptin, 1 mM 2-mercaptoethanol, 50 mM Tris-HCI, pH 8.0 (buffer 1). The column was washed with 20 ml of 0.05 M NaCl/buffer 1, and then the receptor was eluted by a 400 ml linear NaCl gradient (0.05-0.5 M in buffer 1). The peak fractions containing InsP3 binding activities and P400 protein were pooled and were applied to a column of heparin-agarose (0.97 x 7 cm) equilibrated in 0.2% Triton X-100, 10 jtM pepstatin A, 10 IsM leupeptin, 0.1 mM PMSF, 1 mM 2-mercaptoethanol, 50 mM Tris-HCI, pH 8.0 (buffer 2) containing 0.25 M NaCl. The column was washed with 30 ml of 0.25 M NaCl/buffer 2 and then the receptor was eluted with 30 ml of 0.5 M NaCl/buffer 2. The peak fraction of the InsP3 binding activity and P400 protein were pooled and were applied to a column of lentil lectin-Sepharose (0.95 x 2.8 cm) equilibrated in 0.5 M NaCl, 0.2% Triton X-100, 10 yM leupeptin, 10 jsM pepstatin A, 0.1 mM PMSF, 20 mM Tris-HCI, pH 8.0, 1 mM 2-mercaptoethanol (buffer 3). The column was washed with 8 ml of buffer 3 and the receptors were eluted with 0.8 M a-methyl-D-mannoside/buffer 3. The peak fractions of the InsP3 binding activity and P4W) protein were pooled and the proteins were absorbed to a column of Bio Gel HTP (0.7 x 0.8 cm) equilibrated in 0.2% Triton X-100, 0.5 M NaCl, 1 mM 2-mercaptoethanol, 25 mM sodium phosphate, pH 8.0 (buffer 4). The column was washed with 2 ml of buffer 4 and the receptors were eluted with a 10 ml linear sodium phosphate gradient (25-150 mM).

Measurement of [3H]lnsP3 binding Measurement of InsP3 binding was performed with PEG precipitation method. [3H]InsP3 was added to the 50 pA of sample at the concentration of 10 nM. The sample was incubated for 10 min at 4°C and then mixed with 2 pl of 50 mg/ml y-globulin. Fifty microliters of the solution containing 30% PEG6000, 1 mM 2-mercaptoethanol and 50 mM Tris-HCI, pH 8.0, was added to the sample. which was incubated for 5 min at 0°C. Nonspecific binding was measured in the presence of 1 pM cold InsP3. After centrifugation at 10 000 g for 5 min, the precipitate was dissolved in Protosol (NEN) and the radioactivity was measured with a liquid scintillation counter. Monoclonal antibody The monoclonal antibodies against P40) protein were prepared by injecting partially purified mouse P400 protein into a rat and fusing the spleen cells with mouse Sp2 myeloma cells as described elsewhere (Maeda et al., 1988). The hybridomas were cultured in Nissui SFM 101 medium. The culture supernatant was concentrated with an Amicon YM 10 membrane and stored at -800C. Staphylococcus aureus V8 protease digestion Purified proteins (-0.6 pg) were dissolved in 20 tl of 0.5% SDS, 10% glycerol. 0.125 M Tris-HCI, pH 6.8. and this solution was heated in a boiling water bath for 5 min. Two microliters of the enzyme solution containing 5 pg/ml Staphylococcus aureus V8 protease. 0.1 % SDS, 1 mM EDTA, 10% glycerol, 0.125 M Tris-HCI, pH 6.8, was added, and the sample was incubated at 37°C for 3 h. Twenty microliters of the solution containing 3% SDS, 20% 2-mercaptoethanol was added, and the solution was heated in a boiling water bath for 5 min. The sample was subjected to SDS -PAGE and immunoblotting. Electron microscopy The sample was applied to a thinly carbon-coated grid. and then washed with three drops of 1 % uranyl acetate. The grids were dried under vacuum and observed with a Hitachi HU-12A electron microscope. Other methods SDS -PAGE and immunoblotting were performed as described elsewhere (Maeda et al., 1988). Immunohistochemistry was done with avidin-biotinperoxidase complex (ABC) method as described previously (Maeda et al.,

1989a). Autoradiography was performed according to the method of Worley et al. (1987). Protein concentration was measured using the Bio Rad protein assay kit. P4(0) protein was purified as described elsewhere (Maeda et al.,

1988).

Acknowledgements The authors wish to express their great thanks to Professor Jean-Pierre Changeux (Institut Pasteur, Paris) for supporting us in our study of P400 protein and encouraging us to continue the work. We are also grateful to Dr Takashi Wada (Research Institute for Microbial diseases, Osaka University) for electron microscopic analysis and to Drs Yutaka Watanabe, Shoichiro Ozaki and Masato Hirata for the gift of Ins-1, 3, 4-P3. This study was supported by grants from Special Coordination Funds of the Science and Technology Agency of the Japanese Government, from the Japanese Ministry of Education, Science, and Culture.

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Received ont Ju/v 24, 1989; revised on Septeiber 25, 1989

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