Purification, pharmacological characterization and photoaffinity labeling of sigma receptors from rat and bovine brain. David I. Schuster *, Frank J. Arnold 1, ...
BRAIN RESEARCH ELSEVIER
Brain Research 670 (1995) 14-28
Research report
Purification, pharmacological characterization and photoaffinity labeling of sigma receptors from rat and bovine brain David I. Schuster *, Frank J. Arnold 1, Randall B. Murphy Department of Chemistry and Center for Neural Sciences, New York UnicersiO', New York, NY 10003, USA Accepted 13 September 1994
Abstract
The sigma receptor/binding site, found in the brain and periphery, binds haloperidol, ( + )-benzomorphans, N-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) and certain atypical neuroleptics with high affinity. We have succeeded in ca. 6,000-fold purification of protein(s) from rat and bovine cerebellum which display pharmacology characteristic of the sigma receptor. This purification was achieved by affinity chromatography using a Sepharose gel linked to a new high-affinity ligand, (S)-3-(3-methoxyphenyl)-Y-oxo-Y-phenyl-N-propylpiperidine, an analog of (S)-3-PPP. Elution of the affinity column with haloperidol afforded material which, after reconstitution into bimolecular lipid vesicles, was pharmacologically characterized by specific radioligand binding assays using [3H]haloperidol combined with competitive displacement using appropriate selective ligands. Comparison of the relative rank orders of potency of the ligands in these selective sigma receptor assays corresponded well with values obtained with tissue homogenates. The observed enantioselectivity for the binding of SKF-10,047 and cyclazocine suggests that the material purified corresponds to the o-~ receptor subtype. SDS-PAGE indicated that the purified material consisted of two bands of approximate molecular masses 65 and 63 kilodaltons. Photoaffinity labeling of the affinity-purified receptor with [3H]azidoDTG led to incorporation of the label into material of molecular mass 50-70 kDa, by slicing of SDS gels, while similar photolabeling of crude cerebellar homogenates led to exclusive labeling of a 29 kDa polypeptide, as found previously using other tissues. Molecular sizing under non-denaturing conditions indicated the photolabeled species is a labile large receptor complex of mass ca. 300-500 kDa which gradually breaks down upon standing at - 80°C into the lower mass (50-70 kDa) material. The oreceptor ligand binding subunit, which appears to be of the o-~ subtype, appears to be contained within the 29 kDa polypcptide, which may be a subunit of the 63-65 kDa protein, which in turn appears to be a component of a much larger receptor complex. It further appears that the 29 kDa polypeptide is readily dissociable from a largcr photolabeled o- receptor complex in tissue homogenates, but does not dissociate from the photolabcled affinity-purified CHAPS-solubilized ~ receptor.
Keywords: Sigma receptor; Affinity chromatography; 3-PPP; Haloperidol; Photoaffinity labeling; Rat cerebellar receptor; Bovine cerehellar receptor
1. Introduction
The sigma receptor/binding site, originally termed the sigma (~r) opiate receptor, was first proposed by Gilbert and Martin [5] to be a putative opiate receptor subtype, which in the presence of the prototypical agent N-allylnormetazocine (SKF 10,047) produced highly characteristic CNS effects such as dysphoria and
* Corresponding author. Fax: (1) (212) 260-7905. 1 Present address: Lederle Laboratories, Pearl River, NY 10965, USA.
0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 2 3 - 0
autonomic stimulation. These actions were distinct from effects such as analgesia and sedation, produced by morphine and other typical opiates which act at/.L-, 8-, and K-opiate receptors. Su [31] later found that the binding of [3H](+)-SKF-10,047 was insensitive to etorphine, which displaces radioligands from traditional opiate receptor subtypes. The results of studies by Quirion et al. [26] and Musacchio et al. [22,23] further demonstrated that the ~r site was not a traditional opiate receptor. Similarities in the behavioral response of SKF-10,047 and the hallucinogen phencyclidine (PCP) led to a suggestion that they had a common site of action [44]. It later became apparent from autoradiographic and specific radioligand binding studies
D.L Schuster et al. / B r a i n Research 670 (1995) 14-28
that the o- site was distinct from the PCP-binding site, which was identified as a component of the N M D A receptor [26]. The precise functional role of o- receptors in the brain and the periphery remains elusive. Sigma receptors/binding sites exist in pituitary, testis and ovary, adrenal gland, spleen, peripheral blood leukocytes, vas deferens, pineal gland, various gastrointestinal sites, and liver as well as the brain (reviewed in [10,39]). It has been speculated that a receptors may represent a link between the central nervous system and the endocrine and immune systems [33,34,42,43]. The molecular characterization of ~ receptors, and the determination of their possible structural relationship to other membrane associated receptors [24] is thus a subject of considerable current interest. The CNS sigma receptor/binding site has been demonstrated to bind a wide variety of neuroleptics, in particular haloperidol, with high affinity [15,32]. In addition, a number of atypical antipsychotic drugs have much higher affinity for ~r sites than for dopamine D 2 receptors [17,30]. 3-(3-Hydroxyphenyl)-piperidine (3PPP) and a large number of analogous compounds have also been shown to bind with high affinity to the sigma site [13,16], as does di-o-tolylguanidine (DTG) [40]. Specific photoaffinity labeling of cr receptors in guinea pig brain tissues using [3H]azido-DTG lead to incorporation of the label in a 29 kDa protein, observed by SDS-PAGE [12]. Higher molecular mass material (300-400 kDa) which could be eluted on non-denaturing gels was also labeled by [3H]DTG, but only the 29 kDa component could be detected under denaturing conditions. A protein of similar molecular mass (26 kDa) was specifically labeled by [12Sl]azidococaine, a highly selective Cr ligand; labeling was inhibited in the presence of haloperidol, DTG, dextromethorphan and 3-PPP [9]. We have recently succeeded in purifying components of the rat liver ¢r receptor of mass 28, 40 and 65 kDa by affinity chromatography, using an oximino derivative of haloperidol as the affinity ligand [3,27]. The fact that these proteins could be eluted using dextrallorphan and (+)-pentazocine suggests they are components of the ~r~ receptor subtype, characterized by Bowen and coworkers [7,8,26]. A 15 amino acid N-terminal sequence was obtained for the 28 kDa protein which is identical to that of rat cyclophilin A, [28,29] which supports the proposal that ~ receptors provide linkage between the immune system and the CNS [33,34,42,43]. We now describe purification by affinity chromatography of material from rat cerebellum that displays characteristic ~ pharmacology, and which is photolabeled in a o--specific manner by [3H]azido-DTG. The affinity ligand utilized in the present study [1] is a derivative of 3-PPP.
15
2. Materials and methods
2.1. Preparation of tissue Male, Sprague-Dawley rats (150-350 g, Taconic Animal Farms, Germaintown, NY) were surgically anesthetized with CO 2 or ether, rapidly decapitated, and cerebella were dissected on ice. All work with animals was carried out in accord with NIH guidelines. The pooled cerebella were homogenized in 20 vol (w/v) of 50 mM Tris-HC1 buffer, pH 8.0, which contained 100 p.g/ml of bacitracin (Sigma), 250 p.M phenylmethanesulfonyl fluoride (Calbiochem), 200 ~ g / m l Soybean Trypsin inhibitor (Sigma; Type II-S), 250 p.M o-phenanthroline (Baker), 1 m g / m l Bovine Serum Albumin (Sigma; Fraction V, A2153), and 5 mM EGTA. This homogenate was stored until required in a low temperature freezer (-80°C). Samples could be stored for several months without appreciable loss in binding activity (see below). A similar protocol was used to prepare bovine cerebellar homogenates using fresh bovine brains obtained from Max Insel Cohen, Newark, NJ.
2.2. Solubilization Triton-X-100 (Rohm and Haas), Lubrol WX (Sigma), and digitonin (Sigma) solubilization studies were carried out by stirring the homogenate (see above) with 20 vols. (based on original wet weight of tissue) of a 1% (w/v) solution of the detergent in 50 mM Tris-HC1 buffer, pH 7.5 (at 4°C) for a period of 1 h at I°C. This buffer also contained the protease inhibitors at the concentrations specified above. This material was sedimented at 100,000 × g in an L5-45 ultracentrifuge (Beckman) at 4°C for 1 h. The pellet was discarded and the resultant clear supernatant was pharmacologically assayed for ~ binding activity (see below). Solubilization with 3'-[(3-cholamidopropyl)dimethylammonio)]-l-propane sulfonate (CHAPS) was carried out using several different buffer systems for comparative purposes. These were: Buffer A, 50 mM Tris-HCl, 5 mM KC1, 5 mM EDTA, pH 8.0; Buffer B, 50 mM Tris-HCl, 120 mM NaC1, 5 mM KC1, 5 mM EDTA, pH 8.0; Buffer C, 50 mM Tris-HCl, 120 mM NaCI, 5 mM KCI, 1 mM MgCl2, 5 mM EDTA, pH 7.7. The detergent (CHAPS) was employed at a concentration of 10 mM in all cases. The incubation and sedimentation procedures with CHAPS were similar to those used by others in related studies with CHAPS [19] and other detergents [11]. Again, all buffers contained the protease inhibitors at the concentrations specified above.
2.3. Sigma receptor radioligand binding assays Radioligand binding assays for the o, receptor binding site were performed using [3H]haloperidol accord-
16
D.I. Schusteret aL /Brain Research 670 (1995) 14-28
ing to published protocols [15,32]. [3H]Haloperidol (New England Nuclear, 18.2 Ci/mmol) was employed at concentrations ranging from 100 pM to 50 nM for generation of the Scatchard isotherm. For displacement studies, [3H]haloperidol was employed at a concentration of 2 nM. Non-specific binding was defined in the presence of 500 nM unlabeled haloperidol (Janssen). Spiperone (25 nM; Janssen) was included to prevent association of the radioligand with dopamine D 2 receptors. Final protein concentrations (Bradford) were generally about 100/~g in a total volume of 2 ml. Glass fiber filter strips (S and S) were soaked in 0.5% (w/v) polyethyleneimine in distilled water for at least 30 min prior to passing the drug-receptor mixtures through the filters. A Brandel 24-position manifold under mechanical vacuum pump suction was used for these studies. Radioactivity retained on the glass fiber filters was assayed using a Beckman LSC 7500 counter. Data from saturation curves and drug displacements was analyzed with the aid of the LIGAND algorithm [21]. A similar protocol was utilized for the determination and statistical evaluation of binding to reconstituted receptor preparations (see below), except that the concentration of the radioligand was increased to 8 nM. Specific binding ranged from 50 to 80% (see below). Displacing drugs were used at ten concentrations ranging from 0.02 nM to 25 p~M, and assays were done in triplicate.
OCH3
1
OCH3
O
2
OCH9
o
(HO.~.CH2ONH2)2 HCI
OH Aminosepharose CI-4B . EDAC, pH 4 5 3
~ H
N
NOCH20-NH(CH2)2NH'CH'2CH(OH)'CH;O(CH2)4OGH2CH(OH)CH20 ,
verted into the desired affinity ligand 3 (CMO-PPP) in 90% yield by reaction with an equimolar amount of carboxymethyoxyamine hydrochloride (Aldrich) in refluxing ethanol for 4 h. The crude product was purified by flash chromatography on silica gel using 5% methanol/methylene chloride as the eluent. The product appeared as a single spot on thin layer chromatography, and its structure was verified by its characteristic 300 MHz IH NMR spectrum in CDC13 (chemical shifts in ppm relative to tetramenthylsilane): 8.0 (d),7.6 (m), 6.8 (d) [aromatic H's]; 4.8, s, 2H, 0CI-IzCOOH; 3.9 (s), OCH3; 1.9, 2.1, 3.8, multiplets for remaining aliphatic H's.
2.4. Preparation of the affinity ligand 2.5. Preparation of the affinity gel
Racemic 3-(3-methoxyphenyl)-piperidine (1) was prepared from 3-bromoanisole and 3-bromopyridine and then resolved into its enantiomers as described by Wikstr6m et al. [41]. The (R) and (S) enantiomers were then separately converted to the enantiomers of compound 2 by Mannich condensations with acetophenone and formaldehyde. In a typical run, 8.8 mmol of the hydrochloride salt of 1 was heated on a steam bath with 13.5 mmol of paraformaldehyde and 8.8 mmol of acetophenone in absolute ethanol for 3-4 h. The reaction mixture was allowed to cool before ethyl ether was added, leading to precipitation of the hydrochloride salt of 2, which was filtered and recrystallized from diethyl ether. The purified product was isolated in 24% yield, which was not optimized. The 300 MHz ~H NMR spectrum of 2 was completely consistent with the assigned structure. Elemental analysis: Calcd. for C21H26NOzCl: C, 70.08; H, 7.28; N, 3.89. Found for (S)-(+)-2: C, 69.77; H, 7.60; N, 3.42. Found for (R)(-)-2: C, 69.90; H, 7.42; N, 3.60. Radioligand binding assays (see above) demonstrated that the (S)-isomer, prepared from (S)-3-MPP, was a much more potent sigma receptor ligand than the (R)-isomer, ICso 16 nM compared to 201 nM, vs. [3H]haloperidol [3]. Racemic and (S)-(+)-2 were con-
CMO-PPP was coupled to amino-Sepharose gel in a manner very similar to that described by Senogles et al. [29]. Amino-Sepharose was prepared from Sepharose CL-4B by standard protocols. Derivatization was first carried out with excess bis-(1,4)-epoxybutane (Aldrich) in alkaline solution at room temperature. After being washed with water, the epoxyalkyl Sepharose (50 ml) was transformed into amino-Sepharose by incubation with 100 ml of 1 M ethylenediamine in 0.1 M sodium carbonate, pH 10.0, for 18 h at room temperature. The gel was washed successively with at least 10 vols. of water, 0.2 M acetic acid, 50 mM NaOH and then water again until the pH was ca. 5. The CMO-PPP dissolved in dimethyl sulfoxide (70 mg) was coupled with 25 ml of amino-Sepharose gel C1-4B, maintaining the pH at 4.5. 1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDAC) was added (1 g/50 ml gel), the pH was adjusted to 4.5, and the mixture allowed to react with constant shaking for 16 h. Additional EDAC was added and reaction continued for another 10 h. The coupled gel 4 was washed with a 1:1 mixture of dimethylsulfoxide/water (0.5 1/50 ml gel) and 2.5 1 of water, and was stored at 4°C in the presence of 0.02% sodium azide.
D.L Schusteret al. / Brain Research 670 (1995) 14-28 2.6. Affinity chromatography The CHAPS-solubilized rat or bovine cerebellar preparations in Buffer B was loaded onto the affinity gel column (1 cm x 20 cm) using continuous-flow recirculation with a peristaltic p u m p for 16 h at 4°C. The amount of material which was applied to the column corresponded to a concentration of 5 m g / m l total Bradford protein in a total volume of 30 ml. The column was then washed with at least 10 bed vols. of 1 mM C H A P S in Buffer B. No protein with UV absorption at 280 nm was detectable at the end of this time with a column monitor (ISCO) set at its maximum sensitivity. After elution with several additional bed volumes of buffer as above, the column was e[uted with 20 mM haloperidol in Buffer B which also contained 1 mM CHAPS. Fractions of volume 2 ml were collected. Haloperidol was removed from these fractions by dialysis against a large excess volume (4 1) of Buffer B, or by passing the purified material through a Sephadex G-25 minicolumn. Preliminary studies using crude solubilized receptor and [3H]haloperidol indicated that these treatments were sufficient to remove the haloperidol efficiently from the receptor (data not illustrated). Following these procedures, the fractions were examined for pharmacological activity after lipid reconstitution as described below. In some studies sufficient protein was present such that the fractions could be pooled after concentration on an Amicon concentrator, based upon their absorbances at 280 nm.
17
cles. The amount of protein incorporated into the lipid vesicles in the reconstitution procedure was highly variable, which is typical of reconstitution procedures. Therefore the calculated fold-purification represents a lower limit rather than an absolute value. Typically, Bradford assay of reconstituted preparations suggested that 10-30% of the protein present was incorporated into the lipid vesicles. As mentioned above, specific binding of 8 nM [SH]haloperidol to the reconstituted cr receptors varied from preparation to preparation, ranging from 50-80%. This was not unexpected [18]. It should be noted that the lipid vesicles alone do not bind [3H]haloperidol in a saturable manner at nanomolar concentrations. As the binding to the cr receptor is defined in terms of specific displaceable binding (see abowg, low affinity non-specific binding to the lipid vesicles and to the glass fiber filters does not affect determination of specific binding.
2.8. SDS-polyacrylamide gel electrophoresis S D S - P A G E was carried out essentially as described by Laemmli [14]. Uniform 7% acrylamide gels were constructed as vertical slabs (16 cm x 18 cm x 1.5 mm). A 3% stacking gel was applied. Slabs were subsequently fixed and were silver stained using the procedure of Merril et al. [20] with a commercially available kit (Bio-Rad).
2.9. Photoaffinity labeling 2. 7. Membrane reconstitution of affinity purified sigma binding site The procedure was modeled on that of Levitzski [18]. Methylene c h l o r i d e / m e t h a n o l (2:1 v / v ) was degassed with dry N 2 at 4°C for 10 min. A 1:1 mixture of phosphatidyl-ethanolamine (soybean, Avanti) and phosphatidylcholine (crude dog brain extract; Sigma) was then added to achieve a final lipid concentration of 5 m g / m l . Solvent was removed under vacuum at low t e m p e r a t u r e in a rotary evaporator (Biichi). Degassed (dry N 2, 15 min) Buffer B was then added along with bovine serum albumin (Sigma; essentially lipid free) to achieve a final protein concentration of 0.5 m g / m l . The mixture was then sonicated (under N 2 blanket) at 4°C for 10 min using a Fisher sonic dismembrator at a setting of 30. This material was added to an equal volume of the dialyzed purified receptor preparation (see above) and was allowed to stand at 4°C for 60 rain in the presence of bio-beads SM-2 (Bio-Rad) to remove detergent (1 g b e a d s / 1 0 0 mg detergent). Pharmacological activity was then assayed (see above). The sonicated lipid preparation was shown under phase contrast microscopy to contain liposomes which were bimolecular as well as some multilamellar vesi-
Bovine cerebellar homogenates (1 m g / m l protein, 2 ml total volume) were incubated with 10 nM [3H]azidoD T G (102 C i / m m o l ) , obtained from Weber's group, in the absence and presence of displacing drugs (10 /~M haloperidol, D T G or prazosin). This was followed by irradiation for 20 min at 350 nm using a Rayonet Photochemical Reactor (140 w) or at 254 nm (100 w low-pressure mercury arc) for 10 min. Dark controls (i.e. identical solutions not exposed to UV irradiation) were performed at the same time. Sample solutions in pyrex test tubes were maintained at 0-4°C. Photolabeling of affinity-purified rat and bovine cerebellar sigma receptors was done similarly, except that the protein concentration was approximately 100 n g / m l . After photoincorporation of the label, samples were sedimented by spinning at 10,500 rpm for 30 s in an E p p e n d o r f centrifuge. Samples were then denatured and analyzed by S D S - P A G E as described above. Gels were subsequently sliced, dried, digested with 30% hydrogen peroxide and counted by liquid scintillation. Photoincorporation in molecular sizing studies was performed in a similar fashion. After irradiation, samples were applied to a 1 x 100 cm column packed with
D.L Schuster et aL /Brain Research 670 (1~)05)14 2,~
18
Bio-gel A0.5m. M a t e r i a l was e l u t e d with b u f f e r B containing 1 m M C H A P S at a flow r a t e o f 0.5 m l / m i n . O n e h u n d r e d 1 ml fractions w e r e c o l l e c t e d a n d c o u n t e d for r a d i o a c t i v i t y by liquid scintillation, as above.
3. Results 3.1. Solubilization and pharmacological characterization of rat cerebellar sigma receptors T h e relative efficacy of a series o f d e t e r g e n t s to yield p h a r m a c o l o g i c a l l y active sigma r e c e p t o r s by solub i l i z a t i o n of rat c e r e b e l l a r m e m b r a n e s was d e t e r m i n e d using S c a t c h a r d analysis for specific b i n d i n g o f [ 3 H ] h a l o p e r i d o l . T h e results are given in T a b l e 1. T h e z w i t t e r i o n i c d e t e r g e n t C H A P S gave by far the best results. K a v a n a u g h et al. [11] have r e p o r t e d similar results on s o l u b i l i z a t i o n o f ¢r r e c e p t o r s from g u i n e a pig b r a i n m e m b r a n e s . N o t surprisingly, d e t e r g e n t s such as T r i t o n X-100 essentially d e s t r o y the biological activity of t h e r e c e p t o r . A f t e r C H A P S h a d b e e n d e m o n s t r a t e d to be t h e d e t e r g e n t o f choice, d e t a i l e d c o n d i t i o n s o f C H A P S s o l u b i l i z a t i o n w e r e f u r t h e r e v a l u a t e d by varying t h e salt c o n c e n t r a t i o n a n d the pH. It was d e t e r m i n e d that the p H a n d salt c o n c e n t r a t i o n o f Buffer B (see e x p e r i m e n t a l m e t h o d s ) w e r e o p t i m a l , a n d this b u f f e r system was u s e d in all f u r t h e r work. T h e extent of solubilization o f sigma r e c e p t o r s a p p e a r s ( T a b l e 1) to d e p e n d critically on the p a r t i c u l a r m e l a n g e o f salts p r e s e n t in the C H A P S buffer. Crude CHAPS-solubilized rat cerebellar hom o g e n a t e s w e r e first e x a m i n e d p h a r m a c o l o g i c a l l y to
Table 1 Scatchard analysis of binding of [3H]haloperidol to rat cerebella solubilized using different detergents Detergent
a
Lubrol WX Triton X-100 CHAPS (A) CHAPS (B) CHAPS (C)
K d (nM)
Bma x
(fnol/mg protein)
19 ,+ 6 28 + 8 33 _+10 18 _+5 85 + 26
16 _4-5 10 _+4 3,500 _+1,000 4,700 _+1,400 247 _+74
Solubilized rat cerebellar tissue was prepared in the detergents shown below as described in section 2. CHAPS solubilization was carried out using three different buffer systems: (A) 50 mM Tris-HCl, 5 mM KCI, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 120 mM NaCI, 5 mM KCI, 5 mM EDTA, pH 8.0; (C) 50 mM Tris-HCl, 120 mM NaCI, 5 mM KCI, 1 mM MgCI2, 5 mM EDTA, pH 7.7. Binding assays were performed by incubating solubilized material, diluted by 1/4 to give a final detergent concentration of 2.5 raM, with [3H]haloperidol in concentrations ranging from 0.1 to 50 nM. Spiperone (25 nM) was present to prevent association of the radioligand with D 2 dopamine receptors. Non-specific binding was defined using 0.5 /xM haloperidol. Each assay consisted of 10 points each performed in triplicate. Reproducible specific binding data could not be obtained using digitonin solubilized preparations.
Table 2 Affinity of selected ligands fl)r sigma receptor sites labeled by [3H]baloperidol in rat cerebellar homogenates and CHAPS solubilized preparations Compound ( + )-SKF-10,047 ( - )-SKF- 10,047 ( + )-Butaclamol ( - )-Butaclamol Pimozide Perphenazine Prazosin Ketanserin BHT-920 SCH-23,290 Ketamine
IC5o (nM) Homogenates ;'
Solubilized preparation ~'
125,++38 4,000+ 1,200 2,200+ 660 45() _+140 125 _+40 95 + 30 2,500 + 750 > 5,000 > 5,000 > 5.000 > 5,000
212 2.700 3,500 657 nd nd nd nd nd nd nd
Rat cerebellar homogenates were incubated with 2 nM haloperidol in the presence of 25 nM spiperone (see Table 1), and non-specific binding was defined using 0.5 /xM haloperidol. A minimum of 20 points were employed for each displacement curve, and were performed in triplicate. Data were analyzed using LIGAND. ~' Duplicate sets of assays were performed for the first four ligands. h Due to the limited amount of solubilized material, and the good agreement with data on homogenates, these results were not replicated. verify the p r e s e n c e of o- b i n d i n g sites. C o m p e t i t i v e d i s p l a c e m e n t s were p e r f o r m e d as d e s c r i b e d in the M e t h o d s using [ 3 H ] h a l o p e r i d o l as the r a d i o l i g a n d [15], a n d the results are given in T a b l e 2. T h e c h a r a c t e r i s t i c inverse s t e r e o s p e c i f i c i t y of the e n a n t i o m e r s of butaclamol for the ~ site vs. d o p a m i n e D 2 r e c e p t o r s was o b s e r v e d , along with the well e s t a b l i s h e d e n a n t i o s p e c i ficity o f b i n d i n g of the ( R ) a n d ( S ) e n a n t i o m e r s of S K F 10,047. This e n a n t i o s p e c i f i c i t y o p e r a t i o n a l l y defines the r e c e p t o r / b i n d i n g site, a n d in p a r t i c u l a r the cr~ rec e p t o r s u b t y p e as c h a r a c t e r i z e d by Bowen a n d coworkers f r o m b i n d i n g of ( + ) a n d ( - ) b e n z o m o r p h a n s [7,8,38]. S o m e ligands which b i n d to o t h e r r e c e p t o r sites, such as p e r p h e n a z i n e ( D t , D 2) a n d p i m o z i d e (D 2) also s h o w e d m o d e r a t e o- potency, while o t h e r nonsigma ligands (prazosin, BHT-920, k e t a n s e r i n and SCH-23,390) w e r e totally inactive. S i m i l a r assays on the C H A P S (B) solubilized rat c e r e b e l l a r p r e p a r a t i o n s h o w e d the s a m e b i n d i n g e n a n t i o s p e c i f i c i t y with respect to the e n a n t i o m e r s o f b u t a c l a m o l a n d SKF-10,047, a n d i n d e e d n e a r l y identical potency, as shown in T a b l e 2. T h e s e d a t a establish the p r e s e n c e of ~r r e c e p t o r s in the C H A P S solubilized rat c e r e b e l l a r p r e p a r a t i o n s which s e e m to be p r e d o m i n a n t l y of the cr 1 subtype. T h e s e results are similar to those o f K a v a n a u g h et al. [12], who u s e d s o m e w h a t d i f f e r e n t e x p e r i m e n t a l conditions.
3.2. Affinity chromatography A n u m b e r of derivatives of 1, i.e. analogs of 2 with d i f f e r e n t n i t r o g e n substituents, were t e s t e d for their
D.L Schuster et al. /Brain Research 670 (1995) 14-28 Table 3 Affinity of R- and S isomers of 2 for selected receptors Assay
"
D2-Dopaminergic a i-Adrenergic /3-Adrenergic Muscarinic Sigma
R isomer, ICso (nM)
S isomer, ICs0 (nM)
3,800 1,000 23,300 3,900 201 _+60 (TL1 /zM) b
16,600 1,200 24,400 8,900 16_+3(1izM) h
Receptor binding assays were carried out for various receptor sites using established protocols, as follows: D2-dopaminergic, rat striatal homogenates, 0.5 nM [3H]spiperone in the presence of 250 nM unlabeled ketanserin, non-specific binding defined using 1 /xM (+)butaclamol; a~-adrenergic, rat frontal cortical homogenate, 1 nM [3H]prazosin, non-specific binding defined by 100 nM prazosin; /3-adrenergic, rat cerebellar homogenates, 2 nM [3H]dihydroalprenolol, non-specific binding defined by 10/xM alprenolol; muscarinic, rat frontal cortical homogenates, 0.25 nM [3H]QNB, non-specific binding defined by 10 IxM atropine; for sigma assay, see section 2. " Note that only the sigma receptor assays were replicated, due to the limited amount of material available. Both isomers were essentially inactive in the other assays. h Value for binding to a second low-affinity site in two-site fit of the data according to LIGAND.
efficacy as ~ and D 2 receptor ligands ([31]; Arnold, Trampota, Schuster and Murphy, unpublished). Of these, compound 2 was one of the most active at ~r sites, while all of them showed essentially no activity (IC50 > 2,000 nM) at D 2 receptor sites as assayed using 0.5 nM [3H]spiperone binding in bovine striatal homogenates in Tris-saline buffer at p H 7.4. The fact that 2 could be readily synthesized and converted into a derivative which could be easily linked to a solid support also was a factor in its selection as the affinity ligand for receptor purification studies. The R and S isomers of 2 were prepared and separately assayed for pharmacological activity, as seen in Table 3. As can be seen, the S isomer of 2 was 13-fold more active than the corresponding R isomer in the ~r receptor assay, and had no significant affinity for D2, muscarinic, cr 1and #-adrenergic receptor sites. Racemic and S-2 were converted to the corresponding oximino carboxylic acids 3 (CMO-PPP), which were then coupled to amino-Sepharose CL-4B by standard techniques to give an affinity gel matrix of structure 4 (see section 2). The ability of this matrix to purify rat cerebellar ~r receptors was then examined. The CHAPS-solubilized rat cerebellar preparation above was applied to the affinity matrix (see section 2) which was then extensively washed with 1 mM C H A P S in Buffer B until the eluates showed no U V absorption at 280 nm above background levels. This generally required at least 10 bed volumes of the buffer solution. The column was eluted with 20 /zM haloperidol in buffer B containing 1 mM CHAPS, collecting 2 ml fractions. There was an immediate appearance of UV absorption at 280 nm in the first fractions, which decreased gradually upon further elution. Haloperidol
19
itself does not show significant UV absorption at 280 nm at concentrations 10-fold that used in this procedure. The material showing UV absorption was typically eluted using 2 bed vols. of the haloperidol eluent. Fractions of haloperidol eluates with optical density > 0.1 were pooled and concentrated (see section 2). These concentrated fractions were then subjected to S D S - P A G E which upon silver staining revealed two prominent closely spaced bands at M r 63 and 65 kDa, as shown in Fig. 1. In some samples which were allowed to stand for several hours at 4°C even in the presence of protease inhibitors, considerable degradation was observed, as evidenced by the presence of smeared bands in the low molecular mass region. The presence of soybean trypsin inhibitor was essential to obtaining a reasonable yield of the purified proteins. This suggested that small amounts of a protease were probably present on the glassware a n d / o r co-purified along with the sigma binding site. However, the protease was not apparent on the SDS-gels using the silver-staining technique. Similar degradation was also
SIGMA RECEPTOR PURIFIED FROM RAT CEREBELLUM 2
3 205K
116K 92K 66K
45K
Fig. 1. Rat cerebellar sigma receptors on 7.5% polyacrylamide slab gel (16 cm x 18 cm x 1.5 mm) after silver staining (see section 2 for details). Lane 1, affinity purified rat cerebellar sigma receptor. showing two bands at 63 and 65 kDa. Lane 2, CHAPS solubilized rat cerebellar preparation before affinity Chromatography. Lane 3, molecular weight markers, in kDa.
20
D.L Schuster et al. / B r a i n Research 670 (1995) 14 28
observed in samples which had been stored at -80°C for several weeks. Identical treatment of solubilized bovine cerebellar tissue gave similar results, that is, appearance of prominent bands at 63 and 65 kDa in purified protein (data not shown). Extensive degradation was observed when this material was allowed to stand for 24 h at room temperature [1].
3.3. Pharmacology of purified sigma receptor proteins In order to investigate the pharmacology of the affinity-purified proteins, it was necessary, to first desalt the material purified as above to remove the haloperidol used to elute the material from the column. In preliminary studies using [3H]haloperidol to elute the column, we determined that the dialysis conditions were sufficient to remove essentially all the haloperidol (data not illustrated). However, even after desalt±rig by dialysis or by passage through a Sephadex G-25 minicolumn, the protein did not specifically bind [3H]haloperidol. The protein was therefore reconstituted into lipid vesicles containing equal proportions of PC and PE with BSA as the carrier protein, following the methods of Levitzski [18]. The reconstituted protein indeed showed levels of specific binding of [3H]haloperidol between 50% and 80% (data not shown). Results of displacement assays on reconstituted affinity-purified protein derived from approximately 500 fresh rat cerebella given in Table 4 demonstrate that this material possesses characteristic o- receptor pharmacology [9,10,12,13,15-17,30,32,39,40,42,43]. Thus, haloperidol (IC50 3 nM, K i 9 nM) and DTG (IC50 45 nM, K i 9 nM) showed the greatest affinity to the receptor of the ligands tested. Enantiospecific displacement of [3H]haloperidol, characteristic of binding to o- receptors, was shown by the enantiomers of SKF 10,047 (IC50 40 nM for ( + ) , 5,000 for ( - ) ) , butaclamol (IC50 251 nM for ( - ) , 14,000 for ( + ) ) and cyclazocine (ICs0 630 for ( + ) , 5,000 for ( - ) ) . Analysis of the displacement curves for haloperidol, (+)-SKF 10,047 and DTG using the LIGAND algorithm [34] indicates that both high and low affinity binding sites are present in the receptor protein, which probably correspond to the 0-1 and 0-2 receptor subtypes as defined by Bowen and co-workers [7,8,38] and others [25]. Subtype-specific binding assays [8,36], which were developed following completion of this study [31], were not performed. The material purified on the CMO-PPP affinity matrix did not possess dopaminergic, a-adrenergic, #- or/~-opiate or phencyclidine activity as defined by the selective ligands sulpiride, prazosin, etorphine and PCP, respectively. Similar pharmacological characterization of reconstituted affinity-purified bovine cerebellar receptors is
Table 4 Displacement studies on reconstituted affinity-purified rat cerebetlar sigma receptors Displacing ligand
IC5. (nM)
K i (nM)
Haloperidol
3 +_ 1 40 _+4 5,000 _± 1,500 14,1)00 ± 2,240 251 _+40 630 ± 82 5,000 ± 800 45 ± 14 3,{)00 + 340 4,000 22,000 1,000
0.8 + f).5 (6,000) * 58 + 6 (52,000) 58,000 ± 4,800 44,800 ± 7.200 590 + 94 1,750 ± 230 15,600 _+2,500 0 + 3 (25,000) * 670 _+87 ND ND ND
+ )-SKF-10,047 - )-SKF-I 0,047 + )-Butaclamol - )-Butaclamol + )-Cyclazocine - )-Cyclazocine DTG PCP
Etorphine Sulpiride Prazosin
Affinity-purified receptors reconstituted into lipid vesicles (see section 2) were incubated with 8 nM [3H]haloperidol and the displacing drug at concentrations ranging from 0.02 nM to 25 txM. Non-specific binding was defined using 0.5 /~M haloperidol. Each assay consisted of 10 points done in triplicate. Data were analyzed graphically and with the aid of the LIGAND algorithm. Analysis of the binding data for the sigma-active compounds indicated with an asterisk according to a two-site model using the LIGAND program [21] gave the values of K i shown below for binding to the high affinity site; the K, values for binding of these ligands to the low-affinity site are shown in parenthesis.Values of K i for the low-affinity ligands were also obtained from LIGAND using a one site binding model. ND. not determined.
summarized in Table 5. The data are very similar to those for the purified rat preparation. Again, the most potent ligands are haloperidol (IC.~0 1.6 nM, K, 2.1 Table 5 Displacement studies on reconstituted affinity-purified bovine cerebellar sigma receptors Displacing ligand
ICs0 (nM)
K i (nM)
Haloperidol
1.6 ± 0.2 20 _+8 > 25,000 3,100_+ 600 630 ± 69 63 _+7 794 + 95 25 ± 8 12 ± 4 11)0 ± 27 > 25,000 > 25,000 > 25,000 > 25,000
2.1 +_0.2 53 ± 10 ND 7,100± 1,300 620 ± 99 45 ± 5 327 +_31 58 + 8 3.9 + 2 140 _+27 ND ND ND ND
( + )-SKF-10,047 ( - )-SKF-10,047 ( + )-Butaclamol ( - )-Butaclamol ( + )-Cyclazocine ( - )-Cyclazocinc DTG
Tiaspirone Buspirone Etorphine Sulpiride Prazosin PCP
Affinity-purified bovine cerebellar sigma receptors reconstituted into lipid vesicles (see section 2) were incubated with 8 nM [3H]haloperidol and the displacing drug at concentrations ranging from 0.02 nM to 25 ~M, Non-specific binding was defined using 0.5 ~zM haloperidol. Each assay consisted of 10 points done in triplicate. Data were analyzed graphically and with the aid of the LIGAND algorithm. Most of the active compounds fit well to both a one- and two site model as described by the LIGAND program. The 1C50 values for the second low-affinity site ranged from 1,000 to 25,000 nM. ND. not determined.
D.L Schusteret aL/Brain Research670 (1995) 14-28 nM) and D T G (IC50 25 nM, K i 58 nM). T h e enantiospecificity for binding of SKF 10,047, butaclamol and cyclazocine to the purified bovine receptor was the same as that seen for the purified rat receptor, while sulpiride, prazosin, etorphine and P C P again were completely inactive. O t h e r high affinity o- ligands, tiaspirone ( B M Y 14,802) and buspirone [35,36], were also examined for their affinity to the bovine ~r receptor. B M Y 14,802 was f o u n d to be nearly as potent as haloperidol (IC50 12 nM, K d 3.9 nM) while buspirone was considerably less active (IC50 100 nM, K d 140 nM).
3.4. Calculation of fold purification of the purified sigma receptor F o r this p u r p o s e , S c a t c h a r d analysis (using [3H]haloperidol) was p e r f o r m e d on the crude rat cerebellar h o m o g e n a t e , the CHAPS-solubilized preparation and the material purified by a single pass t h r o u g h the C M O - P P P affinity column (after reconstitution). Results are given in Table 6. As can be seen, C H A P S solubilization resulted in ca. 30-fold purification over crude homogenates, and affinity c h r o m a t o g r a p h y afforded at least 200-fold additional purification. As noted earlier, the yield in the reconstitution step is highly variable and is certainly not quantitative. Alt h o u g h the total a m o u n t of protein incorporated could be m e a s u r e d using a radiolabel, this is not necessarily related in a simple m a n n e r to the a m o u n t of pharmacologically active receptor which was functionally reconstituted. Thus, the yields o b t a i n e d f r o m the affinity purification and the resultant fold-purification of ca. 6,000 given in Table 6 are lower limits to the degree of purification actually achieved in this process. T h e purified material derived from bovine cerebellar was submitted for E d m a n amino acid sequencing on an Applied Systems M i c r o s e q u e n c e r at Columbia University Medical Center. N o sequence could be obtained, indicating that this protein is probably N-terminal blocked.
21
SIGMA RECEPTOR PURIFIED FROM RAT H IPPOCAMPUS A
B
C
205K 116K 97K 66K
45K 17K Fig. 2. Rat hippocampal sigma receptors on 7.5% polyacrylamide slab gel (16 cm × 18 cm x 1.5 mm) after silver staining (see section 2 for details). Lane A, molecular weight markers, in kDa. Lane B, sigma receptors affinity-purified from rat cerebellar tissue. Lane C, sigma receptors affinity-purified from rat hippocampal tissue. m e n t in which 50 freshly dissected rat h i p p o c a m p a e were homogenized, solubilized with C H A P S and affinity-purified as described for the cerebellar preparation, two bands were again seen on S D S - P A G E at 63 and 65 k D a (Fig. 2). Judging from relative b a n d intensities on silver staining, it appears that roughly equal amounts of ~ r e c e p t o r were obtained using 50 rat h i p p o c a m p a e as o p p o s e d to 20 rat cerebella, showing the latter is a m o r e convenient source of these proteins. N o receptor protein was visualized on S D S - P A G E using silver staining after similar affinity purification of C H A P S solubilized rat frontal cortex tissue.
3.5. Purification of sigma receptors from other areas of rat brain
3.6. Photoaffinity labeling
Purification of o- receptors was also a t t e m p t e d from the rat h i p p o c a m p u s and frontal cortex. In an experi-
Before using [ 3 H ] a z i d o - D T G as a photoaffinity label for our purified cerebellar ~r receptors, it was consid-
Table 6 Quantification of rat cerebellar sigma receptor enrichment Material Crude rat cerebellar membranes CHAPS membranes Material from one pass through the affinity column after reconstitution into lipid vesicles
Specific activity (fmol/mg protein)
Fold purification
150 _+35 4,664 _+1,166 910,000 + 110,000
1 31 6,066
Data are based upon Scatchard analysis of material at various stages of the purification process using 10 concentrations of [3H]haloperidol, each point done in triplicate. The procedure was repeated three times in each case. Protein concentration was determined by the method of Bradford.
22
D.L Schuster et al. / B r a i n Research 670 (1995) 14-28
ered prudent to first replicate the results reported by Kavanaugh et al. [12] for specific photolabeling of or receptors in guinea pig brain homogenates. Indeed, both rat and bovine cerebellar homogenates reacted with [3H]azido-DTG in a Rayonet Photoreactor (see section 2) specifically by incorporation into a material (presumably polypeptide) of mass 29 + 1 kDa, as determined by slicing of SDS gels (see Fig. 3). The radioligand was not incorporated in an identical sample that was kept in the dark. Photoincorporation into the 29 kDa material was completely blocked in the presence of 10 ~ M concentrations of the ~ receptor ligands haloperidoi, D T G and SKF 10,047, but not by 10/~M prazosin. These findings essentially replicated the report of Kavanaugh et al. [12] and gave us confidence that [3H]azido-DTG is indeed a useful reagent for specific photolabeling of cr receptors.
1,200
1,0001
800 "O c (/) -1
o t"
600
0-
0 400
200
"
65
63
40
29
APPARENT MOLECULAR MASS (kDa) Fig. 4. Photoincorporation of l0 nM [~H]azido-DTG into affinitypurified bovine cerebellar receptors. Completely analogous results were obtained for affinity-purified rat cerebellar receptors. See section 2 for details. O, UV irradiated sample; A, non-irradiated (dark) sample; +, D, I , X, UV irradiation of samples containing 10 p~M haloperidol, DTG, (+)-SKF-10,047 and prazosin, respectively.
%-. "03 to l-v
a. 2 0
65
63
40
33
29
25
APPARENT MOLECULAR MASS (kDa) Fig. 3. Photoaffinity labeling of crude bovine cerebellar homogenates using 10 nM [3Hlazido-DTG. Photolysis in the presence and absence of displacing ligands was performed as described in section 2. Denatured samples were applied to SDS-polyacrylamide gels which were subsequently sliced, dried, digested with 30% hydrogen peroxide and the radioactivity counted using liquid scintillation. +, UV irradiated sample; D, non-irradiated (dark) sample; A, ×, I , samples irradiated in the presence, respectively, of 10 /~M haloperidol, 10 p~M DTG or 10 p.M prazosin Molecular weights based on markers on SDS gels. Completely analogous results were obtained using crude rat cerebellar homogenates.
The photolabeling experiment was then carried out with the purified rat and bovine cerebellar ~ receptor preparations. Photoincorporation in both cases again showed ~r-specificity, in that it was blocked by 10 ~ M haloperidol, D T G and ( + ) - S K F 10,047, but not by 10 ~ M prazosin. However, gel slicing indicated specific photolabeling of material whose mass ranged between 60 and 65 kDa (see Fig. 4). No photolabeling of the 29 kDa protein was observed under these conditions. Kavanaugh et al. [12] used [3H]azido DTG to demonstrate that the o- receptors exists as a molecular complex of size 300-500 kDa. The experiment involved photoincorporation into guinea pig cerebellar homogenates, solubilization and passage through a Sepharose C1-6B molecular sizing column. We performed a similar experiment using freshly purified rat cerebellar cr receptors. After exposure to [3H]azidoD T G as above, the photolabeled material was applied to a 1 × 100 cm column packed with 0.5 m Bio-Gel A and eluted with 1 mM CHAPS in Buffer B. Labeled protein eluted in two bands, a major band at 300-500 kDa and a minor band at 50-70 kDa (Fig. 5). When a
23
D.I. Schuster et al. ,/Brain Research 670 (1995) 14-28
similar experiment was performed on a purified receptor preparation that had been stored for several days at - 8 0 ° C before photolabeling, the relative sizes of the two bands changed significantly, as shown in Fig. 6. The substantial increase in the extent of photoincorporation into the 50-70 kDa band suggests that the complex of mass 300-500 kDa dissociates upon standing even at - 8 0 ° C into the lower mass polypeptide. Using a purified receptor preparation that had been stored for two weeks at -80°C, photoincorporation occurred only into the lower mass material, suggesting that the large complex had completely dissociated under these conditions (Fig. 7). In this case, the extent of photolabeling was reduced by only ca. 50% in the presence of 10 tzM haloperidol, suggesting that some of the receptor complex may have been destroyed on prolonged standing, even at -80°C, or alternatively that some non-specific photolabeling by [3H]azidoD T G was occurring. The former hypothesis is supported by SDS gels of irradiated material (not shown), in which many new lower mass bands are apparent in
1,000 50 - 70 kDa
800 3 0 0 - 500 kDa Z W
IO
600
rl o') c o o
z;
i
'1
400
n
'll
O
200
1,200 55
300- 500 kDa
800
addition to the prominent bands noted for the reconstituted receptor at 63 and 65 kDa. This study was repeated using freshly purified bovine cerebellar ~ receptors using a lamp providing 254 nm light. Again, two bands were observed by gel slicing at 50-70 kDa and 300-500 kDa. Photolabeling under these conditions was completely blocked by 10 IzM haloperidol [1].
W
O
t'r" r~ c 0 o
600
13_
O
95
Fig. 6. Molecular sizing as in Fig. 5 of affinity-purified rat cerebellar sigma receptors that had been stored for 3 days at -80°C before photolabeling with [3H]azido-DTG.
1,000
z
75
FRACTION -#
400
4. Discussion 50 - 70 kDa
200
61
81
101
FRACTION # Fig. 5. Molecular sizing of freshly purified rat cerebellar sigma receptors by photoaffinity labeling with 10 nm [3H]azido-DTG (see section 2 for details). Photoaffinity-labeled material was passed through a Bio-gel molecular sizing column under non-denaturing conditions. The radioactivity inl ml fractions was counted by liquid scintillation.
4.1. Affinity purification of cerebellar sigma receptors Successful purification of protein(s) which have pharmacological properties characteristic of the or receptor as defined by several groups has been achieved using an affinity column constructed from an analog of (S)-3-PPP. Several such analogs which displayed high o- receptor affinity and selectivity were prepared, of which compound 2 was one of the most active. Since the (S)-isomer of 2 was 13-fold more potent as a ~r ligand than the (R)-isomer, (S)-( + )-2 was converted to an oximino derivative which was then covalently linked
24
D.1. Schuster et al. / B r a i n Research 670 (1995) 14-28
3,000 50 - 70 kDa
2,500 "¢o
0 tI--
2,000
z W
~- 1,500
0
c o
o ('3
1,000
Q.
(3
500
J
85
95
105
115
FRACTION # Fig. 7. Molecular sizing as in Fig. 5 of affinity-purified rat cercbellar sigma receptors that had been stored for 2 weeks at -80°C before photolabeling with [3Hlazido-DTG. I , UV irradiated sample; +, sample irradiated in the presence of 10/zM haloperidol.
to amino-Sepharose. This affinity matrix selectively bound o, receptors from CHAPS-solubilized rat and bovine cerebeUar preparations, as determined by measurement of specific [3H]haloperidol binding to solubilized material before and after exposure to the matrix. After extensive washing of the column, additional proteinaceous material specifically eluted from the column using 20 p,M haloperidol in a Tris buffer containing 1 mM CHAPS did not display specific [3H]haloperidol binding after removal by dialysis of the eluting ligand, haloperidol. However, after the eluted and desalted purified protein was reconstituted into lipid vesicles formed from a 1:1 mixture of purified phosphatidylcholine and phosphatidylethanolamine, specific [3H]haloperidol binding was observed. The reconstituted receptor demonstrated characteristic ~ receptor pharmacology: high affinity binding of haloperidol, DTG, (+)-SKF-10,047 and (in the case of the bovine preparation) tiaspirone and buspirone, as well as enantiospecific affinity for the (+)-isomers of benzomorphans (SKF 10,047 and cyclazocine) and the (-)-isomer of butaclamol. These characteristics have been observed for o- receptors in several tissues by several groups using a variety of radioligands: [3H]haloperidol,
[3H]DTG, [3H](+)-3-PPP, [3H](+)-SKF 10,047 and [3H](+ )-pentazocine [7-10,12,13,15-17,30,32,3840,42,43]. Based upon saturation binding analyses, 30-fold purification was achieved on solubilization and at least 200-fold additional purification by one pass through the affinity column. The approximately 6,000-fold overall purification achieved (relative to membrane homogenates) is essentially that required for purification to homogeneity of a protein of mass 65 kDa. If the molecular mass of the intact receptor is larger (see below), somewhat greater fold-purification will be necessary to achieve complete homogeneity. The purified rat and bovine cerebellar ~r receptor shows up consistently on SDS-PAGE as a doublet of bands at 63 and 65 kDa. The fact that two different proteins of nearly identical mass are simultaneously purified using this procedure is not unprecedented. [28] The smaller protein might be a degradation product of the other, or the two bands might represent two glycosylated forms of the same receptor protein. We lean to the latter explanation, even though (as noted by a referee) differing extents of glycosylation might be expected to result in even greater heterogeneity. Although the precise explanation remains to be determined, we find that samples of these purified receptors that have been stored at -80°C for more than five years still show this particular doublet of bands on SDS-PAGE analysis. Subtype-specific radioligand binding assays to differentiate o7 and ~r: receptors, developed by Bowen and co-workers [8,38] following completion of the present study [1], have yet to be carried out on the affinitypurified rat and bovine cerebellar proteins. In a study performed subsequently [3,27], rat liver cr receptors were purified on an affinity matrix constructed from an oximino derivative of haloperidol using a somewhat different experimental protocol. Material eluted from the affinity column by dextrallorphan that specifically bound [3H]haloperidol without reconstitution into lipid vesicles showed bands on SDS-PAGE at 28 and 40 kDa as well as a single band at 65 kDa. This liver preparation did not show the characteristic doublet of bands at 63 and 65 kDa seen for the rat and bovine cerebellar receptors. All attempts to sequence the purified bovine cerebellar receptors by Edman degradation failed, suggesting the protein is N-terminal blocked. It will be necessary to purify larger amounts of material using this affinity matrix in order to obtain peptide fragments by selective enzymatic cleavages.
4.2. Photoaffinity labeling of sigma receptors Kavanaugh ct al. [12] first reported photoaffinity labeling of ~r receptors in guinea pig membranes using
D.I. Schuster et al. /Brain Research 67(1 (1995) 14-28
[3H]azido-DTG, which was shown to be a selective, potent and specific 0- receptor ligand. SDS-PAGE of sodium cholate-solubilized photolabeled material showed that the photolabel was selectively incorporated into a band of M r 29 kDa. Photolabeling was blocked by potent 0- ligands (haloperidol, (+)-3-PPP, D T G and (+)-pentazocine, all at 10 /~M) but not by dopamine, serotonin, scopolamine and y-aminobutyric acid at the same concentration. Molecular sizing of the receptor by chromatography of photolabeled material under non-denaturing conditions showed specific labeling of a material of M r 150 kDa, which was concluded to be a receptor complex. When this material was concentrated and subjected to SDS-PAGE, the radioactivity was found to be associated with a peak at M r 29 kDa. It was therefore concluded [12] that the material of mass 29 kDa probably contains the binding subunit of the guinea pig brain 0- receptor. The fact that the 29 kDa protein was seen when the larger receptor complex was treated with SDS under non-reducing conditions suggests that this protein is not covalently linked to other subunits of the or receptor complex. When Hellewell and Bowen [7] later photolabeled 0receptors in guinea pig brain membranes using [3H]azido-DTG, the label was found to be selectively incorporated into a band that consistently migrated at 25 kDa in SDS gels, which was assumed to be the same protein observed at 29 kDa by Kavanaugh et al. [12]. On the other hand, photolabeling of membranes of undifferentiated PC 12 cells using the same reagent led to two bands in SDS gels at 18 and 21 kDa, which as with the guinea pig preparation was blocked by the presence of 1 /~M haloperidol. Bowen and co-workers [2,8] concluded that there two different types of o- sites are present in guinea pig and rat liver membranes, which differ in mass as well as pharmacology. The PC 12 sites and similar sites, now known as 0-2, in rat liver, rat kidney and clonal cells of neuronal and glial origin, [38] have much lower affinity for the (+)-enantiomers of benzomorphans than do 0-1 sites in guinea pig brain, rat liver and rat kidney membranes as well as NCB-20 cell lines. Since the affinity of ( - ) - b e n z o m o r p h a n s for 0"2 VS. 0-1 sites is essentially unchanged, the enantiospecificity of benzomorphans for 0"2 sites [( - ) > ( + ) ] is reversed from that for 0"1 sites [ ( + ) > ( - ) ] . Both sites retain high affinity for haloperidol, D T G and ( + ) - 3-PPP. Using subsite-specific radioligand binding assays, Hellewell and Bowen [8] concluded that 0"2 sites comprise 75-80% of the total population of 0" receptors in rat liver and rat kidney, and also suggested that the two 0" sites may have different biological functions. Consistent with the concept of two 0" sites is the finding that photolabeling of rat liver membranes with [3H]azido-DTG results in specific labeling of two poly-
25
peptides of mass 25 and 21.5 kDa. Labeling of both polypeptides was blocked by 10 p,M haloperidol, while dextrallorphan at 100 or 500 nM completely blocked labeling only of the 25 kDa polypeptide but had no effect on photolabeling of the 21.5 kDa polypeptide. This supports the proposal that the 25 kDa polypeptide contains the 0-1 binding site while the 21.5 kDa polypeptide contains the 0-2 binding site. Only a single site with M r 29 kDa (0-1) is photolabeled by [3H]azidoD T G in NCB-20 cells, while this same reagent labels two bands in PC12 cells at 18 and 21 kDa (0-2). The results of the present investigation will now be discussed in light of these earlier findings as well as more recent developments. First, we replicated the finding of Kavanaugh et al. [12] that [3H]azido-DTG specifically labels material of M r 29 kDa in crude brain preparations (Fig. 3), in our case rat and bovine cerebellar homogenates. The labeling exhibited characteristic or receptor pharmacology. However, upon photolabelling of the afffinity-purified receptor protein, which displayed characteristic or pharmacology after reconstitution into lipid vesicles, the label was incorporated into material of mass 60-65 kDa (Fig. 4), corresponding to the mass of the proteins detected directly by silver staining of SDS gels. No labeling of a 29 kDa polypeptide was observed under these conditions. The fact that the labeling of the solubilized purified protein was blocked selectively by 10 mM ( + ) - S K F 10,047 as well as haloperidol and D T G (Fig. 4) supports the conclusion that this protein contains a o- binding site, most likely of the 0-1 subtype. The pharmacology of the affinity-purified 63-65 kDa protein(s) was determined by radioligand binding assays after reconstitution into lipid vesicles (Table 5) as well as by the effect of added ligands on photolabeling by [3H]azido-DTG; for the 29 kDa polypeptide, the pharmacological characterization depends only upon the inhibitory effect of ligands on photolabeling. The results indicate that these sites are indistinguishable pharmacologically and implicate the binding site as a 0site. Thus, we conclude that the 29 kDa polypeptide is a subunit of the 65 kDa protein, which itself may be a subunit of a much larger receptor complex with mass of at least 150 kDa, based upon the molecular sizing studies (i.e. photolabeling under non-denaturing conditions) carried out earlier [12] and in the present study. Our work clearly demonstrates that the larger complex (apparently 300-500 kDa) breaks down on standing to give material of mass 50-70 kDa. All of these findings were reproducible using both rat and bovine cerebellar preparations. It is possible but highly unlikely that this large complex could arise by photocrosslinking; if so, it would not be expected that the complex would be so readily dissociable. Based upon Bowen's demonstration [7,8] that both 0-1 and 0-2 sites are photolabeled in rat liver and guinea
26
D.L Schuster et al. / B r a i n Research 070 (1995) 14 2,~
pig brain by [3H]azido-DTG, and the enantioselectivity of (+)-benzomorphans for the ~r site in the 29 kDa and 63-65 kDa cerebellar proteins observed in the present study, we suggest that the these purified proteins contain predominantly al binding sites. Photolabeling of proteins of mass 18-21 kDa, which presumably correspond to cr2 sites, was not observed. The binding site is present in the 29 kDa polypeptide subunit of the 63-65 kDa protein, which appears to be part of a much larger receptor complex of > 150 kDa. We can only speculate as to why the 63-65 kDa protein is photolabeled in the affinity -purified protein while the 29 kDa polypeptide is photolabeled in crude membrane homogenates. It is known from the work of Kavanaugh et al. [12] that in guinea pig brain membrane suspensions the 29 kDa polypeptide becomes dissociated from the large c a . 150 kDa photolabeled ~r receptor complex when the latter is concentrated and then treated with SDS. This does not occur with the CHAPS-solubilized affinity-purified 63-65 kDa rat and bovine cerebellar ~r receptor, which was not concentrated before analysis by SDS-PAGE. One possibility is that some factor (perhaps a protease) present in the homogenates but not in the CHAPS-solubilized preparation induces separation of the 29 kDa subunit from the larger complex on denaturing gels, so that the photolabeled 63-65 kDa protein in homogenates breaks down more readily than the same protein in CHAPS-solubilized preparations. We are assuming that the two bands in SDS gels at 63 and 65 kDa represent two forms of the same receptor protein differing only in extents of glycosylation, which can only be established when these proteins are actually isolated and structurally characterized. We have also recently developed a different affinity matrix constructed from an oximino derivative of haloperidol to purify ~r receptors from rat liver preparations [3,27]. As mentioned earlier, the pharmacologically active affinity-purified protein showed three bands on SDS-PAGE, at 29, 40 and 65 kDa. When this material was concentrated for the purpose of amino acid sequencing, SDS-PAGE showed very prominent bands at 29 and 40 kDa and only a weak band at 65 kDa, suggesting that the former were derived by degradation of the 65 kDa protein a n d / o r an even larger receptor complex. Thus, intact o- receptor complexes seem to be very labile, and break down easily under a variety of conditions into smaller components. A final assignment of the relationship of the various proteins identified as components of the o, receptor in this and related studies will only be possible when the full amino acid sequences of these proteins are determined. There has been only limited progress in this direction. We have recently determined the N-terminal sequence of the 29 kDa affinity-purified polypeptide derived from rat liver membranes, and found it to be
identical to that of rat cyclophilin A, a cytosolic 18 kDa protein [3,27]. Although a 22 kDa membrane-associated cyclophilin has been isolated from rat liver membranes [37], its N-terminal sequence is very different from that determined for the 29 kDa polypeptide [3,27]. It has yet to be determined if any of these proteins can be photolabeled by [3H]azido-DTG in a sigma-specific manner.
5. Summary CHAPS-solubilized rat and cerebellar ~r receptors have been purified at least 6,000 fold relative to membrane homogenates on an affinity matrix linked to an oximino derivative of an analog of 3-PPP which shows high affinity and selectivity for ~r receptors. The affinity-purified cerebellar receptors appear as a doublet at 63 and 65 kDa on SDS-PAGE, which probably represent two forms of the same protein differing in extents of glycosylation. The pharmacologically inactive purified receptor proteins display characteristic ~r pharmacology after reconstitution into lipid vesicles, with ligand affinities and enantioselectivity very similar to those of membrane homogenates and CHAPS-solubilized preparations. Based upon the high affinity of (+)-benzomorphans for the binding site, the site is concluded to be (predominantly) of the ~r~ subtype. Photoaffinity labeling of cerebellar homogenates with [3H]azido-DTG leads to incorporation exclusively into a 29 kDa polypeptide, as reported earlier [12] for photolabeling of guinea pig brain suspensions with this reagent. It has been suggested by others that this polypeptide in guinea pig brain and rat liver and kidney preparations contains the ~r~ binding site [7,8]. However, when the affinity-purified cerebellar receptors which show ~r pharmacology are photolabeled with [3H]azido-DTG, the label is incorporated in materials of mass 60-65 kDa and not into the 29 kDa polypeptide. Molecular sizing of photolabeled non-reduced freshly purified proteins shows that a large molecular complex with M r 300-500 kDa is specifically labeled, but that labeled material of mass 50-7(I kDa becomes increasingly apparent when the purified protein is allowed to stand at - 8 0 ° C before being photolabeled by [3H]azido-DTG. Since the pharmacology of the 29 kDa and 63-65 kDa proteins are indistinguishable, it is concluded that the ~r (probably cr~) binding site is located in the 29 kDa polypeptide, which appears to be a subunit of protein(s) of mass 63-65 kDa, which seem to be derived from an even larger molecular complex. That the 29 kDa polypeptide is not observed in SDS gels or on photolabeling of the purified cr receptor protein suggests that some factor which promotes degradation of the intact ~r receptor in membrane homogenates (most likely a protease) is not
D.L Schuster et al. /Brain Research 670 (1995) 14-28
present in affinity-purified solubilized cerebellar cr receptors. Attempts at amino acid sequencing of the affinitypurified sigma receptor protein(s) by Edman degradation were unsuccessful, suggesting this material is Nterminal blocked. Enzymatic cleavages will be performed when larger quantities of the purified protein are available.
Acknowledgements This work was supported in part by a grant from the Sandoz Foundation. Portions of this work were reported in preliminary form at Meetings of the Society for Neuroscience in 1987 and 1988. We are grateful to Miroslav Trampota for synthesis of the affinity ligand, and to Dr. Eckhard Weber and his group for a gift of [3H]azido-DTG. We also acknowledge the assistance of George Ehrlich in several experiments, and numerous discussions with Drs. Jiirgen Br6sius and Henry Tiedge at Mt. Sinai School of Medicine. We are also grateful to Dr. Wayne Bowen for a preprint of [8].
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