European Journal of Pharmacology - Molecular Pharmacology Seeticm, 225 (I 992) 179-193

179

© !992 Elsevier Science Publishers B.V. All rights reserved 0922-4106/92/$05.00

EJPMOL 90266

Pharmacological characterization of inositol 1,4,5,-trisphosphate binding sites: relation to Ca z+ release Bernard Mouillac, t: nette Devilliers, Serge Jard and Gmes Guillon Centre CNRS-INSERM de Pharmacoiogie-Endocrinologie, M'~;apellien France

Received 31 July 1991, revised MS 22 October 1991, accepted 12 November 1991

Two subcellular fractions, one enriched in plasma membranes and tile other in endoplasmic reticulum membranes, were obtained from WRK~ cells using a combination of differential centrifugations and Pcrcoll gradient fractionation. Specific inositol 1,4,5-trisphosphate (lns(1,4,5)P 3) binding sites were detected in these two preparations. Endoplasmic reticulum membranes exhibited a binding capacity which was about 5-fold higher than that of plasma membranes. Dose-dependent Ins(1,4,5)P 3 binding was determined. Experimental data obtained with endoplasmic reticulum membranes could be adequately fitted with a two-site model (a high-affinity binding site with K a and Bm~× values of 0.7 _+0.15 nM and 12.9 + 5 fmol/mg protein and a low-affinfly binding site with K d and Bmax values ol 44.2 + 14.6 nM and 143 + 43 fmol/mg protein). Both the high- and low-affinity binding sites were selective for Ins(1,4,5)P3. Besides Ins(1,4,5)P 3, Ins(1,3,4,5)P4 also discriminated between the two populations of sites while heparin interacted with the high- and low-affinity binding sites with the same affinity. Ins(l,4,5)P3-induced calcium release from endoplasmic reticulum vesicles was determined by monitoring the calcium concentration in the extravesicular compartment with fura-2. Under experimental conditions where the degradation of Ins(1,4,5)P 3 was reduced (incubation at 0°C). a high-affinity Ins(1,4,5)P3-induced calcium release (apparent K~ct around 20 riM) could be demonstrated. These results suggest that in WRK l cells, the endoplasmic reticulum is a Iaajor site for lns(1,4,5)P 3 action and that the high-affinity binding sites located on the endoplasmic reticulum membranes may contribute to the physiological regulation of the cytosolic free calcium concentration. ,..a- ; Inositol 1,4,5-trisphosf~hate receptors: WRKj cells; Inositol phosphates

1. Introduction The

hydrolysis

of

phosphatidylinositol

4,5-bis-

~h-o~,~,~v,,,ov,,,,,~r,,,~,~phospholipase C is a primary event in the

mechanism of action of hormones, neurotransmitters or other regulator3, molecules (Berridge, 1987). This hydrolysis l i b e r a t e s inositol 1,4,5,-trisphosphate (lns(l,4,5)P 3) and diacytglycerol which both act as second messengers: Ins(1,4,5)P 3 for calcium mobilizati6n (Berridge and Irvine, 1989) and diacylglycerol for activating protein kinase C (Nishizuka et al., 1984). Ins(l,4,5)P 3 has been shown to release sequestered calcium from intracellular stores (Streb et al., 1983) in a wide variety of cells by interacting with a specific receptor (Guillemette et al., 1987). Ins(1,4,5)P 3 binding sites have been characterized in many different cell types from both peripheral tissues and brain (13aukal et al., 1985; Guillemette et al., 1987;

Correspondence to: Gilles Guillon, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier Cedex 5, France. Tel. 67-14.29.23; Fax 67-54.24.32.

Worley et at., 1987a). Two years ago, the Ins(1,4,5)P 3 binding protein was purified from rat cerebellum (Supattapone et ai., 1988a). It is a m e m b r a n e glycoprotein with a relative molecular mass of 250 kDa which interacts with both heparin and concanavalin A and is a substrate for cyclic A M P - d e p e n d e n t protein kinase ($upattapone ct ,&, t988b). Recently, the Ins(1.4,5)P 3 receptor has been cloned (Furuichi et al., 1989) and structural information clearly demonstrated that the same molecule mediates both Ins(t,4,5)P 3 binding and release of calcium (Ferris et ai., 1989). The structure of the Ins(L4,5)P 3 receptor was also shown to be strikingly similar to the recently cloned ryanodine receptor (RR), the calcium channel of the sarcoplasmic reticulum of skeletal muscle (Takeshima et al., 1989). Another important similarity between the lns(1,4,5)P 3 receptor and the R R is that both associate into multimerle structures. T h e r e is agreement that the relative molecular mass ~f solubilized lns(t,4,5)P 3 receptor is around 1000 kDa: suggesting a tetrameric structure, the four subunits possibly surrounding a central Ca z+ channel. Several points concerning the hts(1,4,5)P3 receptors

180 remain controversial: their precise intracellular localization, the existence of multiple subtypes and the discrepancy between tile affinity of the receptors and the potency of Ins(1,4,5)P 3 to release calcium. First, recent immunocytochemical studies (Ross et al., 1980) showed that the Ins(1,4,5)P3 rectptors are mainly located on rough and smooth endoplasmic reticulum, a localization in good agreement with calcium release experiments performed on permeabilized ceils (Streb et al., 1983) or subcellular membrane preparations (Prentki et al., 1984). Other laboratories (Furuichi et al., 1989) have provided evidence for a plasma membrane localization, a conclusion that is supported by electrophysiological data obtained from human Tlymphocytes (Kuno and Gardner, 1987) and Ins(1,4,5)P 3 binding studies on rat hepatocytes (Mauger et al., 1989). A single specialized Ins(1,4,5)P3-responsive organelle (the calciosome) has also been suggested (Volpe et al., 1988). Second, in most cases, binding experiments revealed the existence of a single type of Ins(l,4,5)P 3 binding site with dissociation constant values (K d) in the range of 1-80 nM (Baukal et al., 1985; Guillemette et ah, 1987; Worley et al., 1987b). However, the existence of two Ins(1,4,5)P 3 receptors with different affinities on the same cell type has also been described in rat liver (Sp~it et al., 1986) and bovine pituitary microsomal preparations (Sp~it et al., 1987), and more recently in a crude plasma membrane preparation from rat hepatocytes (Mauger et al., 1989). Therefore, the question was raised as to which receptor is functionally coupled to calcium release. Based on studies performed on permeabilized rat hepatocytes, Mauger et al. (1989) suggested that the low-affinity binding site was responsible for calcium release and the high-affinity binding site could represent a desensitized state of the receptor. Third, a close correlation between lns(I,4,5)P 3 binding and lns(l,4,5)P3-induccd Ca 2+ release has been reported for rat basophilic leukemia cells (Meyer et al., 1988), and on rat cerebellar microsomal fractions (Stauderman et al., 1988). However, in most permeabilized cells and subcetlular fractions, the concentration of Ins(l,4,5)P~ needed to evoke hall~maximal calcium release was found to be in a micromolar range (Bcrridge, 1987). This value is 100to 1000-fold higher than the K d for lns(l,4,5)P 3 binding. Previous studies by our group and others indicated that WRK~ cells represent a convenient system to investigate the different steps involved in receptormediated phospholipase C activation and subsequent calcium mobilization (Guillon et al., 1986a; Kirk et al., 1986; Mouillac et al., 1989, 1990). These cells derived from a chemically-induced mammary tumor, express a large number of phospholipase C-coupled vasopressin receptors of the V~, subtype (Gufllon et at., 1986a). Vasopressin induces a marked inositol phosphate accu-

mulation in intact cells (Kirk et al., 1986) and in acellular preparations derived from these cells (Guillon et al., 1986b). In the present study, we addressed the problem of the location of the putative Ins(1,4,5)P 3 receptors and the relation of lns(1,4,5)P 3 binding to Ins(1,4,5)P3-induced calcium release. For this purpose, 'we developed a fractionation method which enabled us to obtain subcellular fractions enriched in either plasma membranes or endoplasmic reticulum membranes. We also designed experimental conditions in which both Ins(1,4,5)P3 binding and lns(1,4,5)P3-induced calcium release could be studied under similar conditions. Calcium release experiments were performed by using the calcium fluorophore fura-2. This technique allows a rapid determination of calcium release as compared to the use of Ca 45 and wa', more reliable for the functional characterization of an Ins(1,4,5)P3-sensitive calcium channel since these phenomenons were extremely rapid (Champeil et al., 1989). Special attention was paid to the influence of Ins(1,4,5)P 3 degradation on the determination of binding parameters and calcium release

2. Materials and methods

Z I. Chemicals

[3H]Ins(1,4,5)P3 (30-60 C i / m m o l ) was obtained from Amersham, myo-[2-3H]inositol (16.5 C i / m m o l ) was from Comissariat ~ l'Energie Atomique (France). lnositol 1,4,5-trisphosphate (Ins(1,4,5)P3), lns(1,3,4)P3, fura-2 (free acid) and ionomycin were obtained from Calbiochem. Inositol !,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), lns(1,4)P 2, inositol 1-phosphate (Ins(l)P), phosphocreatine and creatine kinase were obtained from f_=ehringer-Mannheim, heparin was from Prolabo. Deoxyribonuclease I and dithiothreitol (DTT) were from Sigma and Percolt was from Pharmacia. Arginine vasopressin (AVP) was from Bachem. AI! other reagents were from Sigma or Merck and of the highest available grade. 2.2. Cell culture

As previously described (Guillon et al., 1986a), W R K 1 cells were grown in monolayer culture (Petri dishes) in Eagle's minimum essential medium (MEM) containing: Earle's salts; glutamine (290 mg/I); penicillin (100 U / r o l l ; streptomycin (100 mg/ml); fetal calf serum (5%, v / v ) and rat serum (2%, v/v). Cells were incubated at 37°C in a humidified atmosphere composed of 95% air and 5% carbon dioxide. The culture medium was changed every 2 days and the experiments were performed 7 days after seeding.

2.3. Preparation of subcellular fractions and ultrastructuml examinations Col~fluent W R K j cells were washed 3 times at 0°C phosphate-buffered saline (PBS) without calcium and magnesium. Cells were then scraped at 0°C with a rubber policeman into the following homogenization medium: KCI, 100 raM; NaCI, 20 mM; KH2PO ~, 5 raM; MgCI 2, 2 raM; Tris-HCl (pH 7.4), 25 raM; EGTA, 2 mM; and DTT, 1 mM. Cells were homogenized at 0°C in a Dounce Potter homogenizer equipped with a loose pestle (18 strokes). This gentle homogenization procedure followed by a 100 × g centrifugation for 5 rain at 0°C allowed the recovery in the supernatant (SN1) of plasma membranes and cytosolic constituents (see below). Plasma membranes were recovered as a 44,000 x g (20 rain at 0°C) pellet of the SN1. Most of the other cellular organeltes were sedimented in the 100 x g pellet PI. This pellet was suspended in 3 mi of the homogenization medium (ranes added. Calcium uptake was initiated by the addition of 2 mM ATP in a volume of 2 JLL Calcium release was determined either at 37°C or DoC after short periods of incubation with the analogue tested (5 s). In the latter case, the fluorimeter cuvette was rapidly equilibrated at DoC and calcium release was initiated by the addition of Ins(1,4,5)P3 in a volume of 2 I-tL Fluorimetric monitoring of free calcium concentration was performed using a Perkin-Elmer LS-5 fluorescence spectrophotometer: the excitation wavelength was 340 or 380 nm

(slit 10 nm) and the emission was recorded at 500 nm (slit 2.5 nm). Calcium uptake and release activities were measured under constant agitation. Each record was calibrated by the addition of known amounts of calcium (2 nmol of Ca z+) to the medium. The free calcium concentration of the medium was calculated from the Fmax and Fmin values obtained by adding excess Ca z+ (l mM) and EGTA pH 8.5 (20 mM) respectively, after treatment with 1 I-tM ionomycin. The free calcium concentration was calculated according to the equation of Grynkiewicz et aL (l985): [Ca z+) = K d X {3 X (R-Rmin)/(Rmax - R) where K d = 225 nM, {3 = Fmin 380 nm/Fmax 380 nm, R max = Fmax 340 nm/Fmax 380 nm, R min = Fmin 340 nm/Fmin 380 nm and R = F 340 nm/F 380 nm. 2.8. Control of Ins(J,4,5)P3 and Ins(J,3,4,5)P4 degradation

Subcellular fractions from WRK 1 cells were incubated for various periods of time in the presence of labeled Ins(l,4,5)P3 (lor 10 nM) or Ins(l,3,4,5)P4 (60 nM) under experimental conditions identical to those used for Ins(1,4,5)P3 binding or Ins(1,4,5)P3-induced calcium release (see above). The reaction was stopped at DoC by adding a mixture of different unlabeled inositol phosphates (inositol; Ins(l)P; Ins(l,4)P z; Ins(l,3,4)P3; Ins(l,4,5)P3 and Ins(l,3,4,5)P4 ; final concentration of each, 0.1 I-tM), and centrifuging the samples at 2000 X g for 10 min at DoC. Labeled inositol phosphates present in the supernatant were separated by a high performance liquid chromatography (HPLC) technique, as previously described (Mouillac et aI., 1989). Briefly, HPLC experiments were performed on a Partisil 10-SAX (25 cm X 0.46 em) anion exchange column. After injection of the sample, the column was washed with distilled water for 10 min to remove any unbound 3H-Iabeled material. Then, the inositol phosphates were eluted by increasing the concentration of ammonium formate (adjusted to pH 3.7 with or~ thophosphoric acid) from 0 to 3 M. The flow rate Jas kept constant at 1.1 mljmin and fractions were taken every 0.5 min. Aquasure II scintillation fluid (l.5 rill) was added in each fraction and the radioactivity counted. All results were corrected for quenching and expressed in dpm. To identify each inositol phosphate peak, a mixture of tritiated standards was run in parallel under the same experimental conditions. Based on these data, we assumed that peaks having similar retention times corresponded. 2.9. Mass measurement of Ins(J, 4, 5)P3

WRK 1 cells were routinely grown in MEM for 5 days. Then, cells were pre-incubated for 10 min at 37°C in a HEPES buffer pH 7.4 which contained 1 mg/ml BSA but no lithium. The cells were then further incu-

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185 TABLE 1 Biochemical characterization of subcellular fractions from WRK1 cells. Subcellular fractions derived from WRK 1 cells were obtained as indicated in Materials and methods. Glt~eose-6-phosphatase a_,-;d5'-nucleolidase activities were measured at 37°C as described in Materials and methods. For each assay, 100 p.g of protein deriving from each subceliular fraction were used. The amount of inorganic phosphate formed was measured by the method of Fiske and Subbarow. The end,me marker activities were expressed as nmoi Pi/mg protein/30 rain. Values are means_+_S.E.M. derived from three different experiments each done in triNica~e. Subcellular fraclions Homogenate 44.7± 3.1(3)

Protein con{ent (mg} 5'-Nucleotidase activi~' (nmol Pi/mg protein/30 rain) Glucose-6-phosphatase activity (nmol Pi/mg protein/30 rain)

t

Plasma membrane 2.1_+ 0.2(3)

0.3(3t

102(1 ±t67131

1(}0 + 30(3)

2511 ±20(3)

120 ± 10(31

93(1 ±120(31

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Endoplasmic reticulum

tog [free Ca 2 + ] Fig. 2. Phospholipase C activities in subce!lular fractions from WRK I cells. WRK 1 cells were grown in the presence of 2 /zCi/ml myo-~23H]inositol as previously described_ Fractions enriched in pIasma membranes ( I , D) and endoplasmic reticulum (e, ©) were prepared as described in Materials and methods. Phospholipase C activities were measured on these two preparations (3{l-80 #g protein/assay~ with ( D, ©) or without ( I , e) I /z M vasopressin + 0.1 mM guanosine triphosphate (GTP} and in the 9resence of the indicated free calcium concentrations. Inositol bis- and trisphosphates which accumulated during a 6 rain incv,bation period at 37°C were measured. Results were expressed in dpm per mg of protein. Values on the graph are means± S.E.M. of quadruplicate determinations derived from a single experiment which was representative of three.

o f t h e l a b e l e d ligand), a d d i t i o n o f a large excess o f u n l a b e l e d Ins(1,4,5)P_~ (I ~zM) led to a r a p i d ( h a l f - t i m e a r o u n d 10 s) a n d a l m o s t c o m p l e t e d i s s o c i a t i o n o f t h e l a b e l e d tigand. D i s s o c i a t i o n rate c o n s t a n [ s (k_ i) o f 3.0 ± 0.2 a n d 4.8 _+ 0.3 m i n - ~ ( t h r e e d i s t i n c t d e t e r m i n a t i o n s ) for e x p e r i m e n t s p e r f o r m e d with 1 a n d t 0 n M [3H]Ins(1,4,5)P3 respectively, w e r e c a l c u l a t e d f r o m s e m i - l o g a r i t h m i c plots o f *he d i s s o c i a t i o n c u r v e s (fig, 3B, inset). T h e s e v a l u e s w e r e s t a t i s t i c a l b ~ d i f f e r e n t (t = 5.0, P < 0 . 0 0 1 ) . I n s ( t , 4 , 5 ) P 3 b i n d i n g to e n d o p l a s m i c r e t i c u l u m m e m b r a n e s was s a t u r a b l e . B i n d i n g o f [3H]l n s ( l , 4 , 5 ) P 3 (1 n M ) was i n h i b i t e d in a d o s e - d e p e n d e n t m a n n e r by i n c r e a s i n g c o n c e n t r a t i o n s o f u n l a b e l e d Ins(1,4,5)P 3 (fig. 4A). R e s i d u a l l a b e l e d Ins(1,4,5)P 3 b i n d i n g in t h e p r e s e n c e e f a l a r g e excess o f cold lns(1,4,5)P 3 (1 , a M ) r e p r e s e n t e d a b o u t 15% o f t h e total b i n d i n g d e t e r m i n e d in t h e a b s e n c e o f u n l a b e l e d ms( . . . . . ," 3- T h e S c a t c h a r d plot o f t h e d o s e - d e i } e n d e n t specific b i n d i n g (fig. 4A, i n s e t ) was cuB, ilinear s u g g e s t ing e i t h e r n e g a t i v e c o o p e r a t M t s in Ins(1,4,5)P 3 b i n d i n g or the existence of several categories of independent b i n d i n g sites. A n a l y s i s o f t h e b i n d i n g d a t a with t h e Ligand cc,-aputer program indicated that experimental d a t a c o u l d b e f i t t e d with a m o d e l involving t h e p r e s e n c e o f ~ o c a t e g o r i e s o f h i g h - a n d low-affinity b i n d i n g sites for I n s ( t , 4 , 5 ) P 3. T h e m e a n K d a n d B~.~ v a l u e s d e d u c e d f r o m five e x p e r i m e n t s identical to t h a t s h o w n ~n fig. 4 A w e r e 0.7_+ 0.1 n M a n d t2.9 ± 5 f m o l / m g p r o t e i n a n d 44.2 + 14.6 n M a n d 143 _+ 43 f m o l / m g p r o t e i n for t h e h i g h - a n d low-affinity b i n d i n g sites r e s p e c t i v e l y ( F i s h e r ' s test, P < 0.05 in five distinct experiments),

Fig. I. Electron micrographs of WRKI cells and different subcet!ular fractions. Ultrathin sections of different preparations were performed as described in Materials and methods and examined in a Jeol 20{~) EX electron microscope: (A)control WRK I cells attached to Petri dishes, ~B) plasma membrane-enriched fraction, (C) band t of the discontinuous Percoll gradient, Le. a mixture of piasma membrane vesicles and endoplasmic reticulum vesicles, (D) endoplasmic reticutum-enrkbed fraction. Bars, t ,am: magnification, x 6tXlt/.

186 Specific l n s ( 1 , 4 , 5 ) P 3 b i n d i n g c o u l d also b e d e t e c t e d in t h e p l a s m a m e m b r a n e f r a c t i o n (fig. 4B). H o w e v e r , the amount of labeled Ins(1,4,5)P 3 bound per mg of p r o t e i n in t h e p r e s e n c e o f 1 n M [3H]lns(1,4,5)P3 w a s

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Fig. 3~ Time course of [3H]lns(1,4,51~ binding to endoplasmic reticulum fractions. Panel A: Association time course. Endoptasmic reticulum preparations (100-150 /zg protein/assay) were incubated for different periods of times at 0°C with 1 nM (O) or 10 nM (e) [3H]lns(1,4,5)P3. At the indicated times, samples were removed and filtered as described in Materials and methods. Data were corrected for non-speciiic binding and expressed as a percentage of maximal specific binding of [3H]Ins(1,4,5)P3 measured after a 15 rain incubation period (288 dpm and 285 dpm for 1 and 10 nM labeled Ins(1,4,5)P 3 respectively). Note that the specific radioactivity of labeled Ins(1,4,5)P 3 was 32.4 and 9.7 Ci/mmol for experiments performed with 1 and 10 nM Ins(1,4,5)P 3 respectively. Values on the graph are means ± S.E.M. of triplicate determinations obtained from one experiment which was representative of three. Panel B: Dissociation time-course. Endoplasmic reticulum preparations (i00-150 /zg protein/assay) were incubated for 15 rain at 0°C in the presence of 1 nM (o) or 10 nM (1) [3Hllns(l,4,5)p3. Dissociation of the labeled |igand was initiated by the addition of 1 I~M unlabeled Ins(1,4,5)P3 (t = 0) and the radioactivity remaining bound (B) was determined at the indicated times. Data were corrected for non-specific binding and expressed as a percentage of maximal specific binding measured at time t = 0. Semi-logarithmic plots of the dissociation curves Ln [(B-Beq)/(B0-Bcq)] vers-:s time (s) are shown in the inset. The apparent equilibrium values for specific binding Betj leading to the best exponential fit close to 0.1 x B 0 were determined empirically. Values on the graph are means± S.E.M. of triplicate determinations obtained from one experiment which was representative of three.

Fig. 4. Dose-dependent [3H]Ins(1,4,5)P3-specific binding to endoplasmic reticulum and plasma membrane preparations. Panel A: Endoplasmic reticulum fractions (188 /zg protein/assay) were incubated for 15 rain at &C in a binding medium containing 1.12 nM labeled lns(1,4,5)P3 (total dpm/assay = 31,200) and increasiilg concentrat~ons of unlabeled Ius(l,4,5)P3. Data were expressed as total dpm bound/mg protein (total binding in the absence of added unlabeled lns(1,4,51P 3 was 422 dpm/assay, non-specific binding measured at 1 ,u.M unlabeled lns(1,4,5)P3 was 65 dpm/assay). Values on the graph are means of quadruplicate determinations derived from a single experiment which was representative of five independent experiments. The Scatchard plot of the data is shown in the inset. Panel B: Plasma membrane fractions (250 /zg protein/assay) were incubated for 15 rain at 0°C in binding medium containing 1 nM labeled Ins(1,4,5)P3 (total dpm/assay = 21,3001 and increasing concentrations of unl-abeled Ins(1,4,5)P3. Data were expressed as total dpm bound/rag protein (total binding in the absence of added unlabeled Ins(1,4,5)P3 was 108 dpm/assay, non-specific binding measured at 1 #.M unlabeled lns(1,4,5)P3 was 65 dpm/assay). Values on the graph are means of quadruplicate determinations derived from a single experiment which was representative of three independent experiments. The Scatchard plot of the data is shown in the inset.

a b o u t 5 t i m e s l o w e r t h a n t h a t m e a s u r e d in t h e e n d o plasmic reticulum fraction. Non-specific binding accounted for about 55-60% of total binding. This high non-specific binding limited the accuracy of the determination of the specific binding. Nevertheless, Ligand a n a l y s i s o f t h e b i n d i n g d a t a a l s o i n d i c a t e d a b e t t e r fit with a two-site model than with a one-site model. Also, the K d value and the maximal binding capacity of the low-affinity binding sites could not be adequately de-

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tration will label preferentially high-affinity binding sites (72% of bound [3H]Ins(1,4,5)P3 attached to highaffinity binding sites) while at a 10 nM concentration the larger part (69%) of bound Ins(1,4,5)P 3 will correspond to binding to the low-affinity binding sites. The data from all experiments were fitted with a two-site model using the Ligand computer program. As shown in fig. 5A and B, [3H]Ins(1,4,5)P3 binding sites exhibited a high selectivity for Ins(1,4,5)P 3, Ins(1)P was almost inactive in inhibiting [3H]Ins(1,4,5)P3 binding. Ins(1,4)P z and the Ins(l,3,4)P 3 isomer inhibited

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