(Received for publication, October 14, 1994; and in revised form, January 17, 1995)
From the
We recently characterized (Moebius, F. F., Burrows, G. G.,
Striessnig, J., and Glossmann H.(1993) Mol. Pharmacol. 43,
139-144) and purified (Moebius, F. F., Hanner, M., Knaus, H. G.,
Weber, F., Striessnig, J., and Glossmann, H.(1994) J. Biol. Chem. 269, 29314-29320) a binding protein for the phenylalkylamine
Ca antagonist emopamil. The emopamil-binding protein
(EBP) acts as a high affinity acceptor for several antiischemic drugs
and thus represents a potential common molecular target for
antiischemic drug action. Degenerate oligonucleotides were synthesized
according to the N-terminal amino acid sequence of purified EBP and
used to amplify a guinea pig cDNA with reverse transcriptase-polymerase
chain reaction and to clone full-length cDNAs from guinea pig and human
liver cDNA libraries. The cDNAs coded for 229 (guinea pig) and 230
(human) amino acid 27-kDa polypeptides without significant sequence
homology with any known protein. However, EBP shared structural
features with pro- and eukaryotic drug transport proteins. The amino
acid identity between human and guinea pig EBP was 73%. Hydrophobicity
plots predicted four transmembrane segments. The C terminus contained a
lysine-rich consensus sequence for the retrieval of type I integral
membrane proteins to the endoplasmic reticulum. The heterologous
expression of human and guinea pig EBP in Saccharomyces cerevisiae demonstrated that the expression of EBP alone is sufficient to
form high affinity drug- and cation-binding domains identical to the
[
H]-emopamil-binding site of guinea pig liver.
Northern and Western blot analysis revealed high abundance of EBP in
guinea pig epithelial tissues as liver, bowel, adrenal gland, testis,
ovary, and uterus and low densities in brain, cerebellum, skeletal
muscle, and heart. EBP is suggested to be the first structurally
characterized member of a family of high affinity microsomal drug
acceptor proteins carrying so called
-binding sites.
The phenylalkylamine Ca antagonist emopamil
labels a high affinity binding protein (emopamil-binding protein, EBP) (
)for a variety of structurally different compounds in
guinea pig liver(1, 2) . Some of these drugs such as
emopamil, ifenprodil, opipramol, trifluoperazine, and chlorpromazine
exert antiischemic effects in animal models of stroke, but their
neuroprotective action is not fully understood at the molecular level.
Since the possibility exists that EBP is involved in ischemia-related
cellular events biochemical studies were undertaken to investigate the
physiological function of EBP. These studies revealed that the EBP is
an integral membrane protein of the endoplasmic reticulum that migrated
with a relative molecular mass of 22.5 kDa in SDS-PAGE(2) .
Ferguson analysis revealed a molecular mass of 27.2 kDa (3) .
Hydrodynamic studies and the extensive characterization of its
drug-binding properties (2, 4) demonstrated striking
similarities with other microsomal drug-binding proteins, so called
receptors (see (5) for review). This led to the proposal
that EBP and sigma receptors are members of a superfamily of high
affinity drug-binding proteins in the endoplasmic reticulum of
different tissues(3) .
Here we report the cloning and heterologous expression of human and guinea pig cDNAs coding for proteins with the pharmacological properties of EBP. We also present the tissue distribution of EBP and EBP-mRNA and discuss the structural features which EBP shares with drug transporters.
Microsomal membranes from different tissues (see legend to Fig. 2B) were prepared as described
previously(3) . Microsomal protein was solubilized in sample
buffer containing 4% (w/v) SDS and 10 mM dithiothreitol and
separated on 13% (w/v) polyacrylamide gels. After electrophoretic
transfer to a polyvinylidene difluoride membrane (Immobilon P,
Millipore), immunostaining with affinity purified
anti-EBP and anti-EBP
was
carried out as described previously (3) .
Figure 2:
Tissue distribution of EBP in guinea pig. A, Northern blot hybridization with a guinea pig EBP cDNA
probe. 15 µg of total RNA/lane were separated on a 1.2% (w/v)
agarose gel and transferred to a nylon membrane (Boehringer Mannheim).
Hybridization with a DIG-labeled guinea pig antisense cDNA probe was
carried out as described under ``Experimental Procedures''
with an alkaline phosphatase-conjugated second antibody and
chemoluminescence detection. EBP was encoded in guinea pig and human
liver by a 1.3-kilobase mRNA. The human EBP hybridized to a lesser
extent due to the sequence difference to the guinea pig cDNA probe. B, Western blot analysis with affinity-purified
anti-EBP antibody. Microsomal membranes were
prepared as described(2) , and 30 µg of microsomal protein
were separated on 13% (w/v) SDS-polyacrylamide gels. Immunoblotting was
performed as described previously(3) . The arrow indicates the migration of EBP. The
(±)[
H]emopamil binding activity was
measured at a ligand concentration of 1.89 nM in parallel.
Nonspecific binding was defined as the binding in the presence of 30
µM ZnCl
to selectively inhibit
(±)[
H]emopamil binding to EBP. The binding
activity expressed as percent binding relative to guinea pig liver
microsomes (1.95 ± 0.17 pmol of
(±)[
H]emopamil bound/mg of protein (n = 2)) is given in parenthesis (means ±
standard deviations, n = 2). The following
abbreviations were used: Li, guinea pig liver (100%); Lh, human liver (not determined); Sp, spleen (5.1
± 0.4); Lu, lung (8.2 ± 0.3); Br, brain
(2.4 ± 0.8); Sm, skeletal muscle (1.2 ± 0.1); Ki, kidney (4.5 ± 0.9); Hm, heart muscle (1.9
± 0.9); Ce, cerebellum (2.1 ± 0.4); Ad,
adrenal gland (15.1 ± 0.1); Oe, oesophagus (5.3
± 1.8); St, stomach (0 ± 0); Il, ileum
(10.8 ± 2.2); Co, colon (20.0 ± 0.4); Pc, pancreas (0 ± 0); Sk, skin (3.6 ±
0.1); Eye (5.0 ± 0.3); Te, testis and epididymidis (9.7
± 1.4); Ov, ovary (8.0 ± 0.2); Ut,
uterus (4.8 ± 2.1).
Figure 1:
A, comparison of the deduced amino acid
sequences of guinea pig (G.P.) and human (H.S.) EBP.
cDNAs were isolated from guinea pig and human liver cDNA libraries. The
four hydrophobic putative transmembrane segments are shaded.
The consensus sequence for phosphorylation by cAMP-dependent
proteinkinase (), the polylysine cluster for retrieval into the
endoplasmic reticulum (underlined,(17, 18) ),
the sequences of the peptides (EBP
and
EBP
) used for antibody raising (bold),
and the amino acid sequences previously determined by Edman degradation
of tryptic peptides A-E of the purified EBP (overlined,(3) ) are indicated. The numbers of the
amino acid residues are given on the right. B, hydrophobicity
analysis of guinea pig EBP. Hydropathy plotting of the EBP amino acid
sequence was performed according to Kyte and Doolittle (19) with an averaging window size of 19 plotted at one-residue
intervals. On the ordinate hydrophobicity and hydrophilicity
are indicated by positive and negative numbers, respectively.
TM1-4 designate the putative transmembrane segments. C,
proposed topology model of guinea pig EBP. Based on the endoplasmic
reticulum retrieval sequence (17, 18) and the above
hydrophobicity analysis of the amino acid sequence the protein is
postulated to have cytoplasmic N and C termini, four transmembrane
segments, and two connecting loops in the lumen of the endoplasmic
reticulum (ER) (
, hydrophobic residues; &cjs2125;,
positively charged residues;
, negatively charged residues;
, others).
Nonspecific [H]emopamil binding was defined
with 30 µM ZnCl
which is known (2) to
selectively block [
H]emopamil binding to EBP. The
distribution of EBP mRNA and immunoreactivity in different tissues
correlated with the binding activity (see Fig. 2A and B and legend to Fig. 2). The highest EBP densities were
found in epithelial tissues as liver, ileum, and colon. Urogenital
tissues as kidney, adrenal gland, testis, ovary, and uterus contained
also high densities of EBP. Lower densities were present in spleen,
oesophagus, stomach, and eyes. No binding activity was detected in
microsomes from pancreas and stomach perhaps due to proteolysis during
incubation at 22 °C. Very low densities were found in excitable
tissues as brain, cerebellum, heart, and skeletal muscle. Since the
microsomes used for Western blot analysis and binding studies are
mainly derived from the endoplasmic reticulum (2) the
differential distribution of EBP rather reflects differences in EBP
density than differences in the cellular endoplasmic reticulum content.
Figure 3:
Characterization of human and guinea pig
EBP expressed in S. cerevisiae. A,
[H]emopamil binding to microsomal membranes from
guinea pig liver (Li), Hep G2 cells (He), and S.
cerevisiae transfected with nonrecombinant vector (Co) or
vector with human (HS
, HS
),
and guinea pig (GP
, GP
) EBP
cDNAs. Microsomal membranes were prepared as described under
``Experimental
Procedures.''(-)-[
H]emopamil binding
was performed at a ligand concentration of 1.1-1.7 nM.
The results shown are the mean of 4-11 experiments. EBP cDNAs
were expressed in S. cerevisiae strain JB811 (obtained from
Kim Nasmyth, Vienna) using the yeast episomal plasmid YEp351 (21) with an ADC1 cassette(9) . Yeast cells were lysed
with glass beads (see ``Experimental Procedures''). HS
and GP
are the entire clone inserts
HS3 and
GP5, respectively; in GP
the 5`-noncoding region, and
in HS
the 5` and the 3`-noncoding region were removed by
PCR (see ``Experimental Procedures''). B,
anti-EBP
immunoblotting. 7 µg of microsomal
membrane protein (He, HepG 2, 50 µg of protein)
were separated on a 13% (w/v) SDS-polyacrylamide gel and analyzed by
immunostaining with anti-EBP
. The higher molecular
mass observed for EBP in Hep G2 cells could be due to higher protein
load. C, saturation analysis of
(-)-[
H]emopamil binding to human EBP
(HS
) expressed in S. cerevisiae. Saturation
analysis was performed by decreasing the specific activity of
(-)-[
H]emopamil by dilution with unlabeled
(-)-emopamil at a protein concentration of 19 µg/ml. A
B
of 75 pmol/mg protein and a K
of 76 nM were obtained. The inset shows
the Scatchard transformation of these data. D,
[
H]azidopamil photoaffinity labeling of
microsomal protein from vector-transfected yeast cells (lane
1), guinea pig liver microsomes (lane 7), and yeast cells
transfected with the human cDNA (HS
).
Photoaffinity labeling was carried out in the absence (lanes
1, 2, 6, and 7) or presence of 0.15
µM of(-)-emopamil (lane 3), opipramol (lane 4), and ifenprodil (lane 5) by incubation of 21
nM [
H]azidopamil with 0.5 mg microsomal
protein/ml. 50 µg of microsomal protein were separated on a 15%
(w/v) polyacryamide gel. For fluorography the gel was equilibrated in
Amplify
(Amersham), dried, and exposed to Kodak X-Omat AR5
(10-day exposure).The migration of EBP is indicated by the arrow. E, chemical cross-linking. Microsomal
membranes from yeast cells expressing guinea pig EBP (GP
) were solubilized in in 1% (w/v) digitonin,
150 mM NaCl, 20 mM K
H
PO4, pH
7.8, at a protein concentration of 4 mg/ml. After dilution in 150
mM NaCl, 20 mM K
H
PO4, pH 7.8,
to a final digitonin concentration of 0.2% (w/v) samples were incubated
2 h at 4 °C in the absence (lane 1) or presence of
chemical cross-linkers (20 mM disuccinimidyl suberate, lane 2; 20 mM SANPAH, lane 3; 10 mM glutaraldehyde, lane 4). After photolysis of SANPAH (3
min, 3 cm distance) with a Sylvania GTE germicide lamp 10 µg of
protein were separated by SDS-PAGE and immunoblotted as described under
``Experimental Procedures.''
Saturation analysis with
[H]emopamil (see Fig. 3C)
revealed similar densities of EBP in microsomes from S. cerevisiae cells expressing EBP (HS
, B
70 ±
4 (n = 3) pmol/mg membrane protein; GP
,
B
29 ± 7 (n = 3) pmol/mg membrane
protein) and guinea pig liver (B
35 pmol/mg membrane
protein,(1) ). The dissociation constants of human (K
15 ± 1 (n = 3)
nM) and guinea pig EBP (K
10 ± 3 (n = 3) nM) expressed in S. cerevisiae were similar to the value measured in guinea pig liver (K
13 nM,(1) ).
The pharmacological profile of the expressed guinea pig and human EBP revealed no major difference (see Table 1) to the EBP in guinea pig liver. Minor decreases in affinity were observed for haloperidol, NaCl (human and guinea pig EBP), and (+)-verapamil (human EBP).
Accordingly, [H]azidopamil photoaffinity
labeling of HS
(see Fig. 3D) reflected the
properties of [
H]emopamil binding, i.e. complete block of labeling in the presence of 0.15 µM of emopamil, opipramol, and ifenprodil. No labeling of a protein
with similar apparent molecular mass was observed in cells transfected
with the expression vector without EBP cDNA.
We previously reported that disuccinimidyl suberate, SANPAH, and glutaraldehyde led to dimerization of the liver EBP(3) . Dimerization after incubation with the above cross-linkers was confirmed for the expressed guinea pig (see Fig. 3E) and human (results not shown) EBP.
EBP is a high affinity binding protein for the antiischemic
phenylalkylamine Ca antagonist
[
H]emopamil and the photoaffinity label
[
H]azidopamil. A variety of compounds with
antiischemic effects in animal models of cerebral ischemia inhibited
[
H]emopamil binding to EBP with high affinity. It
could therefore represent a common molecular target of antiischemic
drug action(2) . This prompted further biochemical studies to
eventually clarify its physiological function. The close
pharmacological and biochemical relationship of EBP with so-called
receptors (5) led to the proposal that EBP and
receptors are members of a superfamily of high affinity microsomal
drug-binding proteins(3) .
The functional expression of the
cloned EBP-cDNAs in S. cerevisiae demonstrated that they
encode proteins which are able to form the
[H]emopamil-binding site. Chemical cross-linking
of the guinea pig and human EBP expressed in yeast suggested that the
protein forms a functional homodimer.
The localization of EBP in the endoplasmic reticulum membrane hampers in vivo demonstration of drug transport due to intracellular drug compartimentalization. In contrast to the P-glycoprotein, the EBP amino acid sequence contains no ATP-binding cassette. Although EBP carries a sodium ion-binding site (1) as in sodium-dependent transporters(26, 27, 28) , the driving force of a potential drug transport by EBP is unknown making in vitro studies difficult.
The molecular cloning of a high affinity
binding protein for phenylalkylamine Ca antagonists
demonstrates a unique primary structure distinct from the drug binding
subunit of L-type Ca
channels and
rules out a relationship with known drug metabolizing enzymes. It was
previously demonstrated that EBP shares many pharmacological and
biochemical features with so-called
receptors(2, 4) . We therefore proposed (3) the existence of a superfamily of microsomal high affinity
drug acceptor proteins comprising binding polypeptides for
[
H]pentazocine,
[
H]ditolylguanidine, and
[
H]SKF10047 and a high affinity
[
H]opipramol-binding site(29) . EBP is
the first structurally characterized member of this family. Of
considerable interest is the molecular organization of the promiscous
drug-binding domain of these proteins in comparison to other
drug-binding domains with a narrower drug specificity, e.g. the phenylalkylamine binding domain of L-type Ca
channels(30) . This can be investigated now by
site-directed mutagenesis employing the efficient expression system
presented here. The comparison of human and guinea pig cDNAs will allow
the cloning of EBP and related proteins from other species to identify
evolutionary conserved motifs important for the function of these
proteins. Furthermore, our work paves the way for the cloning and
disruption of the mouse EBP gene to generate EBP-deficient animals
which could give insight into the pharmacological significance and
biological function of this new family of microsomal drug acceptor
proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z37985 [GenBank]and Z37986[GenBank].