Identification of regions of the α-1 receptor ligand binding site using a novel photoprobe

Article abstract of DOI:10.1124/mol.107.038307

σ Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2′,6′-dimethylmorpholino)3- (4-t-butylpropylamine) (fenpropimorph), a

Full text of DOI:10.1124/mol.107.038307

0026-895X/07/7204-921–933$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 72:921–933, 2007
Vol. 72, No. 4
38307/3255037
Printed in U.S.A.
Identification of Regions of the ␴-1 Receptor Ligand Binding
Site Using a Novel Photoprobe
Arindam Pal, Abdol R. Hajipour, Dominique Fontanilla, Subramaniam Ramachandran,
Uyen B. Chu, Timur Mavlyutov, and Arnold E. Ruoho
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin (A.P., D.F., S.R., U.B.C., T.M.,
A.E.R.); and Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan, Iran (A.R.H.)
Received May 18, 2007; accepted July 10, 2007
ABSTRACT
␴ Receptors, once considered a class of opioid receptors, are liver membranes with high specificity. Furthermore, after cleav-
now regarded as a unique class of receptors that contain ing the specific [125I]IAF-photolabeled ␴-1 receptor in guinea
binding sites for a wide range of ligands, including the drug pig and rat liver membranes and the pure guinea pig ␴-1
1-N(2Ј,6Ј-dimethylmorpholino)3-(4-t-butylpropylamine)
(fen- receptor with EndoLys-C and cyanogen bromide, the [125I]IAF
propimorph), a yeast sterol isomerase inhibitor. Because fen- label was identified both in a peptide containing steroid binding
propimorph has high-binding affinity to the ␴-1 receptor, we domain-like I (SBDLI) (amino acids 91–109) and in a peptide
have synthesized a series of fenpropimorph-like derivatives containing steroid binding domain-like II (SBDLII) (amino acids
with varying phenyl ring substituents and have characterized 176–194). Because a single population of binding sites (R2
ϭ
their binding affinities to the ␴-1 receptor. In addition, we have 0.992) for [125I]IAF interaction with the ␴-1 receptor was iden-
synthesized a carrier-free, radioiodinated fenpropimorph-like tified by (ϩ)-[3H]pentazocine competitive binding with nonra-
photoaffinity label, 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-azido- dioactive [127I]IAF, it was concluded that SBDLI (amino acids
3-[125I]iodo-phenyl)propane ([125I]IAF), which covalently deri- 91–109) and SBDLII (amino acids 176–194) comprises, at least
vatized the ␴-1 receptor (25.3 kDa) in both the rat liver and in part, regions of the ␴-1 receptor ligand binding site(s).
guinea pig liver membranes and the ␴-2 receptor (18 kDa) in rat
The ␴ receptor is a unique receptor that, for the past 30 other known neurotransmitter or hormone receptors
years, has been persistently enigmatic. ␴ receptors were ini-
tially proposed to be subtypes of opioid receptors based on
work performed by Martin and colleagues (1976) to study the
actions of antipsychotic drugs. In these experiments, they
proposed the existence of a ␴-opioid receptor based on the
psychomimetic effects of SKF-10047, which could not be ex-
plained by ␮- or ␬-opioid receptors (Martin et al., 1976). This
hypothesis, however, was later refuted when the ␴ receptor
was shown to be insensitive to naloxone, a common opioid
receptor antagonist (Iwamoto, 1981; Su, 1982; Vaupel, 1983).
As a result, ␴ receptors were reclassified as unique nonopioid
and nonphencyclidine binding sites present in the central
nervous system and peripheral organs that are distinct from
(Quirion et al., 1992).
To date, two subtypes of the ␴ receptor have been identi-
fied, the ␴-1 and ␴-2 receptors, which are distinguishable by
their pharmacology, function, and molecular mass. The ␴-1
receptor was first cloned from guinea pig liver in 1996 (Han-
ner et al., 1996) and subsequently from other sources, includ-
ing human placental choriocarcinoma cells (Kekuda et al.,
1996), human brain (Prasad et al., 1998), rat brain (Seth et
al., 1998; Mei and Pasternak, 2001), and mouse brain (Pan et
al., 1998). The ␴-2 receptor, however, has yet to be cloned.
The ␴-1 receptor has 223 amino acids and shares 30% iden-
tity and 67% similarity with a yeast sterol C8–C7 isomerase,
which is involved in cholesterol synthesis (Moebius et al.,
1997). Unlike this yeast sterol isomerase, however, the ␴-1
receptor does not have any sterol isomerase activity (Hanner
et al., 1996), and it shares no sequence homology with any
known mammalian proteins, including the mammalian
C8–C7 sterol isomerase.
This work was supported by the National Institutes of Health grant R01-
MH065503 (to A.E.R.).
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.038307.
ABBREVIATIONS: SKF-10047, N-allyl-normetazocine; IACoc, 3-iodo-4-azido cocaine; IAF, 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-azido-3-iodo-
phenyl) propane; SBDLI, sterol binding domain-like I; SBDLII, sterol binding domain-like II; TMD, transmembrane domain; CNBr, cyanogen
bromide; PAGE, polyacrylamide gel electrophoresis; DTG, ditolylguanidine; MBP, maltose binding protein; THF, tetrahydrofuran; TLC, thin-layer
chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
921

922
Pal et al.
In mammalian systems, the ␴-1 receptor is ubiquitously
expressed in the central nervous system and peripheral or-
gans of the endocrine and immune systems. Because of its
broad distribution among tissues, it is speculated that the
␴-1 receptor is able to mediate different cellular events such
as modulation of voltage-gated Kϩ channels (Wilke et al.,
1999), calcium release (Hayashi and Su, 2001), regulation of
lipid compartmentalization on the endoplasmic reticulum
(Hayashi and Su, 2003), regulation of cocaine effects (Mc-
Cracken et al., 1999a,b), neuroprotective effects (Lysko et al.,
The synthesis of 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-fluoro-3-
phenyl propionamide (2) and 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-
fluorophenyl-propylamine) (3) is outlined in Scheme 1 and that of the
remainder of the compounds (6-11) is outlined in Scheme 2.
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-fluoro-3-phenyl pro-
pionamide) (2). To a solution of dicyclohexylcarbodiimide (5) (4.5 g,
22 mmol) in CH2Cl2 (120 ml), a mixture of 3-(4-fluoro-3-phenyl)
propionic acid (1) (3.4 g, 20 mmol) in CH2Cl2 (80 ml) was added. After
10 min of stirring at room temperature, 2,6-dimethylmorpholine (22
mmol, 2.5 g) was added drop-wise. The reaction mixture was stirred
at room temperature until the starting acid disappeared (3 h) (TLC,
1992), increase in extracellular acetylcholine levels (Matsuno hexane/EtOAc, 9:1). The solid was removed by filtration, and the
solution was washed with H2O (2 ϫ 100 ml), 10% NaHCO3 (2 ϫ 100
ml) before drying with MgSO4. The solvent was evaporated by rotary
evaporator, and the crude product was purified by column chroma-
tography (silica gel, n-hexane/EtOAc, 9:1) as an oil with 90% yield
(4.77 g, 18 mmol). 1H NMR: ␦ 7.18 (m, 2 H), 6.97 (m, 2 H), 6.05 (S, 1
H, NH), 3.84 (m, 2 H), 3.43 (d, J ϭ 8.1 Hz, 4H), 2.83 (t, 2 H), 2.51 (t,
2 H), 1.21 (d, J ϭ 8.0 Hz, 6 H). 13C: ␦ 173.6, 139.6, 135.0, 129.7 (2 C),
115.9 (2 C), 71.1 (2 C), 57.8 (2 C), 34.5, 31.6, 18.4. Anal. Calcd. for
et al., 1993), and inhibition of proliferative responses to mi-
togens (Paul et al., 1994). ␴-1 Receptor knockout mice are
viable and fertile, showing no overt constitutive phenotype
(Langa et al., 2003). Langa and colleagues did find, however,
that when the knockout mice were injected with the ␴ ligand
(ϩ)SKF-10047, there was abrogation of the hypermotility
response, suggesting a role for ␴-1 receptors in psychostimu-
lant actions, which is further supported by the fact that
methamphetamine also binds to the ␴-1 receptor (Nguyen et
al., 2005).
A unique trait of this receptor is that it has high binding
affinity for an assorted array of naturally occurring com-
pounds such as steroids and neuropeptides, but it has yet to
be determined which, if any, of these compounds are the
endogenous ligands for the ␴-1 receptor. Pharmacological
studies have indicated that this receptor binds to a wide
range of compounds including opiates, antipsychotics, anti-
depressants, antihistamines, phencyclidine-like compounds,
␤-adrenergic receptor ligands, serotonergic compounds, co-
C
15H20FNO2: C, 67.90; H, 7.55; N, 5.28%. Found: C, 68.00; H, 5.67;
N, 6.10%.
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-fluorophenyl-propyl-
amine) (3). In a double-necked round-bottomed flask equipped with
septum and condenser, solution of 1-N-(2Ј,6Ј-dimethyl-
a
morpholino)-3-(4-fluoro-3-phenyl propionamide) (2) (2.65 g, 10 mmol)
was added drop-wise to a stirred solution of LiAlH4 (0.74 g, 20 mmol)
in anhydrous tetrahydrofuran (THF) (20 ml) under argon. TLC ind-
icated that the reaction was almost completed after 15 min at room
temperature. The reaction was driven to completion by brief reflu-
xing (15 min), and the reaction mixture was cooled to room temper-
ature followed by the dilution with 100 ml of THF. The excess LiAlH4
was destroyed by drop-wise addition of water (3 ml), 15% aqueous
caine and cocaine analogs, neurosteroids, and neuropeptides. NaOH (3 ml), and finally water (10 ml). The reaction mixture was
stirred for 30 min at room temperature, and the solids were removed
by filtration. The filtrate was dried with MgSO4 and evaporated by a
rotary evaporator to give pure product as yellow oil in 95% yields
(2.38 g, 9.5 mmol). 1H NMR: ␦ 7.12 (m, 2 H), 6.95 (m, 2 H), 3.84 (m,
2 H), 3.33 (d, J ϭ 8.1 Hz, 4H), 2.38 (t, 2 H), 2.55 (m, 2 H), 1.72 (t, 2
H), 1.21 (d, J ϭ 8.0 Hz, 6 H). 13C: ␦ 160.6, 135.6, 130.0 (2 C), 115.7 (2
C), 71.0 (2 C), 57.8 (2 C), 34.5, 34.6, 32.2 18.6. Anal. Calcd. for
Previous work from our laboratory showed that a cocaine-
based radioiodinated photoaffinity label [125I]3-iodo-4-azido
cocaine ([125I]IACoc) is a high-affinity ligand for the ␴-1
receptor (Kahoun and Ruoho, 1992) and specifically identi-
fied steroid binding domain-like II (amino acids 176–194) as
part of the guinea pig ␴-1 receptor binding site for cocaine
(Chen et al., 2007). In this article, we report the synthesis
and characterization of several fenpropimorph-like ligands
that bind to ␴-1 and ␴-2 receptors as determined in guinea
C
15H22FNO: C, 71.68; H, 8.82; N, 5.57%. Found: C, 71.59; H, 8.90; N,
5.70%.
1-N-(2؅,6؅-Dimethyl-morpholino)-3-phenyl propane (6).
A
pig liver membranes and rat liver membranes, respectively. mixture of 1-bromo-3-phenyl propane (4) (3 mmol, 0.59 g) and 2,6-
dimethylmorpholine (5) (3.6 mmol, 0.41 g) was refluxed overnight,
and the reaction mixture was cooled and purified by column chro-
matography (silica gel, n-hexane/EtOAc, 4:1) in yellow oil in 98%
yield (0.6 g, 2.94 mmol). 1H NMR: ␦ 7.21–7.03 (m, 5 H), 3.67 (m, 2 H),
2.65 (m, 4 H), 2.31 (t, 2 H,), 1.77 (m, 2 H), 1.69 (t, 2 H), 1.18 (d, 6 H).
13C NMR, ␦ 128.46, 126.60, 126.03, 124.12, 73.23, 56.42, 52.25, 39.89,
26.55, 19.31. Anal. Calcd. for C15H23NO: C, 77.21; H, 9.93; N, 6.00%.
Found: C, 77.48; H, 10.03; N, 5.78%.
The synthesis of the carrier-free, radioiodinated fenpropi-
morph-like photoaffinity label, 1-N-(2Ј,6Ј-dimethyl-morpho-
lino)-3-(4-azido-3-[125I]iodo-phenyl)propane
([125I]IAF),
which covalently derivatizes both the ␴-1 and ␴-2 receptors
with high specificity, is reported. In addition,␴-1 receptor
binding site peptides that were specifically derivatized by
[
125I]IAF upon photolysis have been identified both for the
membrane-bound ␴-1 receptor and the pure guinea pig ␴-1
receptor (Ramachandran et al., 2007) using cleavage strate-
gies with EndoLys-C and cyanogen bromide.
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-nitrophenyl) propane
and 1-N-(2؅,6؅-dimethyl-morpholino)-3-(2-nitrophenyl) pro-
pane (7). In a round-bottomed flask, a mixture of 1-N-(2Ј,6Ј-dimeth-
yl-morpholino)-3-phenyl propane (6) (3 mmol, 0.69 g), Bi(NO3)3
⅐
5H2O (1.5 mmol, 0.73 g), and trifluoroacetic anhydride (3 mmol, 0.42
ml) was stirred at room temperature under solvent-free conditions
Materials and Methods
General. Melting points were determined with a Thomas-Hoover
capillary melting point apparatus and are reported uncorrected.
NMR spectra were recorded on a Varian 300 spectrometer with the
free-base form of the compounds except where noted (Varian, Palo
Alto, CA). Spectra were obtained in CDCl3 with tetramethylsilane as
an internal standard. Chemicals were obtained from Aldrich Chem-
ical Co. (Milwaukee, WI). Elemental analysis was performed by the
Research Institute of Petroleum Industry (Tehran, Iran). Frozen rat
and guinea pig livers were obtained from Pel-Freez (Rogers, AR).
Scheme 1. Schematic diagram for the synthesis of compounds 2 and 3.

Mapping of ␴-1 Receptor Ligand Binding Site
923
(Hajipour et al., 2003, 2005a,b; Hajipour and Ruoho, 2005a,b). After was separated, and the aqueous layer was extracted with ethyl
approximately 5 min, the starting material, 1-N-(2Ј,6Ј-dimethyl-mor- acetate (2 ϫ 25 ml). The combined ethyl acetate solution was dried
pholino)-3-phenyl propane (6), was consumed (determined by TLC (MgSO4), decolorized with activated charcoal (2 g), and filtrated. The
developed in n-Hex/EtOAc, 4:1). The product was isolated by precip- solvent was evaporated with a rotary evaporator to afford 1-N-(2Ј,6Ј-
itation in CH2Cl2 (20 ml). The solvent was evaporated by a rotary dimethyl-morpholino)-3-(4-amino-3-iodo-phenyl) propane (9) as a
evaporator, and the red residue was purified by column chromatog- yellowish oil, 90% yield (2.7 mmol, 1.01 g). The crude product was
raphy (silica gel, n-hexane/EtOAc, 4:1). The final product (yellow oil) used for the next step without purification.
consisted of a mixture of 2- and 4-nitrobenzene isomers in a ratio of
9:1 (the isomers were not separated). The yield of the reaction was
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-azido-3-iodo-phenyl)
propane (10). In a round-bottomed flask, 1-N-(2Ј,6Ј-dimethyl-
90% (2.7 mmol, 0.75 g). 1H NMR: ␦ 7.14–8.23 (m, 4 H), 3.67 (m, 2 H), morpholino)-3-(4-amino-3-iodo-phenyl) propane (9) (3 mmol, 1.12 g)
2. 75 (m, 0.4 H), 2.71 (m, 3.6 H), 2.33 (m, 2 H), 1.79 (m, 2 H), 1.69 (m, was dissolved in 20% HCl (v/v) and allowed to stand for 5 min at 0°C.
2 H), 1.16 (d, 0.6 H), 1.13 (d, 5.4 H). 13C NMR, ␦ 147.25, 144.12, NaNO2 (3.6 mmol, 0.21 g, in 5 ml of H20) was then added, and the
134.15, 130.25 128.46, 126.60, 0.126.03, 125.38, 124.12, 73.23, 59.70, reaction mixture was stirred at room temperature for 30 min. An
59.40, 58.09, 56.42, 52.25, 39.89, 28.66, 27.78, 26.55, 19.31. Anal. aqueous solution of NaN3 (3.6 mmol, 0.23 g, in 5 ml of H20) was
Calcd. for C15H22N2O3: C, 64.73; H, 7.97; N, 10.06%. Found: C, 64.52; added drop-wise to the reaction mixture. The reaction was stirred at
H, 8.13; N, 9.87%.
room temperature in the dark for 30 min and then extracted with
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-aminophenyl)
pro- ethyl acetate (3 ϫ 30 ml). The combined ethyl acetate extracts were
pane (8). The mixture of 1-N-(2Ј,6Ј-dimethyl-morpholine)-3-(4-nitro- dried with MgSO4, and the solvent was evaporated using a rotary
benzene) propane and 1-N-(2Ј,6Ј-dimethyl-morpholine)-3-(2-nitro- evaporator to afford an orange oil. The crude product was purified by
benzene) propane (7) (3 mmol, 0.83 g) was reduced to the amino column chromatography (silica gel, initially toluene/Et2NH, 20:1,
derivatives using Pd/C (10%, 0.20 g) in methanol and H2 gas over- followed by toluene/Et2NH, 4:1). Yield, 95%, (2.85 mmol, 1.14 g),
night. The reaction mixture was filtered to give 1-N-(2Ј,6Ј-dimethyl- Yellowish oil. 1H NMR: ␦ 8.40 (d, J ϭ 1.8, 1 H), 8.20 (dd, J ϭ 1.8, 8.4
morpholino)-3-(aminophenyl) propane (8) as a mixture of 2- and 1 H), 7.2 (d, J ϭ 8.4, 1 H), 3.75 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2 H,),
4-aminobenzene isomers in 96% yield (2.88 mmol, 0.71 g). The 1-N- 2.22 (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). 13C NMR, ␦ 158.90, 139.10,
(2Ј,6Ј-dimethyl-morpholine)-3-(4-aminobenzene) propane isomer was 135.25, 130.20, 128.10, 127.40, 71.61, 59.73, 58.43, 32.84, 26.46,
separated from 1-N-(2Ј,6Ј-dimethyl-morpholine)-3-(2-aminobenzene) 19.30. Anal. Calcd. for C15H22IN4O: C, 44.90; H, 5.53; N, 13.96%.
propane by column chromatography (silica gel, n-hexane/EtOAc, 4:1) Found: C, 45.11; H, 5.79; N, 13.74%.
(90%, 0.64 g).Yellow oil. 1H NMR: ␦ 7.13 (d, J ϭ 8.4, 2 H), 6.63 (d, J ϭ
8.4, 2 H), 4.80 (br, 2 H, NH2), 3.75 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-azidophenyl) propane
(11). This reaction was performed as described above using 1-N-
H,), 2.22 (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). 13C NMR, ␦ 129.93, (2Ј,6Ј-dimethyl-morpholino)-3-(4-aminophenyl) propane (8) (3 mmol,
126.24, 118.46, 115.24, 71.73, 59.69, 58.43, 32.84, 26.46, 19.30. Anal. 0.74 g) as the starting material. The crude product was purified by
Calcd. for C15H24N2O: C, 72.54; H, 9.74; N, 12.28%. Found: C, 72.68; column chromatography as described above (silica gel, first toluene/
H, 9.86; N, 12.68%.
Et2NH, 20:1, and then toluene/Et2NH, 4:1) to yield 1-N-(2Ј,6Ј-di-
methyl-morpholino)-3-(4-azidophenyl) propane (11) in 96% yield
1-N-(2؅,6؅-Dimethyl-morpholino)-3-(4-amino-3-iodo-phenyl)
propane (9). In a mortar, a mixture of 1-N-(2Ј,6Ј-dimethyl-morpho- (0.26 g, 2.88 mmol), m.p. 128–131. 1H NMR, ␦ 7.92 (d, J ϭ 8.4, 2 H),
lino)-3-(4-aminophenyl) propane (8) (3 mmol, 0.74 g), Me4NϩICl2
7.23(d, J ϭ 8.4, 2 H), 3.72 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2 H,), 2.22
Ϫ
(3.6 mmol, 1.0 g) was ground with a pestle under solid-state condi- (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). 13C NMR, ␦ 142.13, 135.25,
tions for 30 min to afford 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-ami- 130.20, 128.40, 71.61, 59.73, 58.43, 32.84, 26.46, 19.30. Anal. Calcd.
no-3-iodo-phenyl) propane (9). The reaction mixture was dissolved in for C15H23N4O: C, 65.43; H, 8.42; N, 20.35%. Found: C, 64.29; H,
a mixture of water and ethyl acetate (100 ml, 1:1). The organic layer 8.59; N, 20.54%.
Scheme 2. Schematic diagram for the synthesis of com-
pounds 6, 7, 8, 9, 10, and 11.

924
Pal et al.
Radiosynthesis of [125I]IAF. Radioactive Na[125I] (5 mCi) in 14 fusion protein was then purified on an amylose column and cleaved
␮l of 0.1 M NaOH was neutralized by adding 14 ␮l of 0.1 M HCl and with Factor Xa at room temperature for 36 to 48 h. The ␴-1 receptor
then diluted with 50 ␮l of 0.5 M sodium acetate buffer, pH 5.6. with a six-histidine tag at the C terminus was collected on an Ni2ϩ
1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-amino-phenyl) propane (10 ␮l column and eluted using 0.25 M imidazole in wash buffer (50 mM
of 2.5 mg/ml in 0.5 M sodium acetate buffer, pH 5.6) was then added
sodium phosphate, pH 8.0, and 0.3 M NaCl) containing 1% Triton
to the reaction. Iodination was initiated by adding 30 ␮l of Chlora- X-100. The protein that was eluted from the Ni2ϩ column was further
mine-T (1 mg/ml in water) and continued for 15 min at room tem-
perature. The reaction was terminated by the addition of 100 ␮l of
Na2S2O5 (5 mg/ml in water). The pH of the reaction was adjusted to
approximately 9 by adding 20 ␮l of 1 M NaOH. The reaction mixture
was then extracted three times with 1 ml of ethyl acetate. The pooled
extracts were evaporated under N2 to approximately 50 ␮l and
streaked on a 0.25-mm thick silica gel (60/254) plate (10 ϫ 20 cm),
which was developed with a solvent system of toluene/diethylamine
(4:1 v/v). 1-N-(2Ј,6Ј-Dimethyl-morpholino)-3-(4-amino-3-[125I]iodo-
phenyl)propane was detected by autoradiography using X-Omat film
(Rf ϭ 0.63) (Eastman Kodak, Rochester, NY), and the corresponding
silica gel was extracted three times with 1 ml of ethyl acetate and
stored at Ϫ20°C (yield, 1.5 mCi, 30%).
purified by incubating with an agarose resin coupled with anti-Maltose
binding protein antibody (binding capacity of approximately 0.4 mg for
MBP per milliliter) at 4°C for 18 to 24 h. After incubation was complete,
the mixture was centrifuged at 4000 rpm at room temperature, and the
supernatant contained pure guinea pig ␴-1 receptor containing a six-
histidine tag at the C terminus.
␴ Receptor Binding Assays. The binding affinities of the newly
synthesized compounds for the ␴-1 and ␴-2 receptors were deter-
mined using competitive binding assays as described previously
(Matsumoto et al., 1995; Nguyen et al., 2005). Assays for ␴-1 recep-
tors were performed using (ϩ)-[3H]pentazocine (10 nM) in guinea pig
liver membranes (25 ␮g/well) incubated at 30°C for 1 h with several
The ethyl acetate extract of 1-N-(2Ј,6Ј-imethyl-morpholino)-3-(4-
amino-3-[125I]iodo-phenyl)propane was evaporated to dryness under
an N2 stream. H2SO4 (50 ␮l of 3%) was added to the tube, vortexed,
and kept on ice for approximately 15 min. To this reaction mixture,
10 ␮l of 1 M sodium nitrite was added and maintained on ice in the
dark for 30 min. Sodium azide (50 ␮l of 1 M) was added, and the
reaction was allowed to proceed on ice in the dark for 30 min. The
reaction was terminated by the addition of 0.5 ml of 10% sodium
bicarbonate and then extracted three times with 1 ml of ethyl ace-
tate. The extracts were pooled and back-extracted with 0.5 ml of
water. The extract was then reduced to approximately 50 ␮l under
an N2 stream and streaked on a 0.25-mm thick silica gel (60/254)
plate (10 ϫ 20 cm), which was developed in toluene/diethylamine
(20:1). The major radioactive reaction product, [125I]IAF, migrated
with an Rf value of 0.81. The material was detected by autoradiog-
raphy, and the corresponding silica gel was extracted three times
with ethyl acetate and stored at Ϫ20°C overnight. The ethyl acetate
extract was centrifuged the next day at 24,000 rpm for 10 min to
remove dissolved silica (appeared as a solid white precipitate). The
radioactive product comigrated with authentic nonradioactive 1-N-
(2Ј,6Ј-imethyl-morpholino)-3-(4-azido-3-iodo-phenyl) propane pre-
pared as described above. The overall yield for the synthesis of
[
125I]IAF varied between 18 and 20%.
Preparation of Rat Liver and Guinea Pig Liver Membranes.
Minced frozen rat livers or guinea pig livers (65 g) were thawed in
100 ml of homogenization buffer (10 mM phosphate buffer, pH 7.4,
containing 0.32 M sucrose, 1 M MgSO4, 0.5 M EGTA, 1 mM phenyl-
methylsulfonyl fluoride, 10 ␮g/ml leupeptin, 1 ␮g/ml pepstatin A,
and 10 ␮g/ml p-toluenesulfonyl-L-arginine methyl ester) and then
homogenized on ice with a homogenizer (setting 6, four bursts of 10-s
each; Polytron; Kinematica, Basel, Switzerland) followed by a glass
homogenizer (Teflon pestle by six slow passes at 3000 rpm). The
homogenized tissues were then centrifuged at 17,000g for 10 min.
The supernatants were recentrifuged at 100,000g for 1 h. The mi-
crosomal pellets were resuspended in homogenization buffer, snap-
frozen with dry ice/ethanol, and stored at Ϫ80°C at a final protein
concentration of 20 mg/ml.
Preparation of Pure Guinea Pig ␴-1 Receptor. Pure guinea pig
␴-1 receptor was prepared as reported previously (Ramachandran et al.,
2007). In brief, the maltose binding protein (MBP)-guinea pig ␴-1 re-
ceptor fusion protein with a Factor Xa cleavage site linking the MBP
and ␴-1 receptor and a six-histidine tag on the C terminus of ␴-1
receptor was expressed in Escherichia coli. The E. coli biomass was
collected by centrifugation at 3000 rpm for 30 min, sonicated for 15 min
at 4°C, centrifuged at 100,000g for 1 h at 4°C and the pellet resus-
pended, and extracted for 3 h with stirring at 4°C in 20 mM Tris, pH 8.0,
0.2 M NaCl, 1 mM 2-mercaptoethanol, and 1 mM EDTA supplemented
with a protease inhibitor cocktail and Triton X-100. The extract was
centrifuged at 100,000g for 1 h at 4°C. The supernatant containing the
Fig. 1. A, structures of the different fenpropimorph derivatives used in
competitive binding assays. B, inhibition of (ϩ)-[3H]pentazocine binding
by fenpropimorph derivatives in guinea pig liver membranes. Competi-
tive binding curves against 10 nM (ϩ)-[3H]pentazocine were generated
for the fenpropimorph derivatives, and data points were fit to a one-site
nonlinear regression curve. Haloperidol (5 ␮M) was used to determine
nonspecific binding. The KD values for compounds were calculated using
GraphPad Prism software (version 4.0c) and are listed in Table 1. Com-
pound 2 was found not to bind to the ␴-1 receptor (KD Ͼ 100 ␮M).
Compounds 3 and 11 had KD values of 84 Ϯ 21.9 and 72.3 Ϯ 14.9 nM,
respectively. Compounds 6, 7, and 8 had low affinities for ␴-1 receptor
and had KD values of 301 Ϯ 61.7, 242 Ϯ 47, and 1500 Ϯ 390 nM,
respectively. IAF (compound 10) had moderate affinity to the ␴-1 receptor
with a KD value of 194 Ϯ 27.5 nM.

Mapping of ␴-1 Receptor Ligand Binding Site
925
concentrations of competing ligands showed in Fig. 1A. After incu- buffer and separated by 16.5% SDS-Tricine/PAGE. The peptides
bation, the guinea pig liver membranes were harvested on a 0.5% were again identified by wet-gel autoradiography, excised, and
polyethylenimine-treated Whatman GF/B filters using a Brandel cell eluted with water as described above. The eluted peptides were
harvester (Brandel, Gaithersburg, MD). The assay for determining concentrated (to approximately 30 ␮l) and subjected to cyanogen
the ␴-2 binding property of IAF was performed using rat liver mem-
branes (25 ␮g/well) and 30 nM [3H]ditolylguanidine (DTG) in the
presence of (ϩ)-pentazocine (100 nM). Haloperidol (5 ␮M) was used
to determine nonspecific binding for both ␴-1 and ␴-2 receptor bind-
ing assays. Radioactivity on the filters was detected by liquid scin-
tillation spectrometry using NEN formula 989 as scintillation cock-
tail (PerkinElmer Life and Analytical Sciences, Waltham, MA). The
values were fit to a nonlinear regression curve using the software
GraphPad Prism version 4.0c (GraphPad Software Inc., San Diego,
CA), and reported dissociation equilibrium constants, KD, were cal-
culated using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).
Photoaffinity Labeling of the Rat and Guinea Pig Liver
Membrane ␴-1 Receptor. Rat or guinea pig liver membranes (200
␮g/tube) were suspended in a final volume of 0.1 ml of incubation
buffer (50 mM Tris-HCl, pH 7.4) in the presence or absence of the
protecting drugs and incubated at 37°C for 20 min. [125I]IACoc (Ka-
houn and Ruoho, 1992; Chen et al., 2007) or [125I]IAF was added to
the membrane suspensions to a final concentration of 1 nM (1% ethyl
acetate, final concentration) and again incubated for 15 min at 37°C.
The tubes containing membrane suspensions were then placed in an
ice-cold water bath in the photoreactor, and the membrane suspen-
bromide (CNBr) digestion separately.
CNBr Digestion of the ␴-1 Receptor Peptides. The concen-
trated peptides were mixed with 70 ␮l of CNBr solution (0.15 M in
70% formic acid solution) for 15 to 18 h at room temperature by
tumbling. The reaction mixtures were then diluted with 1 ml of
water, lyophilized, and separated by 16.5% SDS-Tricine/PAGE. Gels
were stained-destained, dried, and the radiolabeled peptides were
visualized by PhosphorImaging.
Results
Chemistry. The synthesis of fenpropimorph derivatives 2
and 3 is outlined in Scheme 1. 1-N-(2Ј,6Ј-dimethyl-morpho-
lino)-3-(4-fluoro-3-phenyl propionamide) (2) was obtained by
activating the carbonyl group of 3-(4-fluoro-3-phenyl) propi-
onic acid (starting material is designated as compound 1)
with dicyclohexylcarbodiimide followed by stirring at room
temperature with 2,6-dimethyl morpholine. The crude prod-
uct was purified by silica gel column chromatography using
sions were diluted 10-fold with ice-cold incubation buffer containing n-hexane and ethyl acetate mixture at a ratio of 9:1 as the
20 mM ␤-mercaptoethanol immediately before photolysis. Activation
of the photoaffinity labels was accomplished by exposure to a high-
pressure AH-6 mercury lamp (Advanced Radiation Corporation,
Santa Clara, CA) from a distance of 10 cm for 6 s. After photolysis,
membrane suspensions were centrifuged at 100,000g for 1 h at 4°C,
and the pellets were resuspended in 0.1 ml of incubation buffer.
Proteins were separated by 16.5% SDS-tricine/PAGE and visualized
by PhosphorImager (GE Healthcare, Chalfont St. Giles, Bucking-
hamshire, UK).
solvent system. 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-fluoro-
phenyl-propylamine) (3) was obtained by reduction of (2)
with lithium aluminum hydride (LiAlH4) in anhydrous THF
under argon. After 15-min incubation at room temperature
and a 15-min reflux, the LiAlH4 was destroyed by aqueous
base at room temperature, and solids were removed by fil-
tration. The filtrate yielded pure product as a yellow oil.
The synthesis of fenpropimorph derivatives 4 to 11 is out-
lined in Scheme 2. 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-phenyl
propane (6) was obtained by refluxing a mixture of 1-bromo-
3-phenyl propane (4) and 2,6-dimethylmorpholine (5) over-
night, and the reaction mixture was purified by column chro-
matography (silica gel, n-hexane/EtOAc, 4:1) as a yellow oil
in 98% yield. The 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-phenyl
propane (6) was reacted with Bi(NO3)3 in trifluoroacetic an-
hydride under solvent-free conditions to afford a mixture of
1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-nitrophenyl) propane
and 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(2-nitrophenyl) pro-
Photoaffinity Labeling of the Pure Guinea Pig ␴-1 Receptor.
The pure guinea pig ␴-1 receptor (10 ␮g/tube) in 100 ␮l of incubation
buffer containing 20 mM ␤-mercaptoethanol was incubated at 37°C for
20 min in the presence or absence of the protecting ␴-1 ligands. [125I]IA-
Coc or [125I]IAF was then added to each tube to a final concentration of
1 nM (1% ethyl acetate, final concentration) and again incubated for 15
min at 37°C as described above. The tubes were then placed in the
ice-cold water bath of the photoreactor and photolysed as described
above for 6 s. After photolysis, 5ϫ SDS-PAGE sample buffer (25 ␮l/tube)
was added before separation by 16.5% SDS-Tricine/PAGE and finally
visualized by PhosphorImaging.
EndoLys C Digestion of the Photolabeled ␴-1 Receptor. pane (7) in a ratio of 90:10 in 100% yield (1H NMR analysis).
Before the enzymatic cleavage with the EndoLys C, the ␴-1 receptor
was photolabeled separately (10 ␮g of pure protein in 100 ␮l of
incubation buffer per tube or 1 mg of rat and guinea pig liver
membrane separately in 1 ml of incubation buffer per tube) in the
presence and absence of the protector (10 ␮M haloperidol) using both
The mixture of 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-nitro-
phenyl) propane and 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(2-
nitrophenyl) propane (7) was reduced to 1-N-(2Ј,6Ј-dimethyl-
morpholino)-3-(4-aminophenyl)propane and 1-N-(2Ј,6Ј-dime-
thyl-morpholino)-3-(2-aminophenyl)propane (8) in methanol
with Pd/C-H2 in 96% yield. The 1-N-(2Ј,6Ј-dimethyl-
morpholino)-3-(4-aminophenyl)propane was separated from
1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(2-aminophenyl)propane
by column chromatography (silica gel, n-hexane/EtOAc, 4:1).
Reaction of 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-a-
minophenyl) propane (8) with Me4NϩICl2Ϫ under solid-state
conditions afforded 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-a-
mino-3-iodo-phenyl) propane (9) as a yellowish oil. The crude
1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-amino-3-iodophenyl)
propane (9) was reacted with HNO2 and then NaN3 to pro-
duce 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-azido-3-iod-
ophenyl) propane (10) in excellent yields after purification.
The 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-azidophenyl)
[
125I]IACoc (1 nM) and [125I]IAF (1 nM) as described above. Photo-
labeled samples were purified by electrophoresis on a 15% SDS-
polyacrylamide gel and visualized by wet gel (unstained) autoradiog-
raphy. The autoradiogram was used as a template to excise the
specifically labeled and the protected ␴-1 receptor from the gel with
a razor blade. The gel slices were minced and placed into a 1.5-ml
microcentrifuge tube containing 1 ml of water. The tubes were main-
tained overnight at 4°C, and the gel slurries were transferred to spin
columns (Bio-Rad Laboratories, Hercules, CA). The eluted ␴-1 recep-
tor in the supernatant extract was collected by centrifugation at
1600g for 5 min. In general, more than 80% of the photolabeled ␴-1
receptor was recovered in the aqueous supernatant (as measured by
radioactivity). The eluted materials were concentrated to 100 ␮l by
lyophilization and incubated with 0.25 ␮g of EndoLys C (Promega,
Madison, WI) per tube overnight at room temperature. The enzy-
matic digestions were terminated by adding 5ϫ SDS-PAGE sample propane (11) was also prepared by reacting 1-N-(2Ј,6Ј-dime-

926
Pal et al.
thyl-morpholino)-3-(4-aminophenyl) propane (8) with HNO2 were used to covalently derivatize the ␴ receptors from rat
and then with NaN3 in excellent yield. microsomes (Fig. 2A), and specific labeling of both the ␴-1
Binding of Fenpropimorph Derivatives to ␴-1 and and ␴-2 receptors was observed. The ␴-1 receptor was shown
␴-2 Receptors. All fenpropimorph derivatives (structures to be a 25.3-kDa band, which was protected by nonradioac-
shown in Fig. 1A) were tested for binding affinities to ␴-1 tive (ϩ)-pentazocine (100 nM), haloperidol (10 ␮M), DTG (25
receptors in guinea pig liver membranes, and IAF (compound ␮M), and fenpropimorph (10 ␮M), whereas the ␴-2 receptor
10) binding to the ␴-2 receptor was determined in rat liver photolabeling at 18 kDa was protected by haloperidol, DTG,
membranes. The binding affinities of these compounds were and fenpropimorph but not by the ␴-1-selective ligand, pen-
determined by competition of (ϩ)-[3H]pentazocine (10 nM) tazocine (Fig. 2B). Compared with the robust ␴-1-specific
with high affinity and selectivity to the ␴-1 receptor. For IAF labeling of the cocaine-based radioiodinated photoaffinity la-
binding to the ␴-2 receptor, 30 nM [3H]DTG was used in the bel, [125I]IACoc, the fenpropimorph-like photoprobe [125I]IAF
presence of nonradioactive (ϩ)-pentazocine (100 nM).
specifically derivatized both the ␴-1 and ␴-2 receptors (Fig.
The curves obtained for the ␴-1 receptor competition as- 2A). Several additional specifically photolabeled bands are
says are shown in Fig. 1B, and the results are summarized in also detected at 96.7, 130, and 147.3 kDa, as assessed from a
Table 1. Two of the seven compounds presented (3 and 11) plot of Rf versus log molecular mass of the standards (Fig. 2A,
had high affinities for the ␴-1 receptor with KD values of less inset). The high molecular mass bands present appeared
than 100 nM. The iodo-azido derivative or IAF (compound 10) after photolysis with both [125I]IACoc and [125I]IAF (Fig. 2A)
had KD values of 194 Ϯ 27.5 nM and 2.78 Ϯ 1.5 ␮M for the ␴-1 and were each protected by (ϩ)-pentazocine, haloperidol,
and the ␴-2 receptor, respectively. Linear regression curve DTG, and fenpropimorph (Fig. 2B). A band at 19 kDa was
fitting indicated that all the compounds, including IAF, fit to also highly specifically labeled with [125I]IAF (Fig. 2, A and
a single population of binding sites for the ␴-1 receptor with B). Photolabeling of this 19-kDa band was not protected by
R2 values between 0.951 and 0.992 (Table 1).
(ϩ)-pentazocine or haloperidol but was partially protected by
Photoaffinity Labeling of ␴ Receptors in Rat Liver DTG (Fig. 2B) and completely protected by nonradiolabeled
Microsomal Membranes. Both [125I]IACoc and [125I]IAF fenpropimorph.
TABLE 1
␴-1 Receptor affinity (KD) and one-site linear regression analysis of fenpropimorph derivatives as determined in guinea pig liver membranes
KD and R2 values of these compounds were calculated using GraphPad Prism software, version 4.0c.
R2 Value
Ligands
Structures
␴-1 KD Ϯ S.E.M.a
(One-Site
Linear Regression)
nM
2
Ͼ200,000
0.951
3
6
84 Ϯ 21.9
0.978
0.986
301 Ϯ 61.7
7
242 Ϯ 47.0
1500 Ϯ 390
194 Ϯ 27.5
0.988
0.978
0.992
8
10
11
72.3 Ϯ 14.9
0.986
a Mean of triplicate experiments Ϯ S.E.M.

Mapping of ␴-1 Receptor Ligand Binding Site
927
Photoaffinity Labeling of ␴ Receptors in Guinea Pig haloperidol. The 25.3-kDa band, which showed specific label-
Liver Microsomal Membranes. Both [125I]IACoc and ing by both photoprobes, was excised (region outlined by the
[
125I]IAF specifically and covalently derivatized the ␴-1 re- broken lines) and eluted with water. Generally, 80% of the
ceptor from guinea pig liver membranes (Fig. 3A, right).
[
125I]iodine-photolabeled ␴-1 receptor was eluted.
Using both probes, the ␴-1 receptor was detected as a 25.3-
Before EndoLys C cleavage, the starting specifically pho-
kDa band, which was protected by haloperidol (10 ␮M). Com- tolabeled ␴-1 receptors, as eluted from the gels, are shown in
pared with the rat liver membranes (Fig. 3A, left), the ␴-2 the autoradiogram in Fig. 3C (lanes 1 and 2, 5 and 6, 9 and
receptors and the high molecular mass bands were not ob- 10, and 13 and 14). EndoLys-C cleavage, as depicted the
served with either photoprobe in the guinea pig liver mem- cleavage at lysine 142 in Fig. 3B, resulted the eluted [125I]io-
branes.
dine photolabeled ␴-1 receptor into two large fragments of
EndoLys C Digestion of [125I]IACoc and [125I]IAF 16.3 and 9 kDa (Fig. 3C, lanes 3, 7, 11, and 15). Specificity of
Specifically Photolabeled ␴-1 Receptor in Rat and the photoprobe (i.e., haloperidol protection) and subsequent
Guinea Pig Liver Microsomal Membranes. Figure 3A cleavage is further shown in Fig. 3C (lanes 4, 8, 12, and 16).
demonstrates labeling of the rat liver and guinea pig liver No EndoLys-C cleavage was observed at lysine 60 in the
membranes with [125I]IACoc and [125I]IAF. The labeling of guinea pig ␴-1 receptor.
the ␴-1 receptor in both membrane preparations was highly
As reported previously (Chen et al., 2007), [125I]IACoc spe-
specific (compare lanes 1, 3, 5, and 7 with 2, 4, 6, and 8, cifically labeled the SBDLII domain (amino acids 170–195) at
respectively, in Fig. 3A) in the presence and absence of 10 ␮M Asp188, which corresponds to the 9-kDa fragment (Fig. 3C,
Fig. 2. A, comparison of photolabeling
the ␴-1 and ␴-2 receptor using
[
125I]IACoc and [125I]IAF. Rat liver
microsomes (200 ␮g/lane) were photo-
labeled using 1 nM [125I]IACoc and
[
125I]IAF separately with no protector
(Ϫ) and in the presence of 100 nM
(ϩ)-pentazocine (P). Both photoprobes
labeled the ␴-1 receptor and three
high molecular mass (ϩ)-pentazocine-
protectable protein bands. The molec-
ular masses of these three bands were
calculated from the standard curve
obtained by plotting Rf values against
the Log(molecular mass) of various
known protein standards, which
ranged between 205 and 14 kDa (in-
set). B, [125I]IAF labeling of rat liver
microsomes. Photolabeling of rat liver
membranes (200 ␮g/lane) with 1 nM
[
125I]IAF with no protector (Ϫ) and in
the presence of 100 nM (ϩ)-pentazo-
cine (P), 10 ␮M haloperidol (H), 25 ␮M
DTG (D), and 10 ␮M fenpropimorph
(F). Photolabeling was performed as
described under Materials and Meth-
ods, and the proteins were separated
by SDS-Tricine/PAGE.

928
Pal et al.
lanes 3 and 4, 11 and 12). The same SBDLII domain-contain- ment, indicating simultaneous labeling of the ␴-1 receptor
ing peptide (9 kDa) was also labeled with [125I]IAF (Fig. 3C, region, which includes SBDLI (amino acids 91–109).
lanes 7 and 8, 15 and 16). However, it is noteworthy that the
EndoLys-C Cleavage of the Photolabeled Pure
labeling was also specifically located in the 16.3-kDa frag- Guinea Pig ␴-1 Receptor. A similar pattern of EndoLys-C
Fig. 3. A, comparison of photolabeling of rat liver and guinea pig liver membranes using both [125I]IACoc and [125I]IAF. Rat liver membranes (lanes
1–4) and guinea pig liver membranes (lanes 5–8) were photolabeled (200 ␮g/lane) using 1 nM [125I]IACoc (lanes 1 and 2, 5 and 6) and [125I]IAF (lanes
3 and 4, 7 and 8) separately in the absence of 10 ␮M haloperidol (lanes 1, 3, 5, and 7) and in the presence of 10 ␮M haloperidol (lanes 2, 4, 6, and 8)
and visualized by PhosphorImaging. Both [125I]IACoc and [125I]IAF specifically photolabeled the ␴-1 receptors in both membrane preparations. The
␴-1 receptor either photolabeled or protected from photolabeling was excised out separately (excised regions showed by the broken lines) and eluted
from the gel as reported under Materials and Methods for subsequent treatment with EndoLys-C. B, schematic diagram of EndoLys-C cleavage of rat
and guinea pig ␴-1 receptors (25.3 kDa) to produce fragments of 16.3 and 9 kDa. C, phosphorimage visualization of EndoLys-C cleavage of the
photolabeled rat and guinea pig ␴-1 receptor. EndoLys-C treatment of specifically photolabeled, gel-eluted ␴-1 receptor from rat liver membranes
(lanes 1–8) and guinea pig liver membranes (lanes 9–16) produced two peptides as depicted in Fig. 3B. In all cases, the specific labeling was
determined by comparing the photolabeling of the ␴-1 receptor in the absence of 10 ␮M haloperidol (lanes 1, 3, 5, 7, 9, 11, 13, and 15) and in the
presence of 10 ␮M haloperidol (lanes 2, 4, 6, 8, 10, 12, 14, and 16). Using [125I]IACoc (lanes 1–4 and 9–12) as the photoprobe, the specific radiolabel
in the ␴-1 receptor from both species (lanes 1 and 2 for rat and lanes 9 and 10 for guinea pig) were found entirely on the smaller 9-kDa peptides (lanes
3 and 4, 11 and 12) after the cleavage with EndoLys-C, indicating labeling of SDBLII-containing peptides. However, both rat and guinea pig ␴-1
receptor, when specifically photolabeled with [125I]IAF (lanes 5 and 6 for rat and 13 and 14 for guinea pig), showed the specific label on the both the
16.3- and 9-kDa fragments (lanes 7 and 8 for rat and lanes 15 and 16 for guinea pig) upon cleavage with EndoLys-C, indicating labeling of both SBDLI
and SBDLII regions.

Mapping of ␴-1 Receptor Ligand Binding Site
929
cleavage of the pure guinea pig ␴-1 receptor (Fig. 4B) was tions, consistent with the SBDLI- and SBDLII-containing
also observed after photolabeling by
125I]IACoc and peptides. There may be weak cleavage at lysine 60 in the
125I]IAF, as shown previously in Fig. 3C for the guinea pig pure receptor, as indicated in Fig. 4C (lane 12) at 9.3 kDa,
[
[
liver membrane ␴-1 receptor. In these experiments, the pure because cleavage at position 60 would produce a peptide from
␴-1 receptor (Fig. 4B, lanes 1and 2) was cleaved by En- residues 60 to 142, which also contains the SBDLI (91–109)
doLys-C and produced the same two polypeptides (Fig. 4B, region.
lanes 3 and 4), as assessed by Coomassie staining, but the
CNBr Cleavage of the 16.3- and 9.8-kDa Peptides De-
peptide containing SBDLII now migrated at 9.8 kDa instead rived from EndoLys-C Cleavage of the Photolabeled
of the 9-kDa position (from the membrane-bound receptor) Pure Guinea Pig ␴-1 Receptor. Before cleavage by cyano-
because the pure guinea pig ␴-1 receptor contains six histi- gen bromide, the 16.3- and 9.8-kDa peptides, as shown with
dine residues at the C terminus. Specific photolabeling by Coomassie staining (Fig. 5B, lanes 1–4), was eluted from SDS
[
125I]IACoc and [125I]IAF, as illustrated in the autoradio- gels. For both [125I]IACoc and [125I]IAF-labeled peptides, the
gram in Fig. 4B, lanes 5 and 6 and lanes 10 and 11, again Coomassie staining patterns were identical, and therefore,
showed that the cleavage of the photolabeled receptor with only the staining pattern for the [125I]IACoc-labeled peptides
EndoLys-C produced the expected [125I]IACoc-labeled 9.8- is shown for both EndoLys-C cleavage (Fig. 5B) and subse-
kDa peptide containing SBDLII (Fig. 4B, lanes 7 and 8). On quent CNBr cleavage (Fig. 5C). Because the CNBr cleavage
the other hand, EndoLys-C cleavage of the [125I]IAF photo- experiments required more peptide material, the photolabel-
labeled receptor showed labeling of both the 16.3- and the ing intensity as shown in Fig. 5B (lanes 5–12) was more
9.8-kDa peptides (Fig. 4B, lanes 12 and 13) indicating, as was intense than observed in Fig. 4B but produced qualitatively
the case for the membrane-bound receptor (Fig. 3C), that and quantitatively the same relative results. Specific photo-
[
125I]IAF labeled the ␴-1 receptor binding site at two posi- labeling of [125I]IACoc was protected only at the 9.8-kDa
Fig. 4. A, schematic diagram of En-
doLys-C cleavage of the pure six histi-
dine tagged guinea pig ␴-1 receptors. B,
PhosphorImaging visualization of En-
doLys-C cleavage of the pure guinea pig
␴-1 receptor photolabeled with [125I]IA-
Coc and [125I]IAF. Both [125I]IACoc-
photolabeled (lanes 5–8) and [125I]IAF-
photolabeled (lanes 9–12) pure guinea
pig ␴-1 receptor showed a similar En-
doLys-C cleavage pattern as shown pre-
viously in Fig. 3C. In all cases, the spe-
cific labeling was determined by
comparing the photolabeling of ␴-1 re-
ceptor in the absence of 10 ␮M haloper-
idol (lanes 5, 7, 9, and 11) and in the
presence of 10 ␮M haloperidol (lanes 6,
8, 10, and 12). Lanes 1 to 4 showed the
Coomassie staining pattern of the
[
125I]IACoc-photolabeled ␴-1 receptors
(lanes 5–8). The Coomassie staining for
the [125I]IAF-photolabeled ␴-1 recep-
tors was identical and is not shown
here. Upon treatment with EndoLys-C,
the specifically
[
125I]IACoc-photola-
beled receptors (lanes 5 and 6) resulted
in two peptides with the molecular
masses of 16.3 and 9.8 kDa. The specific
radiolabel from [125I]IACoc was located
on the SBDLII region (9.8 kDa) (lanes 7
and 8). The specific [125I]IAF-labeled
␴-1 receptor (lanes 10–11), upon treat-
ment with EndoLys-C, showed radiola-
bel on both the SBDLI (16.3 kDa) and
SBDLII (9.8 kDa) regions (lanes 12 and
13). An additional specific radiolabeled
band at 9.3 kDa (lane 12 and 13) was
observed with [125I]IAF consistent with
the cleavage at the position 60 by En-
doLys-C, which contains the SBDLI do-
main.

930
Pal et al.
peptide containing the SBDLII domain (compare lanes 5 and pure guinea pig ␴-1 receptor at position 60, which yielded a
6 with 7 and 8, respectively, in Fig. 5B), which is again similar fragment containing SBDLI (lane 10, Fig. 5B).
consistent with the pure guinea pig ␴-1 receptor results (Fig.
As expected, cyanogen bromide cleavage of the [125I]IACoc
4B) and data reported previously showing specific [125I]IA- photolabeled 9.8-kDa fragment (Coomassie staining shown
Coc photolabeling of COS7 cells transfected with guinea pig in lanes 2 and 4 in Fig. 5C) produced the specifically photo-
␴-1 receptor (Chen et al., 2007). Consistent with the results labeled 6.8-kDa fragment (compare lanes 6 and 8 in Fig. 5C),
from membrane-bound ␴-1 receptor, [125I]IAF specifically la- whereas no specific labeling of the CNBr-cleaved 16.3-kDa
beled both the 16.3-kDa peptide containing SBDLI and the fragment (Coomassie staining shown in lanes 1 and 3 in Fig.
9.8-kDa peptide containing SBDLII (compare lanes 9 and 10 5C) by [125I]IACoc was seen (compare lanes 5 and 7). CNBr
with 11 and 12, respectively, in Fig. 5B). Specific label incor- cleavage of the [125I]IAF-labeled 9.8-kDa band (Coomassie
poration by [125I]IAF was also observed again in the 9.3-kDa staining represented in lanes 2 and 4 in Fig. 5C) also pro-
peptide consistent with partial EndoLys-C cleavage of the duced the specifically labeled 6.8-kDa fragment (compare
Fig. 5. A, schematic diagram of CNBr
cleavage of the 16.3- and 9.8-kDa En-
doLys-C cleaved peptides from pure
guinea pig ␴-1 receptors. B, gel-eluted
EndoLys-C cleaved fragments of the
pure guinea pig ␴-1 receptor specifi-
cally photolabeled with
[
125I]IACoc
and [125I]IAF. Because both [125I]IA-
Coc-photolabeled (lanes 5–8) and
[
125I]IAF-photolabeled (lanes 9–12)
EndoLys-C cleaved peptides showed
similar Coomassie staining patterns,
only the Coomassie staining pattern
of the [125I]IACoc-labeled peptides are
shown (lanes 1–4). In all cases, the
specific labeling was determined by
comparing the photolabeling of ␴-1 re-
ceptor in the absence of 10 ␮M halo-
peridol (lanes 1 and 2, 5 and 6, and 9
and 10) and in the presence of 10 ␮M
haloperidol (lanes 3 and 4, 7 and 8,
and 11 and 12). C, PhosphorImaging
visualization of CNBr cleavage of the
16.3- and 9.8-kDa EndoLys-C cleaved
peptides photolabeled with [125I]IA-
Coc and
[
125I]IAF. Because both
[
125I]IACoc- and
[
125I]IAF-photola-
beled CNBr cleaved peptides showed
similar Coomassie staining patterns,
only the Coomassie staining pattern
for the [125I]IACoc labeled peptides
are shown (lanes 1–4). In all cases,
the specific labeling was determined
by comparing the photolabeling of the
pure ␴-1 receptor in the absence of 10
␮M haloperidol (lanes 1 and 2, 5 and
6, and 9 and 10) and in the presence of
10 ␮M haloperidol (lanes 3 and 4, 7
and 8, and 11 and 12). Upon treat-
ment with CNBr, the 16.3-kDa pep-
tides (lanes 1 and 3) resulted in two
peptides with molecular masses of
10.5 and 5.5 kDa (lanes 2 and 4), as
depicted in Fig. 5A. The 10.5- and 5.5-
kDa peptides were not specifically ra-
diolabeled when [125I]IACoc was used
as the photoprobe (compare lanes 5
and 7). However, the same 16.3-kDa
peptide that was specifically photola-
beled with [125I]IAF contained specific
label in the SBDLI region (5.5-kDa
fragments) (compare lane 9 with 11).
Specific [125I]IACoc and [125I]IAF la-
beling that was found on the 9.8-kDa
fragments upon treatment with CNBr
was found on the 6.8-kDa fragments
containing the SBDLII regions (com-
pare lanes 6 and 10 with 8 and 12,
respectively).

Mapping of ␴-1 Receptor Ligand Binding Site
931
lanes 10 and 12 in Fig. 5C). On the other hand, CNBr cleav- labeled, which was protectable by fenpropimorph but not by
age of the [125I]IAF-labeled 16.3-kDa fragment (Coomassie other traditional ␴ ligands. The appearance of the selective
staining depicted in lanes 1 and 3 in Fig. 5C) produced the
[
125I]IAF labeling of this 19-kDa band raises the possibility
specifically labeled 5.5-kDa fragment (compare lanes 9 and that this band may represent the emopamil binding protein-
11 in Fig. 5C) containing the SBDLI domain. All of these like protein previously identified by Moebius et al. (2003).
results together support the conclusion that the [125I]IAF
The position of [125I]IACoc binding of the guinea pig ␴-1
photoprobe inserts into peptides containing the sequences receptor has been identified previously in the third hydro-
identified as the SBDLI (amino acids 91–109) and SBDLII phobic region of the ␴-1 receptor, which has high sequence
(amino acids 176–194).
homology to the yeast and fungal sterol isomerase (Chen et
al., 2007). The specific amino acid that was identified as a
photo-insertion site with [125I]IACoc was Asp188. We have
designated this region as SBDLII (amino acids 176–194)
Discussion
Fenpropimorph, a yeast sterol isomerase inhibitor, has (Chen et al., 2007). From similar sequence homology consid-
been found previously to bind with high affinity to the guinea erations, a second region of the guinea pig and rat ␴-1 recep-
pig ␴-1 receptor (Ki ϭ 0.005 nM) (Moebius et al., 1997). This tor (amino acids 91–109), which matches the sequence of the
is perhaps not surprising, because the ␴-1 receptor shows yeast sterol isomerase, has been designated as SBDLI (Chen
sequence homology with the C8–C7 yeast sterol isomerase et al., 2007).
(Moebius et al., 1996; Jbilo et al., 1997; Moebius et al., 1997).
The specific [125I]IACoc and [125I]IAF photolabeled ␴-1
In an effort to find specific, high-affinity ligands and photo- receptors in the natural milieu of the membranes (i.e., in
probes for the ␴-1 and ␴-2 receptors, we have synthesized guinea pig and rat liver membranes) resulted in two identical
various derivatives of fenpropimorph as outlined in Schemes peptides having molecular masses of 16.3 and 9 kDa after
1 and 2. The affinities of these fenpropimorph derivatives cleavage with the protease EndoLys-C (Fig. 3C). The halo-
were determined by competition with (ϩ)-[3H]pentazocine for peridol protectable label incorporation for both [125I]IACoc
binding to the ␴-1 receptor. The affinity of IAF (compound 10) and [125I]IAF was in the SBDLII containing peptide (9 kDa).
for the ␴-2 receptor was determined by competitive binding of In addition, the haloperidol-protectable photolabeling was
[3H]DTG in the presence of nonradioactive (ϩ)-pentazocine. also found in the 16.3 kDa fragment that contains SBDLI
Compound 2 was found not to bind to the ␴-1 receptor (KD Ͼ with [125I]IAF used as the photoprobe (Fig. 3C). The amount
100 ␮M), presumably because its amide group traps the of specific [125I]IAF labeling between the 9- and 16.3-kDa
nitrogen’s lone pair needed for optimal binding (Ablordeppey fragments seemed to differ between the rat liver membrane
et al., 2000). Compounds 3 and 11, on the other hand, had the ␴-1 receptor and guinea pig liver membrane ␴-1 receptor
highest binding affinities to the ␴-1 receptor with KD values (compare lanes 7,8, 15, and 16 in Fig. 3C), which perhaps
of less than 100 nM. IAF (compound 10) bound with nearly could be caused by subtle differences in the binding site of the
14.3-fold higher affinity to the ␴-1 receptor (KD ϭ 194 Ϯ 27.5 ␴-1 receptor in the membrane preparations from the two
nM) over the ␴-2 receptor (KD ϭ 2.78 Ϯ 1.5 ␮M) and was species. An additional observation in the EndoLys-C cleavage
derivatized as a radioiodinated photoaffinity label. Figure 1, experiments that was repeatedly observed was that the
A and B, show that [125I]IAF does indeed photolabel the ␴-1 guinea pig ␴-1 receptor, which contains a lysine at position
and ␴-2 receptors in a specific and protectable manner. The 60, was not cleaved by the enzyme as would be predicted. The
␴-1 receptor at 25.3 kDa is protected by nonradioactive (ϩ)- reason for the lack of cleavage by EndoLys-C in guinea pig
pentazocine, DTG, haloperidol, and fenpropimorph, whereas ␴-1 receptor at position 60 is not readily explained but may
the ␴-2 receptor at 18 kDa is protected by all of the com- be caused by a conformational constraint or otherwise lack of
pounds listed except for the ␴-1-specific ligand (ϩ)-pentazo- access for the proteolytic enzyme. On the other hand, the rat
cine. Figure 1A illustrates that the photoaffinity label previ- ␴-1 receptor contains an arginine at position 60 and is, there-
ously synthesized in our laboratory, [125I]IACoc (Kahoun and fore, not expected to be cleaved by EndoLys-C.
Ruoho, 1992), derivatized the ␴-1 receptor specifically. It is
interesting that, as shown in Fig. 2, A and B, high molecular
The pure guinea pig ␴-1 receptor, photolabeled with both
[
125I]IACoc and [125I]IAF, also showed similar EndoLys-C
mass bands are present, which were derivatized by both cleavage patterns (Fig. 4B) compared with guinea pig and rat
radioiodinated photoaffinity labels and were protected by liver membranes (showed in Fig. 3C). The specific [125I]IACoc
(ϩ)-pentazocine, haloperidol, DTG, and fenpropimorph. The labeling was found only on the 9.8-kDa peptide that contains
molecular masses of these protein bands were identified as the SBDLII (Fig. 4B, lanes 7 and 8), and the specific [125I]IAF
96.7, 130, and 147 kDa, with ligand binding properties con- labeling was found on both the 16.3- and 9.8-kDa peptides
sistent with the ␴-1 receptor. It is significant the ␴-1 receptor (Fig. 4B, lanes 11 and 12). When further cleaved with cyano-
contains two GXXXG motifs, which occur with high fre- gen bromide, the [125I]IAF-labeled 16.3 and 9.8-kDa peptides
quency in membrane proteins that favor helix-helix interac- resulted in peptides of molecular masses of 5.5 kDa (amino
tions (Brosig and Langosch, 1998; Russ and Engelman, 2000; acids 94–142) and 6.8 kDa (amino acids 171–229) that con-
Kleiger et al., 2002; Polgar et al., 2004). Further investiga- tained the SBDLI and the SBDLII domains, respectively, and
tion is required to characterize these high molecular mass were radiolabeled (Fig. 5C, lanes 9 and 10) as described in
bands and determine whether they are homo- or hetero- Fig. 5A. Both the competitive binding data (Fig. 1 and Table
oligomers of the ␴-1 receptor or whether they interact with 1) for IAF versus (ϩ)-[3H]pentazocine and the IAF photo-
other membrane proteins.
probe data indicate a single binding site or two binding sites
In addition to the highly specific labeling of the ␴-1 (25.3 with equal affinity. Together, these observations suggest that
kDa) and ␴-2 (18 kDa) receptors in rat liver membranes (Fig. the ␴-1 receptor contains a haloperidol-protectable ligand
2, A and B), it was observed that a 19-kDa protein was also binding site(s) for IAF that is composed, at least in part, of

932
Pal et al.
the SBDLI and SBDLII domains. The photoreactive azido program (available at http://www.pondr.com/background-
group in [125I]IAF may interact with both the SBDLI and
.html) acquisition of the ␴-1 receptor that evaluates relative
SBDLII domains because of the flexibility of the molecule.
[
LII region because of the structural rigidity and restricted
motion of the tropane ring.
As reported previously (Aydar et al., 2002), the ␴-1 receptor
contains an even number of transmembrane sequences be-
cause both the N and C termini lie on the same side of a
biological membrane. Hydrophobicity analysis of the ␴-1 re-
ceptor (using the prediction program available at http://ww-
order versus disorder profiles of a protein. Using this analy-
sis (Fig. 6A), the hydrophobic regions proposed as TMDI and
TMDII and the SBDLI and SBDLII regions are highly or-
dered and possibly feature an ␣-helical structure. Therefore,
in Fig. 6, B and C, the hydrophobic regions are diagrammed
as cylinders to depict ␣ helices.
125I]IACoc, on the other hand, interacts only with the SBD-
In summary, we report the synthesis of various fenpropi-
morph derivatives and their binding affinities as ligands for
the ␴-1 receptor. We have also demonstrated that the radio-
iodinated photoaffinity label [125I]IAF is able to specifically
photolabel ␴-1 and ␴-2 receptors. Photolabeling with
w.ch.embnet.org/software/TMPRED_form.html)
identifies
three hydrophobic regions as illustrated in Fig. 6A. The
alignment of the ␴-1 receptor sequence reasonably aligns the
first hydrophobic region with transmembrane domain I
(TMD I) and the second hydrophobic sequence with TMD II.
From sequence alignment, the sequence of the amino acids
90 to 110 of the ␴-1 receptor matched with high fidelity to the
yeast and fungal sterol isomerase (Hanner et al., 1996; Chen
et al., 2007). This region partially overlaps putative TMD II
and constitutes SBDLI (amino acids 91–109). The third hy-
drophobic region, which aligns with the SBDLII region, is the
location of Asp188, which is specifically photolabeled by
[
125I]IAF and [125I]IACoc further identified high molecular
mass protein complexes that were not completely dissociated
upon SDS-PAGE analysis. The data suggest that ␴-1 recep-
tors may exist as oligomers or interact with protein partners
either constitutively or through binding of ligands. Based on
the simultaneous specific photolabeling of SBDLI- and SBD-
LII-containing peptides in the guinea pig ␴-1 receptor by
[
125I]IAF and the fact that there is haloperidol-protectable
binding of IAF to the ␴-1 receptor, the data reported in this
article support the conclusion that SBDLI and SBDLII com-
[
125I]IACoc (Chen et al., 2007). Additional support for this
positioning of TMD segments is consistent with a PONDR prise at least portions of the ␴-1 receptor ligand binding
Fig. 6. A, SBDLI and SBDLII regions
of the ␴-1 receptor. Hydrophobic re-
gions of the protein, which are pre-
dicted by the TMPred program (avail-
able at http://www.ch.embnet.org/
software/TMPRED_form.html)
and
the PONDR program (available at
http://www.pondr.com/background-
.html), were used to predict the trans-
membrane segments and areas of or-
der and disorder. Three highly
ordered hydrophobic regions are
shown, the latter two of which contain
the highly conserved SBDLI and SB-
DLII regions, respectively. B, a puta-
tive model of the ␴-1 receptor illus-
trates a possible spatial arrangement
of the ligand binding site in which the
cylinders depict the hydrophobic re-
gions as putative
␣ helices and
[
125I]IAF as ƒ. C, an alternative
model for IAF interaction with the ␴-1
receptor is depicted in B.

Mapping of ␴-1 Receptor Ligand Binding Site
933
Lysko PG, Gagnon RC, Yue TL, Gu JL, and Feuerstein G (1992) Neuroprotective
effects of SKF 10,047 in cultured rat cerebellar neurons and in gerbil global brain
ischemia. Stroke 23:414–419.
site(s) and suggest possible folded structures as portrayed in
Fig. 6, B and C.
Martin WR, Eades CG, Thompson JA, Huppler RE, and Gilbert PE (1976) The effects
of morphine- and nalorphine- like drugs in the nondependent and morphine-
dependent chronic spinal dog. J Pharmacol Exp Ther 197:517–532.
Matsumoto RR, Bowen WD, Tom MA, Vo VN, Truong DD, and De Costa BR (1995)
Characterization of two novel sigma receptor ligands: antidystonic effects in rats
suggest sigma receptor antagonism. Eur J Pharmacol 280:301–310.
Matsuno K, Matsunaga K, Senda T, and Mita S (1993) Increase in extracellular
acetylcholine level by ␴ ligands in rat frontal cortex. J Pharmacol Exp Ther
265:851–859.
McCracken KA, Bowen WD, de Costa BR, and Matsumoto RR (1999a) Two novel
sigma receptor ligands, BD1047 and LR172, attenuate cocaine-induced toxicity
and locomotor activity. Eur J Pharmacol 370:225–232.
McCracken KA, Bowen WD, and Matsumoto RR (1999b) Novel sigma receptor
ligands attenuate the locomotor stimulatory effects of cocaine. Eur J Pharmacol
365:35–38.
Mei J and Pasternak GW (2001) Molecular cloning and pharmacological character-
ization of the rat sigma1 receptor. Biochem Pharmacol 62:349–355.
Moebius FF, Bermoser K, Reiter RJ, Hanner M, and Glossmann H (1996) Yeast
sterol C8–C7 isomerase: identification and characterization of a high-affinity
binding site for enzyme inhibitors. Biochemistry 35:16871–16878.
Moebius FF, Fitzky BU, Wietzorrek G, Haidekker A, Eder A, and Glossmann H
(2003) Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol
delta8-delta7 isomerase activity. Biochem J 374:229–237.
Moebius FF, Reiter RJ, Hanner M, and Glossmann H (1997) High affinity of sigma
1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological
relationship with the yeast sterol C8–C7 isomerase. Br J Pharmacol 121:1–6.
Nguyen EC, McCracken KA, Liu Y, Pouw B, and Matsumoto RR (2005) Involvement
of sigma (sigma) receptors in the acute actions of methamphetamine: receptor
binding and behavioral studies. Neuropharmacology 49:638–645.
Pan YX, Mei J, Xu J, Wan BL, Zuckerman A, and Pasternak GW (1998) Cloning and
characterization of a mouse sigma1 receptor. J Neurochem 70:2279–2285.
Paul R, Lavastre S, Floutard D, Floutard R, Canat X, Casellas P, Le Fur G, and
Breliere JC (1994) Allosteric modulation of peripheral sigma binding sites by a
new selective ligand: SR 31747. J Neuroimmunol 52:183–192.
Acknowledgments
We acknowledge insights and assistance from Dr. Michael K.
Sievert and from Dr. Anupama Gopalakrishnan for proofreading of
the manuscript.
References
Ablordeppey SY, Fischer JB, and Glennon RA (2000) Is a nitrogen atom an important
pharmacophoric element in sigma ligand binding? Bioorg Med Chem 8:2105–2111.
Aydar E, Palmer CP, Klyachko VA, and Jackson MB (2002) The sigma receptor as a
ligand-regulated auxiliary potassium channel subunit. Neuron 34:399–410.
Brosig B and Langosch D (1998) The dimerization motif of the glycophorin A
transmembrane segment in membranes: importance of glycine residues. Protein
Sci 7:1052–1056.
Chen Y, Hajipour AR, Sievert MK, Arbabian M, and Ruoho AE (2007) Character-
ization of the cocaine binding site on the sigma-1 receptor. Biochemistry 46:3532–
3542.
Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1)
and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction. Biochem Pharmacol 22:3099–3108.
Hajipour AR, Adibi H, and Ruoho AE (2003) Wet silica-supported permanganate for
the cleavage of semicarbazones and phenylhydrazones under solvent-free condi-
tions. J Org Chem 68:4553–4555.
Hajipour AR, Kooshki B, and Ruoho AE (2005a) Nitric acid and phosphorus pentox-
ide supported on silica gel as a mild and efficient system for the oxidation of
benzylic alcohols under solvent-free conditions. Org Prep Proced Int 37:585–589.
Hajipour AR, Kooshki B, and Ruoho AE (2005b) Nitric acid in the presence of
supported P2O5 on silica gel: an efficient and novel reagent for oxidation of
sulfides to the corresponding sulfoxides. Tetrahedron Lett 46:5503–5506.
Hajipour AR and Ruoho AE (2005a) Methyltriphenylphosphonium peroxydisulfate
and iodine as mild reagents for the iodination of activated aromatic compounds.
Org Prep Proced Int 37:279–283.
Hajipour AR and Ruoho AE (2005b) Nitric acid in the presence of P2O5 supported on
silica gel—a useful reagent for nitration of aromatic compounds under solvent-free
conditions. Tetrahedron Lett 46:8307–8310.
Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kempner E, and
Glossmann H (1996) Purification, molecular cloning, and expression of the mam-
malian ␴1- binding site. Proc Natl Acad Sci U S A 93:8072–8077.
Hayashi T and Su TP (2001) Regulating ankyrin dynamics: Roles of ␴-1 receptors.
Proc Natl Acad Sci U S A 98:491–496.
Polgar O, Robey RW, Morisaki K, Dean M, Michejda C, Sauna ZE, Ambudkar SV,
Tarasova N, and Bates SE (2004) Mutational analysis of ABCG2: role of the
GXXXG motif. Biochemistry 43:9448–9456.
Prasad PD, Li HW, Fei YJ, Ganapathy ME, Fujita T, Plumley LH, Yang-Feng TL,
Leibach FH, and Ganapathy V (1998) Exon-intron structure, analysis of promoter
region, and chromosomal localization of the human type 1 sigma receptor gene.
J Neurochem 70:443–451.
Quirion R, Bowen WD, Itzhak Y, Junien JL, Musacchio JM, Rothman RB, Su TP,
Tam SW, and Taylor DP (1992) A proposal for the classification of sigma binding
sites. Trends Pharmacol Sci 13:85–86.
Ramachandran S, Lu H, Prabhu U, and Ruoho AE (2007) Purification and charac-
terization of the guinea pig sigma-1 receptor functionally expressed in Escherichia
coli. Protein Expr Purif 51:283–292.
Hayashi T and Su TP (2003) ␴-1 Receptors (␴1 binding sites) form raft-like microdo-
mains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic
reticulum lipid compartmentalization and export. J Pharmacol Exp Ther 306:718–
725.
Iwamoto ET (1981) Locomotor activity and antinociception after putative mu, kappa
and ␴ opioid receptor agonists in the rat: influence of dopaminergic agonists and
antagonists. J Pharmacol Exp Ther 217:451–460.
Russ WP and Engelman DM (2000) The GxxxG motif: a framework for transmem-
brane helix-helix association. J Mol Biol 296:911–919.
Seth P, Fei YJ, Li HW, Huang W, Leibach FH, and Ganapathy V (1998) Cloning and
functional characterization of a sigma receptor from rat brain. J Neurochem
70:922–931.
Jbilo O, Vidal H, Paul R, De Nys N, Bensaid M, Silve S, Carayon P, Davi D, Galiegue
S, Bourrie B, et al. (1997) Purification and characterization of the human SR
31747A-binding protein. A nuclear membrane protein related to yeast sterol
isomerase. J Biol Chem 272:27107–27115.
Su TP (1982) Evidence for ␴ opioid receptor: binding of [3H]SKF-10047 to etorphine-
inaccessible sites in guinea-pig brain. J Pharmacol Exp Ther 223:284–290.
Vaupel DB (1983) Naltrexone fails to antagonize the sigma effects of PCP and SKF
10,047 in the dog. Eur J Pharmacol 92:269–274.
Kahoun JR and Ruoho AE (1992) [125I]Iodoazidococaine, a photoaffinity label for the
haloperidol- sensitive ␴ receptor. Proc Natl Acad Sci U S A 89:1393–1397.
Kekuda R, Prasad PD, Fei YJ, Leibach FH, and Ganapathy V (1996) Cloning and
functional expression of the human type 1 sigma receptor (hSigmaR1). Biochem
Biophys Res Commun 229:553–558.
Wilke RA, Mehta RP, Lupardus PJ, Chen Y, Ruoho AE, and Jackson MB (1999) ␴
Receptor photolabeling and ␴ receptor-mediated modulation of potassium chan-
nels in tumor cells. J Biol Chem 274:18387–18392.
Kleiger G, Grothe R, Mallick P, and Eisenberg D (2002) GXXXG and AXXXA:
common alpha-helical interaction motifs in proteins, particularly in extremo-
philes. Biochemistry 41:5990–5997.
Langa F, Codony X, Tovar V, Lavado A, Gimenez E, Cozar P, Cantero M, Dordal A,
Hernandez E, Perez R, et al. (2003) Generation and phenotypic analysis of sigma
receptor type I (sigma 1) knockout mice. Eur J Neurosci 18:2188–2196.
Address correspondence to: Dr. Arnold E. Ruoho, Department of Pharma-
cology, University of Wisconsin Medical School, 1300 University Avenue, Mad-
ison, WI 53706. E-mail: aeruoho@wisc.edu