©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phenylalkylamine Ca Antagonist Binding Protein
MOLECULAR CLONING, TISSUE DISTRIBUTION, AND HETEROLOGOUS EXPRESSION (*)

(Received for publication, October 14, 1994; and in revised form, January 17, 1995)

Markus Hanner (§) Fabian F. Moebius (§) Florian Weber (¶) Manfred Grabner Jörg Striessnig Hartmut Glossmann (**)

From the Institut für Biochemische Pharmakologie, Universität Innsbruck, Peter Mayr Strasse 1, A-6020 Innsbruck, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [^3H]-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.


INTRODUCTION

The phenylalkylamine Ca antagonist emopamil labels a high affinity binding protein (emopamil-binding protein, EBP) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

(-)[^3H]Emopamil (67 Ci/mmol), (±)[^3H]emopamil (49 Ci/mmol), (-)-[^3H]azidopamil (87 Ci/mmol), and the unlabeled phenylalkylamines were kindly provided by Knoll A.G. (Ludwigshafen, Germany). Sigma ligands were a gift of Dr. Traber (Tropon, Cologne, Germany). Other chemicals were obtained from the following sources: opipramol, Ciba-Geigy (Vienna, Austria); Bradford protein reagent, electrophoresis reagents and molecular weight markers, Bio-Rad; restriction enzymes and polymerases, Promega (Vienna, Austria); oligonucleotide synthesis reagents, Millipore (Vienna, Austria); all other chemicals, Sigma (Vienna, Austria).

PCR Amplification of EBP cDNA Fragments

The N-terminal sequence of purified guinea pig EBP was obtained by Edman degradation (peptide A,(3) ). Degenerate oligonucleotides encoding amino acid residues PLHPYW (5`-CC(CGAT)TT(CGAT)CA(TC)CC(CGAT)TA(TC)TGG-3` (sense primer)) and DHFVPN (5`-TC(GA)TT(CGAT)GG(CGAT)AC(GA)AA(GA)TG(GA)TC-3` (antisense primer)) were synthesized and used in the polymerase chain reaction (PCR) (Bio-med, Thermocycler 60). First strand cDNA was synthesized from guinea pig total RNA with an oligo(dT) primer (Pharmacia Biotech. Inc., first strand cDNA synthesis kit). PCR was performed with 35 cycles at low stringency (1 min at 94 °C, 0.5 min at 37 °C, 1.5 min at 72 °C). The amplified products were separated on a 20% (w/v) polyacrylamide gel. Products with a size of 50- 60 bp were eluted into destilled water (12 h, 37 °C), cloned into pCRII vector (Invitrogen) and sequenced. One clone with a 56-bp insert coded for the amino acid sequence PLHPYWPRHLRLDHFVPN (residues 7-24) of peptide A. The cDNA for EBP was amplified (1 min at 94 °C, 0.5 min at 56 °C, 1.5 min at 72 °C) with an oligonucleotide according to the amino acid sequence HLRLDHF (5`-ACCTGCGGCTGGA(TC)CA(CT)TT-3` (sense primer)) in conjunction with the oligo(dT) primer (3`-antisense primer from the first strand cDNA synthesis kit, Pharmacia Biotech Inc.). After cloning into the pCRII vector, one clone (C7, 940 bp) was sequenced with the dideoxy termination method (6) using standard and specific sequencing primers. This clone coded for a protein containing all partial peptide sequences previously determined by Edman degradation (3) .

Library Construction and Screening

An oligo(dT)-primed cDNA library was constructed in phage gt10 using size-selected (>500 bp) poly(A) RNA derived cDNA from human and guinea pig liver. Recombinant phages were plated on Escherichia coli C600 hfl at a density of 60,000 plaque-forming units/135-mm plate. After plaque formation, plaques from five plates were transferred to Biodyne B membrane (Pall) and hybridized with a C7-specific antisense cDNA probe labeled with digoxigenin 11-dUTP (DIG) following a single strand-labeling PCR protocol(7) . From 30 to 40 hybridizing plaques/plate 10 plaques from each library were isolated. The cDNA inserts were excised with EcoRI and subcloned into pBluescript IISK+ (Stratagene). The longest cDNAs were sequenced on both strands as described above. DNA and protein sequence analysis were performed with the GCG sequence analysis software package (Genetics Computer Group Inc., Madison, WI).

RNA Preparation and Northern Blot Analysis

Total RNA from liver, kidney, adrenal gland, lung, brain, cerebellum, spleen, heart, and skeletal muscle were prepared as described(8) . 15 µg of each RNA were separated on a 1.2% (w/v) agarose gel in 30% (v/v) formaldehyde and blotted. RNAs were probed with a DIG-labeled specific antisense probe (guinea pig cDNA, nucleotides 227-709). After hybridization (42 °C, 50% (v/v), formamide) blots were washed at low stringency (0.2 times SSC (1 times SSC is 0.15 M NaCl, 0.01 M sodium citrate, pH 7.0), 0.1% (w/v) SDS at 22 °C), and bands were detected according to the DIG protocol using chemolumiscence detection (Boehringer Mannheim).

Expression of EBP in Saccharomyces cerevisiae

Guinea pig and human EBP cDNAs were subcloned into the yeast episomal plasmid YEp351ADC1(9) . Truncated cDNAs carrying a 5`-HindIII restriction site and AAA triplet before the initial ATG (10) (human and guinea pig EBP) and a 3`-NotI restriction site behind the stop codon (human EBP) were generated with PCR. PCR products were cloned into pBluescript IISK+ and sequenced before subloning into YEp351ADC1. Transformation of S. cerevisiae JB811 was performed as described(11) . Cells were harvested at an OD of 1.2 and lysed with glass beads in 50 mM Tris-HCl (pH 7.4, 37 °C), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride. The lysate was spun for 5 min at 500 times g (4 °C), and the supernatant was pelleted for 45 min at 100,000 times g (4 °C). This microsomal pellet was washed with 0.5 M KCl, 0.15 M Tris HCl (pH 8, 4 °C), centrifuged at 100,000 times g (4 °C), resuspended in 5% (v/v) glycerol, 20 mM Tris HCl (pH 7.4, 37 °C) at a protein concentration of 3-6 mg/ml, and frozen in liquid N(2).

Immunological Techniques

A polyclonal antiserum was raised in New Zealand White rabbits against a synthetic peptide corresponding to the C terminus of human EBP with an additional N-terminal lysine residue (peptide EBP). The production of a sequence directed antibody against the N terminus of guinea pig EBP (anti EBP) was described previously(3) . Peptide synthesis, coupling to bovine serum albumin, immunization, enzyme-linked immunosorbent assay, and affinity purification of antisera were performed as described(12) .

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 (±)[^3H]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(2) to selectively inhibit (±)[^3H]emopamil binding to EBP. The binding activity expressed as percent binding relative to guinea pig liver microsomes (1.95 ± 0.17 pmol of (±)[^3H]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).



Binding Assays, Photoaffinity Labeling, and SDS-PAGE

These were performed according to standard procedures(2, 3) . Additional experimental detail is given in the figure legends. Protein concentrations were determined according to Bradford(13) , using bovine serum albumin as a standard. Binding parameters (IC, slope factor (n(H)), K(d), and B(max)) were calculated by non-linear curve fitting to the general dose-response equation (14) (inhibition data) or a rectangular hyberbola (saturation data). Data are given as means ± standard deviations.

Hep G2 Cell Culture

Hep G2 cells were grown in Hanks' modified Dulbecco's modified Eagle's medium in 4.5% (w/v) glucose. Cells were trypsinized, collected, and microsomal membranes were prepared as described above.


RESULTS

Cloning of EBP cDNAs

guinea pig and human liver gt10 libraries were screened with the 940-bp EBP cDNA obtained by reverse transcriptase-PCR (see ``Experimental Procedures''). From each library 10 hybridizing clones were isolated. After characterization by restriction enzyme mapping the longest clones (1005-bp clone GP5 for guinea pig EBP and 1073-bp clone HS3 for human EBP) were subjected to sequencing. The deduced amino acid sequences of human and guinea pig EBP are shown in Fig. 1A.


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 (bullet), 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; bullet, negatively charged residues; circle, others).



Sequence Analysis

The open reading frame of guinea pig and human cDNAs coded for 229 and 230 amino acid residues corresponding to a molecular mass of 26.683 and 26.356 Da, respectively. The polyadenylation signal AATAAA of the human EBP cDNA occurred 239-244 nucleotides downstream from the stop codon of the putative EBP and 13 nucleotides upstream from a putative poly(A) tail (not shown). Although the 5` non-translated regions of both cDNAs (nucleotides 1-114 of human EBP) were 64% homologous (results not shown) the first methionine codon was flanked by a consensus sequence for the initiation of translation (15) indicating that it was indeed the start codon. The N-terminal amino acid sequence of the purified EBP determined by Edman degradation only lacked the initial methionine residue(3) . The identity and similarity (determined as described in (16) ) of the amino acid sequences between human and guinea pig EBP were 73 and 85%, respectively (see Fig. 1A). Sequence comparison in protein and DNA databases (Swiss Prot, EMBL/GenBank) showed no significant homology (>20%) with known sequences. Both EBPs contained potential phosphorylation sites for protein kinases A (see Fig. 1A) and C (not shown). The C termini of both proteins were heterologous. They contained a polylysine motif (KVMKSKGK in guinea pig and KAKSKKN in human) known to mediate the retrieval of type I integral membrane proteins into the endoplasmic reticulum(17, 18) . Hydropathy plots according to Kyte and Doolittle (19) computed with a window of 19 amino acid residues predicted four transmembrane segments (TMS) (see Fig. 1B). The connecting loop between TMS3 and TMS4 was also highly hydrophobic (see Fig. 1B). The TMS2 and TMS3 contained two glutamate residues conserved in human and guinea pig EBP whereas the TMS1 and TMS4 contained no charged residues (see Fig. 1A). All cysteine residues were localized in the TMS2 and 3 (see Fig. 1A). A high content of aromatic amino acid residues in the transmembrane segments was determined as described in (20) and was 28 and 23% for guinea pig and human EBP, respectively. The topology model of EBP (Fig. 1C) predicts that most of the protein is buried in the lipid bilayer. From the endoplasmic reticulum retrieval sequence for type I integral membrane proteins(17, 18) , we conclude that the N and C termini face the cytoplasm. In this model the potential phosphorylation site for cAMP-dependent protein kinase (see Fig. 1, A and C) faces the lumen of the endoplasmic reticulum. Protein kinase A did not phosphorylate the detergent-purified EBP (results not shown).

Northern and Western Blot Analysis of Tissue Distribution in Comparison to [^3H]Emopamil Binding

The tissue distribution of EBP was studied in guinea pig by comparing the mRNA levels as well as EBP immunoreactivity with the microsomal [^3H]emopamil binding activity. In Northern blots a 1.3-kilobase mRNA was detected in different tissues (Fig. 2A). No other band hybridized under conditions of low stringency indicating that RNAs with high sequence homology are either absent or present only at lower abundance. The migration of EBP immunoreactivity in different tissues was indistinguishable from liver (Fig. 2B). Immunolabeling was specifically blocked by 0.03 µM of synthetic peptide (results not shown).

Nonspecific [^3H]emopamil binding was defined with 30 µM ZnCl(2) which is known (2) to selectively block [^3H]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.

Expression of EBP in S. cerevisiae

To clarify if EBP alone is able to form the high affinity emopamil binding site and to investigate if the isolated homologous human cDNA also forms a high affinity emopamil acceptor, we expressed both cDNAs. Several mammalian cell lines (e.g. PC12, COS-7 (not shown) and Hep G2 (see Fig. 3, A and B)) were found unsuitable for this purpose due to the presence of endogenous [^3H]emopamil binding activity and anti-EBP immunoreactivity. In contrast, the S. cerevisiae strain JB811 lacked both activities (Fig. 3, A and B) and was therefore used for expression with the yeast episomal plasmid YEp351ADC1(9, 21) . The complete cDNA (GP(0) and HS(0), see Fig. 3, A and B) did not yield detectable expression. We therefore deleted the 5`-untranslated region (GP(1), HS(1), see Fig. 3, A and B) which resulted in high expression of [^3H]emopamil binding activity (Fig. 3A) and anti-EBP immunoreactivity (Fig. 3B). Since comparable mRNA levels were found for GP(0) and GP(1) as well as HS(0) and HS(1) (results not shown) this was not due to differences in transcription or mRNA stability. Instead it could indicate that the highly homologous 5`-nontranslated region of both cDNAs represses translation in S. cerevisiae. With both truncated cDNAs, expression levels similar to the density in guinea pig liver microsomes were observed as quantitated by [^3H]emopamil binding activity (see Fig. 3A) and anti-EBP immunoreactivity (see Fig. 3B). The apparent molecular mass of the expressed protein in SDS-PAGE was indistinguishable from the EBP in guinea pig liver microsomes (see Fig. 3B). The identity of the expressed proteins was further confirmed with a sequence-directed antibody (anti-EBP) specific for the human EBP (see Fig. 1A). This antibody (not shown) as well as anti-EBP (see Fig. 3B) specifically recognized EBP in the human hepatoma cell line Hep G2 and the expressed human EBP whereas no cross-reactivity with guinea pig EBP was observed (results not shown).


Figure 3: Characterization of human and guinea pig EBP expressed in S. cerevisiae. A, [^3H]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(0), HS(1)), and guinea pig (GP(0), GP(1)) EBP cDNAs. Microsomal membranes were prepared as described under ``Experimental Procedures.''(-)-[^3H]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(0) and GP(0) are the entire clone inserts HS3 and GP5, respectively; in GP(1) the 5`-noncoding region, and in HS(1) 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 (-)-[^3H]emopamil binding to human EBP (HS(1)) expressed in S. cerevisiae. Saturation analysis was performed by decreasing the specific activity of (-)-[^3H]emopamil by dilution with unlabeled (-)-emopamil at a protein concentration of 19 µg/ml. A B(max) of 75 pmol/mg protein and a K of 76 nM were obtained. The inset shows the Scatchard transformation of these data. D, [^3H]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(1)). 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 [^3H]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^R (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(1)) were solubilized in in 1% (w/v) digitonin, 150 mM NaCl, 20 mM K(x)H(y)PO4, pH 7.8, at a protein concentration of 4 mg/ml. After dilution in 150 mM NaCl, 20 mM K(x)H(y)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 [^3H]emopamil (see Fig. 3C) revealed similar densities of EBP in microsomes from S. cerevisiae cells expressing EBP (HS(1), B(max) 70 ± 4 (n = 3) pmol/mg membrane protein; GP(1), B(max) 29 ± 7 (n = 3) pmol/mg membrane protein) and guinea pig liver (B(max) 35 pmol/mg membrane protein,(1) ). The dissociation constants of human (K(d) 15 ± 1 (n = 3) nM) and guinea pig EBP (K(d) 10 ± 3 (n = 3) nM) expressed in S. cerevisiae were similar to the value measured in guinea pig liver (K(d) 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, [^3H]azidopamil photoaffinity labeling of HS(1) (see Fig. 3D) reflected the properties of [^3H]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.


DISCUSSION

EBP is a high affinity binding protein for the antiischemic phenylalkylamine Ca antagonist [^3H]emopamil and the photoaffinity label [^3H]azidopamil. A variety of compounds with antiischemic effects in animal models of cerebral ischemia inhibited [^3H]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) .

EBP Forms the High Affinity Emopamil-binding Domain

The purification of EBP from guinea pig liver allowed us to determine its N-terminal sequence by Edman degradation(3) . This enabled us to clone its cDNA from guinea pig and human liver cDNA libraries. The cDNAs coded for proteins with a calculated molecular mass of 26.7 and 26.4 kDa, respectively. This molecular mass of approximately 27 kDa was in agreement with the relative molecular mass of 27.2 kDa determined by Ferguson analysis(3) .

The functional expression of the cloned EBP-cDNAs in S. cerevisiae demonstrated that they encode proteins which are able to form the [^3H]emopamil-binding site. Chemical cross-linking of the guinea pig and human EBP expressed in yeast suggested that the protein forms a functional homodimer.

EBP Carries an Endoplasmic Reticulum Retrieval Sequence

Our previous finding of a subcellular localization of EBP in the endoplasmic reticulum (2) was substantiated by the presence of a lysine-rich consensus sequence in the C terminus of human and guinea pig EBP. This motif has been demonstrated in other proteins to be a signal sequence for the retrieval of type I integral membrane proteins into the endoplasmic reticulum(17, 18) . Therefore the N and C termini of EBP must face the cytoplasm (Fig. 1C).

EBP Resembles Drug Transporters

Since the primary structure of EBP lacked similarity with available protein sequences no conclusion about its biological function could be drawn. However, EBP shares structural features with bacterial and eukaryontic drug transporting proteins like the bacterial drug pump smr(22) . Similar to smr EBP has four putative transmembrane segments and contains two conserved glutamate residues in TMS2 and 3. In smr a glutamate residue seems to be involved in the transport of cationic amphiphilics(23) . Another prominent feature of EBP is its high content of aromatic amino acid residues (>23%) in its transmembrane segments. A similarly high content (18.4%) is characteristic (20) for the P-glycoprotein, a drug pump of the plasma membrane. These aromatic amino acid residues have been suggested to be involved in the drug transport by the P-glycoprotein(20) . Like P-glycoprotein EBP has a broad drug specificity preferring drugs containing positively charged amines and lipophilic side chains as found in compounds from different pharmacological classes(1, 2, 24) . Although the tissue distribution of EBP and the P-glycoprotein are not identical both are mainly expressed in epithelial tissues (Fig. 2, A and B(25) ).

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 alpha(1) 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 [^3H]pentazocine, [^3H]ditolylguanidine, and [^3H]SKF10047 and a high affinity [^3H]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.


FOOTNOTES

*
This work was supported by a Boehringer-Ingelheim fellowship (to F. F. M.), the Dr. Legerlotz Foundation (to J. S.), and Grant S6601 (to H. G.) and Grant P9351 (to J. S.) from the Fonds zur Förderung der Wissenschaftlichen Forschung, Austria. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
Both authors contributed equally to this work.

Part of a doctoral thesis to be presented to the Medical Faculty of the University of Innsbruck.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: EBP, emopamil-binding protein; B(max), maximal density of binding sites; K(x)H(y)PO(4), K(2)HPO(4)/KH(2)PO(4); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SANPAH, N-succinimidyl-6(4`-azido-2`-nitrophenylamino)hexanoate; TMS, transmembrane segment; bp, base pair(s); DIG, digoxigenin 11-dUTP.


ACKNOWLEDGEMENTS

We thank Drs. I. Graziadei and W. Vogel for the gift of Hep G2 cells, Dr. Peter Kaiser for the yeast expression plasmid YEp351ADC1, M. Froschmayr for helpful advice on protein expression in S. cerevisiae, and M. Holzer for skilled technical assistance.


REFERENCES

  1. Zech, C., Staudinger, R., Mühlbacher, J., and Glossmann, H. (1991) Eur. J. Pharmacol. 208, 119-130 [Medline] [Order article via Infotrieve]
  2. Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann, H. (1993) Mol. Pharmacol. 43, 139-148 [Abstract]
  3. Moebius, F. F., Hanner, M., Knaus, H. G., Weber, F., Striessnig, J., and Glossmann, H. (1994) J. Biol. Chem. 269, 29314-29320 [Abstract/Free Full Text]
  4. Moebius, F. F., Burrows, G. G., Hanner, M., Schmid, E., Striessnig, J., and Glossmann, H. (1993) Mol. Pharmacol. 44, 966-971 [Abstract]
  5. Walker, J. M., Bowen, W. D., Walker, F. O., Matsumoto, R. R., De Costa, B., and Rice, K. C. (1990) Pharmacol. Rev. 42, 355-402 [Medline] [Order article via Infotrieve]
  6. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  7. Tautz, D., H ü lskamp, M., and Sommer, R. J. (1992) in In situ hybridization (Wilkinson, E. D., ed) pp. 61-73, Oxford University Press, Oxford, United Kingdom
  8. Chirgwin, J., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  9. Kaiser, P., Mansour, H. A., Greeten, T., Auer, B., Schweiger, M., and Schneider, R. (1994) FEBS Lett. 350, 1-4 [CrossRef][Medline] [Order article via Infotrieve]
  10. Hamilton, R., Watanabe, C. K., and DeBoer, H. A. (1987) Nucleic Acids Res. 15, 3581-3593 [Abstract]
  11. Hill, J., Ian, K. A., Donald, G., and Griffiths, D. E. (1991) Nucleic Acids Res. 61, 5791
  12. Striessnig, J., Glossmann, H., and Catterall, W. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9108-9112 [Abstract]
  13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  14. DeLean, A., Munson, P. J., and Rodbard, D. (1978) Am. J. Physiol. 4, E97-E102
  15. Kozak, M. (1989) J. Cell Biol. 108, 229-241 [Abstract]
  16. Dayhoff, M. O., Schwartz, R. M., and Orcut, P. C. (1978) Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed) Vol. 5, National Biomedical Research Foundation, Silver Spring, MD
  17. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1990) EMBO J. 9, 3153-3162 [Abstract]
  18. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1993) EMBO J. 12, 317-333
  19. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  20. Pawagi, A. B., Wang, J., Silverman, M., Reithmeier, R. A. F., and Deber, C. M. (1994) J. Mol. Biol. 235, 554-564 [CrossRef][Medline] [Order article via Infotrieve]
  21. Hill, J., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) Yeast 2, 163-167 [Medline] [Order article via Infotrieve]
  22. Grinius, L., Dreguniene, G., Goldberg, E. B., Liao, C.-H., and Projan, S. J. (1992) Plasmid 27, 119-129 [Medline] [Order article via Infotrieve]
  23. Grinius, L. L., and Goldberg, E. B. (1994) J. Biol. Chem. 269, 29998-30004 [Abstract/Free Full Text]
  24. Zamora, J. M., Pearce, H. L., and Beck, W. T. (1988) Mol. Pharmacol. 33, 454-462 [Abstract]
  25. Croop, J. M., Raymond, M., Haber, D., Devault, A., Arceci, R. J., Gros, P., and Housman, D. E. (1989) Mol. Cell. Biol. 9, 1346-1350 [Medline] [Order article via Infotrieve]
  26. Hagenbuch, B., Stieger, B., Foguet, M., Lübbert, H., and Meier, P. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10629-10633 [Abstract]
  27. Markovich, D., Forgo, J., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8073-8077 [Abstract/Free Full Text]
  28. Chowdhury, J. R., Novikoff, P. M., Chowdhury, N. R., and Novikoff, A. B. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2990-2994 [Abstract]
  29. Ferris, C. D., Hirsch, D. J., Brooks, B. P., Snowman, A. M., and Snyder, S. H. (1991) Mol. Pharmacol. 39, 199-204 [Abstract]
  30. Catterall, W. A., and Striessnig, J. (1992) Trends Pharmacol. Sci. 13, 256-262 [CrossRef][Medline] [Order article via Infotrieve]
  31. Griep, M. A., and McHenry, C. S. (1992) J. Biol. Chem. 267, 3052-3059 [Abstract/Free Full Text]
  32. Matsumoto, M., Scheller, M. S., Zornow, M. H., and Strnat, M. A. P. (1993) Stroke 24, 1228-1234 [Abstract]
  33. Block, F., Szabo, L., Jaspers, R. M. A., Heim, C., and Sontag, K. -H. (1993) Acta Neurol. Scand. 88, 35-40 [Medline] [Order article via Infotrieve]
  34. Okiyama, K., Smith, D. H., Thomas, M. J., and McIntosh, T. K. (1992) J. Neurosurg. 77, 607-615 [Medline] [Order article via Infotrieve]
  35. Elger, B., Seega, J., and Raschack, M. (1994) Eur. J. Pharmacol. 254, 65-71 [Medline] [Order article via Infotrieve]
  36. Chatelain, P., Gremel, M., and Brotelle, R. (1987) Eur. J. Pharmacol. 144, 83-90 [Medline] [Order article via Infotrieve]
  37. Zivin, J. A., Kochhar, A., and Saitoh, T. (1989) Brain Res. 482, 189-193 [CrossRef][Medline] [Order article via Infotrieve]
  38. Rao, T. S., Cler, J. A., Mick, S. J., Ragan, D. M., Lanthorn, T. H., Contreras, P. C., Iyengar, S., and Wood, P. L. (1990) Neuropharmacology 29, 1199-1204 [Medline] [Order article via Infotrieve]
  39. Gotti, B., Duverger, D., Bertin, J., Carter, C., Dupont, R., Frost, J., Gaudiliere, B., MacEnzie, E., Rousseau, J., Scatton, B., and Wick, A. (1988) J. Pharmacol. Exp. Ther. 247, 1211-1221 [Abstract]

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