m E JOURNAL OF BIOLOGICAL CHEMISTRY (4 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 3, Issue of January 25, pp. 1530-1534,1988 Printed in U.S.A.

Solubilization, Purification, and Characterization of an Inositol Trisphosphate Receptor*

(Received for publication, July 30, 1987)

Surachai Supattapone, Paul F. Worley, Jay M. BarabanS, and Solomon H. Snyder8 From the Departments of Neuroscience, Pharmacology and Molecular Sciences, Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Inositol 1,4,5-trisphosphate is a second messenger of the phosphoinositide system which can mobilize cal- cium from intracellular stores. Rat cerebellum is an abundant source of a receptor for inositol 1,4,5-tris- phosphate (Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S . , and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136). In this study we have sol- ubilized and purified this receptor to apparent homo- geneity from rat cerebellum. Crude membrane, deter- gent-solubilized, and purified receptor preparations display similar selectivity for inositol 1,4,5-trisphos- phate over other inositol phosphates. The purified re- ceptor is globular with a Stokes’ radius of approxi- mately 10 nm. Electrophoretic analysis reveals one protein band with an M, of 260,000. While binding is reversibly inhibited by 300 I1M calcium in particulate fractions and detergent-solubilized membranes, the purified protein is not inhibited by calcium concentra- tions up to 1.5 mM. Inhibition by calcium is reconsti- tuted by addition of detergent-solubilized cerebellar membranes, but not by the cytosolic fraction of cere- bellum.

The phosphoinositide second messenger system provides a transduction mechanism for a multiplicity of hormones, neu- rotransmitters, and growth factors (1-5). In the phosphoino- sitide cycle, phosphatidylinositol 4,5-bisphosphate is con- verted to diacylglycerol and inositol 1,4,5-trisphosphate (InsP3)’ following receptor activation (1, 6). InsPa is thought to bind to an intracellular membrane site to release calcium (1, 7). Direct evidence for InsP3 receptor proteins has been obtained through the demonstration of saturable, high affinity binding sites for radiolabeled InsP3 in permeabilized hepato- cytes and neutrophils as well as membrane fractions from the anterior pituitary, liver, and adrenal cortex (8-11). We have demonstrated high affinity saturable binding of [3H]InsP3 to membrane fractions from the rat cerebellum (12, 13). In both brain tissue and peripheral tissues the receptor demonstrates

* This work was supported in part by United States Public Health Service Grants MH-18501 and DA-00266, Physician Scientist Award AG-00256 (to P. F. W.), Research Scientist Award DA-00074 (to S. H. S.), National Institutes of Health Training Grant GM-07309 (to S. S. ) , the Lucille P. Markey Charitable Trust, and a grant from the Laboratories for Therapeutic Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Lucille P. Markey scholar. §To whom all correspondence and reprint requests should be

addressed. The abbreviations used are: Inspa, inositol 1,4,5-trisphosphate;

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- resis; pCMBS, p-chloromercurobenzoyl sulfonate.

high selectivity for InsP3 in contrast to other inositol phos- phates, paralleling the relative ability of these substances to release calcium. InsPB binding levels in the cerebellum are several hundred times greater than in peripheral tissues, permitting a detailed characterization of the receptor (13). In the present study we have solubilized the [3H]InsP3 receptor from rat cerebellar membranes, purified the protein to appar- ent homogeneity, and characterized some of its properties.

EXPERIMENTAL PROCEDURES

Materiak [3H]Ins-1,4,5-P3 (3.6 Ci/mmol) was obtained from Du Pont-New

England Nuclear. InsP3 was obtained from Amersham Corp. or Sigma, InsP, was obtained from Amersham Corp. Inositol-1,3,4,5-P4 was generously provided by Drs. R. Irvine and M. Berridge (Cambridge, United Kingdom). Phospholipase A, from bee venom, type I phos- pholipase C from Clostridium perfringens, and type 1 phospholipase D from cabbage were obtained from Sigma. Lectins were purchased from Boehringer Mannheim. Proteases were purchased from Wor- thington. All other reagents were obtained from Bio-Rad or Sigma.

rH]InsP3 Binding in Crude Mernbr~nes-[~H]InsP~ binding to rat cerebellar membranes was assayed as described previously (12, 13). For routine studies, cerebella from adult male Sprague-Dawley rata (200-300 g) were homogenized (Polytron setting 9,lO s) in 50 volumes of ice-cold buffer A (50 mM Tris-HCI, pH 8.3, 1 mM EDTA, 1 mM 8- mercaptoethanol), pelleted by centrifugation (35,000 X g for 10 min), and resuspended in 50 volumes of buffer A. Binding assays contained -10 mg wet weight of tissue (=0.6 mg of protein) and 2.5 nM [3H] InsPa in a total volume of 1 ml of buffer A. Assays were incubated 10 min at 4 “C followed by centrifugation at 10,000 X g for 5 min (Beckman Microfuge 12) and aspiration of the supernatant. Pellets were suspended in 5 ml of Formula 963 liquid scintillation mixture (Du Pont-New England Nuclear) and their radioactivity determined. Nonspecific binding was determined in the presence of 1 p M InsP3. The stability of [3H]InsP3 was assessed for each incubation condition using open-column anion-exchange chromatography (Dowex AG 1- X8) (14, 15). In certain experiments, InsP3 phosphatase activity was assayed by chromatographically monitoring the conversion of [3H] InsP3 to [3H]InsP2 (15).

rH]InsP3 Binding in Detergent-solubilized Sarnple~-[~H]InsP~ binding to soluble fractions was assayed by the method of spun- column chromatography. Tissue samples solubilized in buffer A con- taining detergent were incubated with 12.5 nM [3H]InsP3 in a total volume of 200 p1 for 10 min at 4 “C, then loaded onto 1-ml columns packed with 70 g/liter Bio-Gel P-10 resin and centrifuged at 1000 X g for 6 min. Five ml of liquid scintillation mixture were added to the void volume of these columns and radioactivity was determined. [3H] InsP3 binding was linear with tissue concentration from 0 to 2 mg/ ml in crude detergent-solubilized membranes.

Other Methods Protein was determined by the method of Bradford (16). Sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (17). Gels were stained either by Coomassie Brilliant Blue or silver (18). InsP, 5-phosphatase activity was assayed by a standard protocol (15).

1530

Inositol Trisphosphate Receptor Purification 1531

RESULTS

Effects of Reagents and Enzymes on p H ] I m P 3 Binding to Rat Cerebellar Membranes-Prior to attempting solubilization of the receptor, we evaluated the influence of various reagents and enzymes (Table I). [3H]InsP3 binding to intact cerebellar membranes is abolished by 1 mM para-chloromercurobenzoyl sulfonate (pCMBS), an effect which is prevented by pretreat- ment with 1 mM P-mercaptoethanol, suggesting a role for sulfhydryl groups in receptor binding. Dithiothreitol (1 mM) or 1 mM 8-mercaptoethanol alone each provide limited stim- ulation of binding.

[3H]InsP3 binding is abolished by a-chymotrypsin and tryp- sin, consistent with the receptor being a protein. Extraction with high salt does not alter [3H]InsP3 binding to membranes, suggesting that the receptor is an integral membrane protein. Denaturing treatments such as 1 M urea or heating at 60 "C for 1 h also eliminate binding. [3H]InsP3 binding is unaffected by bacterial preparations of phospholipase C , D, or Az. Soy- bean or lima bean lectins diminish binding somewhat sug- gesting that the receptor might be a glycoprotein. Additional support for this notion comes from the adherence of purified receptor to concanavalin A columns (see below).

Solubilization of [3H]InsP3 Binding Sites by Detergents- We treated membrane fractions with detergent solutions for 30 min at 4 "C and examined [3H]InsP3 binding in the super- natant and particulate fractions. In the absence of detergent, all [3H]InsP3 binding is recovered in the membrane fraction with no detectable binding in the supernatant. [3H]InsP3 binding appears to be inactivated by sodium dodecyl sulfate, cholate, or deoxycholate. Some detergents, such as digitonin and Tween, provide very little solubilization. The most exten- sive solubilization occurs with 1% Triton X-100, which pro- vides virtually complete solubilization of receptor binding and full recovery of the binding in original membrane prepara- tions. Three percent Triton appears to degrade binding some- what with a lower recovery in the supernatant fraction. At

TABLE I Effects of enzymes and reagents on rHlInsP3 binding

Rat cerebella were homogenized in ice-cold 50 mM Tris-HC1, pH 8.3. The homogenate was then centrifuged at 20,000 X g for 15 min at 4 "C and the supernatant decanted. Membranes were resuspended in 50 mM Tris, pH 8.3, and aliquot8 containing 0.5 mg of protein were treated with various reagents and enzymes as indicated. After appro- priate time intervals, samples were centrifuged at 10,000 X g in a Beckman Microfuge 12 for 5 min at 4 "C, the supernatant was aspirated, and membranes were assayed for [3H]InsP3 binding (see "Experimental Procedures"). The experiment was conducted twice in triplicate and data presented are mean values which varied less than 20%.

Binding treatment [3H]InsP3 binding

% control pCMBS (1 mM) 0 pCMBS (1 mM) + 8-mercaptoethanol (2 mM) 110 0-Mercaptoethanol(1 mM) 120 Dithiothreitol (1 mM) 130 a-Chymotrypsin (1 unit, 15 min, 25 "C) 0 Trypsin (0.1 unit, 5 min, 25 "C) 100 Trypsin (0.1 unit, 15 min, 25 "C) 40 Trypsin (0.1 unit, 45 min, 25 "C) 0 Heat (1 h, 60 "C) 0 Urea (1 M) 20 Soybean lectin (0.3 mg/ml) 50 Lima bean lectin (0.6 mg/ml) 70 Phospholipase C (1.25 units, 10 min, 25 'C) 100 Phospholipase D (1.25 units, 10 min, 25 'C) 100 Phospholipase A, (1.5 units, 10 min, 25 "C) 100 NaCl(1 M) 100 KC1 (1 M) 100

0.1% Triton X-100, only one-fourth as much [3H]InsP3 bind- ing is solubilized as with 1% detergent. The solubilization by Triton is dependent upon tissue concentration with optimal solubilization and recovery at 50 mg/ml wet weight, while at 500 mg/ml wet weight the extent of solubilization is reduced by 90%.

To determine if the binding site apparently solubilized by 1% Triton X-100 is contained in slowly sedimenting mem- branes, we centrifuged a solubilized preparation at 120,000 x g for 2 h. Following this treatment all the binding is recovered in the soluble fraction with none remaining in the particulate material.

The binding site solubilized with 1% Triton X-100 displays the same properties as the binding site in membrane fractions. Scatchard analysis indicates a single binding site with a KO of about 80 nM in both membrane and solubilized preparations (Table 11; Fig. 1). The inositol phosphate specificity of binding is also the same in solubilized membrane fractions with InsP3 being the only inositol phosphate displaying potent inhibition of binding (Table 111).

In most studies of [3H]InsP3 binding to the purified recep- tor, 1% Triton X-100 is employed. However, even when the detergent concentration is reduced to 0.02%, [3H]In~P3 bind-

TABLE I1 rHlInsP3 binding parameters

Saturation binding experiments were performed as described under "Experimental Procedures" utilizing centrifugation assays for crude membrane samples and spun-column assays for solubilized and pu- rified samples. Displacement of [3H]InsP3 binding by increasing concentrations of nonradioactive InsP3 allowed calculation of KD and E,. by Scatchard transformation. Typically, 10% of the total [3H] InsP, was bound. This experiment was performed twice in triplicate and data presented are mean values which varied less than 20%.

Preparation KO B , Hill (motein) coefficient

nM pmollmg Crude membranes 80 5 1 .o Triton-solubilized 85 6.25

Purified receptor 100 4500 membranes

1 2 3 4 5 Bound ( p m o l / u g )

FIG. 1. Scatchard analysis of [SH]InsPS binding to purified InsPs receptor. Saturation of [3H]InsP3 binding was examined with purified InsP, receptor by utilizing spun-column assays. Assays were conducted with 0.1 pg of pure InsP, receptor, 12.5 nM [3H]InsP3, and various concentrations of nonradioactive InsP,. Less than 10% of the total [3H]InsP3 was bound in all determinations.

1532 Inositol Trisphosphute Receptor Purification TABLE I11

Inhibition of pH]ImP3 binding by various inositol derivatives, heparin, and calcium

[‘HIInsPS binding to crude membranes was assayed as described under ”Experimental Procedures.” [3H]InsP3 binding to detergent- solubilized and purified samples was assayed by the spun-column assay (see “Experimental Procedures”). Calcium was buffered by EDTA. Data are means of four determinations which varied less than 20%.

[SH]InsP3 binding

membrane solubilized Purified

% of control

Crude Detergent

Control 100 100 100 50 nM Ins-1,4,5-P3 86 63 64 100 nM Ins-1,4,5-P3 59 56 52 1 p~ Ins-1,4,5-P3 14 26 10 1 p~ Ins-1,3,4,5-P3 96 95 98

10 p~ Ins-l-P1 92 92 95 10 p~ Ins(SO& a4 116 103 10 pg/ml heparin 54 59 40 200 nM Ca2+ 98 92 100 10 Ca2+ 18 10 500 p~ Ca2+ 12 0 102 1.5 mM Ca2+ 102

10 pM InSPs 85 91

ing to solubilized fractions is retained. Purification of [3HJZsP3 Binding Site-We initially ho-

mogenized cerebellar membranes in a buffer containing 1 mM EDTA and 1 M NaCl. The membranes were then subjected to three additional washes with a buffer containing 1 mM EDTA without salt. This washing procedure provides essentially 100% yield of [3H]InsP3 binding in the membranes with none apparent in the soluble fraction. Solubilization of washed membranes with 1% Triton X-100 provides complete recovery of ligand binding in the soluble fraction (Table IV).

The [3H]InsP3 binding site adheres to DEAE-cellulose and is retained on the column after a wash with 0.1 M NaC1.

Binding activity is eluted with 0.175 M NaCl providing a 5- fold purification with high levels of recovery.

Earlier we showed that heparin potently inhibits [3H]InsP3 binding to membrane fractions (13). Accordingly, we adsorbed the eluate from the DEAE-cellulose column to a heparin- agarose column. The receptor adheres to the column following a wash with 0.25 M NaCl but is eluted with 0.5 M NaCl providing a 160-fold purification with an 80% yield. The receptor binds to concanavalin A-Sepharose and can be eluted with 1 M a-methylmannopyranoside, indicating that it is a glycoprotein.

Further fractionation of the receptor on a gel filtration column (Sephacryl S-400) affords no further purification. The binding site elutes somewhat earlier than thyroglobulin indi- cating an approximate Stokes radius of 10 nm. Electron microscopic examination of the purified protein indicates that it is globular in nature.2 For a globular protein, this Stokes’ radius suggests a native molecular weight of approximately 1,000,000. This value may be an overestimate since Triton X- 100 bound to the protein would tend to increase its apparent Stokes’ radius.

We examined the binding protein purified through the heparin-agarose step by SDS-PAGE analysis (Fig. 2). A pro- tein band of 260,000 is the only one apparent on the gel. This protein band comigrates with [3H]InsP3 binding activity in fractions eluted from the Sephacryl S-400 column (data not shown).

Based on the Bmax in crude cerebellar membranes of 5 pmol/ mg protein and the purification of goo-fold, we calculate a molecular weight of 230,000 for a receptor which binds InsP, stoichiometrically. This value corresponds well with the value from SDS-PAGE analysis. Stoichiometric binding of InsP3 is supported by our earlier observation of a Hill coefficient of one for [3H]InsP3 binding to cerebellar membranes (13).

Properties of the Purified Imp3 Receptor Protein-The binding characteristics of the purified receptor are closely

S. Supattapone and S. Snyder, manuscript in preparation.

TABLE IV Purification of the PH]ImP3 receptor from rat cerebellar membranes

Sixteen rat cerebella were homogenized in 20 volumes of ice-cold buffer A containing 1 M NaCl and the homogenate was centrifuged at 20,000 X g for 15 min. The supernatant was decanted and the pellet was homogenized in 20 volumes of ice-cold buffer A and centrifuged at 20,000 X g for 15 min. This extraction in buffer A was repeated two more times. The final pellet is referred to as “cerebellar membranes.” These cerebellar membranes were resuspended in 50 mg/ml wet weight with buffer A containing 1% Triton X-100. This mixture was incubated for 30 min on ice and then centrifuged at 120,000 X g for 2 h. The supernatant was removed and its salt concentration was adjusted to 0.1 M NaCl by addition of a concentrated salt solution. This solution was then passed over a 70-ml DEAE-cellulose column equilibrated in buffer A with 1% Triton and 0.1 M NaC1. The column was washed with 200 ml of column buffer and eluted by increasing the salt concentration to 0.175 M NaC1. During elution, the outflow of the DEAE-cellulose column was hooked in series to a 3-ml heparin-agarose column. Following elution of the DEAE-cellulose column, the two columns were disconnected and the heparin-agarose column was washed with 20 ml of buffer A 0.1% Triton and 0.25 M NaCl and eluted with 3 ml of 50 mM Tris, pH 7.7, 1 mM @mercaptoethanol, 0.1% Triton, 0.5 M NaC1. The eluate was applied to a 0.5 X 1-cm column of concanavalin A-Sepharose, washed with 10 ml of 50 mM Tris, pH 7.7, 1 mM 8-mercaptoethanol, 0.1% Triton, 0.1 M NaC1, and then eluted in 3 ml of the same buffer with 1 M a-methylmannopyranoside. The eluate was applied to a 1.5 X 50-cm column of Sephacryl S-400 equilibrated in buffer A with 0.1% Triton. The gel filtration column was run at 0.1 ml/min and 3-ml fractions were collected. All operations were carried out at 4 ‘C. Binding assays were performed as described under “Experimental Procedures” utilizing 12.5 nM [3H]InsP3. This purification scheme has been replicated 5 times. Data presented are from a typical experiment.

Fraction Protein [3H]InsPs Specific activity binding Purification Yield

w pmol P n W m g -fold % Cerebellar membranes 30 8.8 0.29 1 100 1% Triton X-100 solubilization 28 10 0.37 1.25 116 DEAE-cellulose 4 7.4 1.8 6.25 83 Heparin-agarose 0.020 5.9 290 1000 66 Concanavalin A 0.010 3.0 300 1030 34 Seuhacrvl S-400 0.005 1.3 270 900 15

Inositol Trisphosphate Receptor Purification 1533

A. B. C.

1200

116

192

166

FIG. 2. SDS-PAGE analysis of purified InsPs receptor. A 3- 17% exponential gradient polyacrylamide gel stained with Coomassie Blue. A , 40 pg of crude Triton X-100-solubilized cerebellar mem- branes. B, 2 pg of purified InsP3 receptor. C, molecular weight markers: myosin (200 kDa), 8-galactosidase (116 kDa), phosphorylase b (92 kDa), and bovine serum albumin (66 kDa). Silver staining displayed no additional protein bands.

similar to those of the initial detergent-solubilized fractions and the membrane-bound receptors. Scatchard analysis re- veals a single binding component with a KO of about 100 nM, similar to the membrane-bound and detergent-solubilized preparations (Table 111). The relative potencies of inositol phosphates are the same in competing for [3H]InsP3 binding to purified, detergent-solubilized and membrane-bound frac- tions (Table 111). InsP3 provides 50% inhibition at concentra- tions slightly below 100 nht and 75-90% inhibition at 1 p ~ . By contrast, 1 pM InsP, 10 pM InsP, 10 pM InsP1, and 10 p~ inositol hexasulfate all do not alter [3H]In~P3 binding to any of the receptor preparations. Heparin inhibits ['HH]InsP3 binding to a similar extent in the three receptor preparations.

One striking difference between the purified and unpurified receptor involves the effect of calcium. While 500 pM calcium reversibly inhibits [3H]InsP3 binding in the particulate and detergent-solubilized receptors, it has no effect on binding to the purified receptor protein.

The dependence upon pH is the same in purified, detergent- solubilized, and particulate receptors. As reported previously (13), [3H]InsP3 binding increases 6-fold between pH 7.0 and 8.5 and remains constant between pH 8.5 and 9.5 (data not shown).

We were concerned about the possibility that InsP, binds to the InsP3 5-phosphatase. This appears to be unlikely for the following reasons: 1) the affinity of InsP3 for the receptor binding sites is nearly 1000 times greater than the half- saturating concentration of InsP3 for the phosphatase (11- 13); 2) heparin does not inhibit the phosphatase but does displace InsP3 binding (13); 3) the molecular weight for the [3H]InsP3 binding site is markedly different from that of the InsP3 phosphatase (15); 4) the InsP3 phosphatase is largely soluble, while the InsP3 receptor binding site is entirely par- ticulate. To further examine this question, we fractionated detergent-solubilized crude cerebellar membranes on a hepa- rin-Sepharose column. Eighty percent of the phosphatase activity is eluted at 0.1 M NaCl and another 20% at 0.25 M NaC1. No [3H]InsP3 binding is eluted a t either of these con- centrations, while increasing the NaCl concentrations to 0.5

M elutes all [3H]InsP3 binding. Taken together with the other results, we conclude that the [3H]InsP3 receptor binding site is distinct from InsP3 phosphatase.

Tissue Factors Mediating the Sensitivity of pH]lnsP3 Bind- ing to Calcium-Calcium potently inhibits [3H]InsP3 binding to membrane-bound and detergent-solubilized fractions in a reversible fashion. Inhibition by calcium can be reversed either by washing membranes with 1 mM EDTA or by adding 1 mM EDTA after treatment with 500 p~ CaC12. The revers- ibility of inhibition by calcium rules out the possibility that calcium merely activates a protease that degrades ligand binding activity.

Following purification of the InsP3 receptor protein, cal- cium (50-500 p ~ ) fails to inhibit [3H]InsP3 binding (Table V). We wondered whether purification of the receptor removes a tissue component which confers sensitivity of the InsP3 receptor to inhibition by calcium. Accordingly, we examined various preparations for their ability to restore sensitivity of the purified receptor to calcium. Calmodulin is well known to bind calcium potently, but neither 2.8 nor 28 pg/ml calmod- ulin in the presence or absence of calcium affects [3H]InsP3 binding to purified receptors. Furthermore, the purified recep- tor fails to adhere to a calmodulin-agarose column either in the presence or absence of 1 mM CaCl,.

Soluble supernatant fractions of the cerebellum also fail to confer calcium sensitivity to InsPB binding. By contrast, de- tergent-solubilized membrane fractions confer calcium sensi- tivity on InsP3 binding in a concentration-dependent fashion. The solubilized membrane fractions are capable of restoring completely the sensitivity of InsP, binding to inhibition by calcium (Table V). This inhibition can be completely reversed by subsequent addition of 1 mM EDTA. The potency of calcium in inhibiting [3H]InsP3 binding to purified receptors in the presence of solubilized cerebellar extract is the same as in intact membrane receptor preparations (data not shown). The factor which confers calcium sensitivity appears to be a protein as its activity is destroyed by treatment with trypsin or heating (Table V). Membranes from heart, liver, testes,

TABLE V Inhibition of pH]InsP3 binding to purified Imp3 receptor

preparations by calcium The InsP3 receptor was purified as described in the legend to Table

IV. [3H]InsP3 binding was assayed by the spun-column technique as described under "Experimental Procedures." Data are given for 1.5 mM added calcium (500 p~ final concentration in the presence of 1 mM EDTA) although similar results were obtained for a range of calcium concentrations (1 p~ to 2 mM final concentrations). Cere- bellar membranes were solubilized with 1% Triton in buffer A for 30 min at 4 "C. The solubilized samples were then centrifuged at 20,000 X g for 1 h at 4 "C and the supernatant passed over a 3-ml heparin- agarose column to remove the InsP3 receptor. Heat treatment of membranes occurred at 60 "C for 1 h. For trypsin treatment, 1 unit of enzyme was incubated for 15 min at 25 "C at which time 5 units of soybean trypsin inhibitor were added to terminate proteolysis. This set of experiments has been performed three times with the same results.

[aH]InsPS binding (cpm)

500 pM calcium No calcium Addition

Purified InsP3 receptor alone 1500 1480 +Calmodulin 28 pg/ml 1480 1470

2.8 pg/ml 1490 1480 +Cerebellar soluble fraction 1000 1000

+Heparin-absorbed solubilized 20 1500

+Heat-treated membranes (1 mg/ml) lo00 1490 +Trypsin-treated membranes (1 mg/ml) 1500 1470

(10 mg/ml)

cerebellar membranes (1 mg/ml)

1534 Inositol Trisphosphate Receptor Purification

and kidney solubilized with 1% Triton do not reconstitute calcium sensitivity.

DISCUSSION

In this study we have purified the Imps receptor-binding protein to apparent homogeneity from rat cerebellum. The purified receptor protein appears as a band of 260,000 on SDS-PAGE. The potent and reversible inhibition by calcium of Inspa binding to the receptor in its membrane-bound state or after initial detergent solubilization fits with a role for the InsP, receptor in mobilizing calcium from intracellular stores. Calcium does not inhibit InsP, binding to the purified receptor protein, but addition of a detergent-solubilized membrane preparation restores the inhibitory effect of calcium, suggest- ing that in intact membranes the InsP3 receptor is closely associated with a calcium-binding protein which regulates the InsP3 receptor. In peripheral tissues [3H]InsP3 binding to membrane fractions is not inhibited by calcium (8-ll), imply- ing that the postulated calcium-binding protein in cerebellar membranes does not occur in peripheral tissues or is not linked in these tissues to InsP, receptors. Since the InsPs- binding protein of peripheral tissues has not been solubilized and purified, we do not know whether it represents the same protein as the receptor we have purified from the brain.

Three groups (19-21) have previously described cerebellum- specific proteins resembling the IP3 receptor in molecular weight and tissue concentration. One of these proteins, PCPP 260 (19), is a substrate for protein kinase A (CAMP-dependent protein kinase). In preliminary work, we have observed phos- phorylation of the InsP, receptor by protein kinase A, but not protein kinase C or Ca*+/Calmodulin-dependent protein ki- nase. Phosphorylation of the InsP, receptor by protein kinase A might provide a mechanism for “cross-talk’’ between the CAMP and phosphoinositide second-messenger systems.

Acknowledgments-We thank Nancy A. Bruce and Dawn C. Dod- son for secretarial assistance. We thank Drs. Robin Irvine and Michael Berridge for donation of inositol phosphates and Paul Green- gard, J. Steiner, and G. V. Bennett for helpful discussions.

1. 2. 3. 4.

5. 6. 7.

8.

9.

10.

11.

12.

13.

14.

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