against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a .... and a Xbal-site-containing antisense primer, FS-3' ( G C n.
Eur. J. Biochem. 225, 1157-1168 (1994) 0 FEBS 1994
Olfactory receptor proteins Expression, characterization and partial purification Uri GAT, Elina NEKRASOVA, Doron LANCET and Michael NATOCHIN Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, Israel
(Received May 13/July 7, 1994) - EJB 94 0687/2
A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation with a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in v i m , followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.
The superfamily of olfactory receptor proteins [l, 21 is likely to underlie the detection of odorant ligands, thus constituting the molecular basis of the sense of smell. Olfactory receptors are seven-transmembrane-domain receptors, as suggested earlier on the basis of considerable evidence that odorant signals are transduced via GTP-binding proteins (Gproteins) on the cilia of olfactory sensory neurons to activate either the CAMP or the phosphoinositide cascades [3-71. Different olfactory receptors are believed to recognize different odorants, and odor coding is afforded by the specific expression of one or a few olfactory receptor types in each sensory neuron [8 - 151. The first members of the olfactory receptor gene superfamily were cloned from rat and shown to encode several subfamilies [l]. Later, nearly 100 additional olfactory receptor genes were identified in several species [ l l , 16-19]. A systematic sequence classification by standard methods [20] shows a minimum of eight olfactory receptor families, each constituting one or more subfamilies, leading to the notion that the olfactory receptor gene repertoire is a multigene suCorrespondence to D. Lancet, Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot IL-76100, Israel Abbreviations. G-protein, guanyl-nucleotide-binding protein; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter. Enzyme. PNGase F, N-glycosidase F (EC 2.2.2.18). Note. The novel nucleotide sequence mentioned here has been submitted to the EMBL, GenBank and DDBT Data Bank(s) and is available under accession number(s) X80671.
perfamily. Members of this superfamily share at least 35% amino acid sequence identity, as well as several structural features and sequence motifs that clearly distinguish them from the other seven-transmembrane-domain receptors [2]. Many of the olfactory receptors studied are expressed specifically in olfactory epithelium [l, 9, 17, 211, but some are also expressed in the testis [16, 211 Most of the information available to date is based on studies of olfactory receptor genes and mRNAs, with only a few results addressing the nature and cellular disposition of the candidate olfactory receptor proteins. A 50-kDa polypeptide in rat olfactory cilia was shown to undergo odorant-dependent phosphorylation [22]. A similarly sized species, which upon deglycosylation was reduced to about 30 kDa, was identified by immunoblotting with olfactory-receptorspecific antibodies [23]. A protein of 40 kDa was visualized by immunoblotting analysis of dog sperm cells with antibodies against the N-terminal sequence of a dog olfactory receptor protein (DTMT). Immunolabelling with anti-(olfactory receptor) IgG against specific olfactory receptor types in rat shows restricted labeling at the ciliary surface of the sensory neurons [15, 231. Subcellular segregation of the dog olfactory receptor DTMT is also seen in sperm cells and in transfected mammalian COS-7 host cells, as visualized using anti-(N terminus) IgG [21]. The odorant specificities and functional properties of individual olfactory receptor proteins are largely unknown. A rat olfactory receptor cDNA clone (OR5), expressed by the baculovirus system in insect cells, showed odorant activation of the phosphoinositide cascade [ 181. Antibodies against the
1158 same receptor were shown to inhibit this cascade in olfactory cilia [23]. As a prerequisite for functional characterization, the present study reports the expression of two rat olfactory receptor cDNAs in vitro and in eukaryotic cells. The polypeptide products are analysed in terms of size, glycosylation, membrane insertion and disposition, using sequence-specific antibodies. Partial immunoaffinity purification of one of the olfactory receptor proteins is also described.
MATERIALS AND METHODS Molecular biology reagents Restriction endonucleases and modification enzymes used were from Boehringer Mannheim or from New England Biolabs (NEB). T7 RNA polymerase was kindly provided by Dr Oleg Denisenko, Protein Research Institute, Pushchino, Russia. cDNA clones encoding the olfactory receptors FS, F12 and I3 in pBluescript were a kind gift from Dr Linda Buck, Harvard Medical School, USA. The bacterial expression vector pET8c-FlagI, bearing the Flag epitope tag, was from Dr Michael Karin, UCSD, La Jolla, USA.
Cell culture Sf9 (Spodoptera Jrugiperda) cells (ATCC accession number CRL 1711) were propagated at 27°C in TNM-FH medium prepared from Grace's medium (Sigma) supplemented with 3.3 g/l each of yeastolate and lactalbumin hydrolysate (both from Difco) and with 10% fetal calf serum (Biolabs), Gentamicin (SO pg/ml, Sigma) and AmphotericinB (2.5 pg/ml, Gibco) as described in [24]. Sf9 cells were grown either a5 monolayers in flasks or in shaker flasks agitated at 100-1.50 rpm. Wild-type Autographa californica nuclear polyhedrosis virus was included in the Baculogold transfection kit that was purchased from Pharmingen Inc. A virus containing the 8-galactosidase gene 12.51 was a gift from Dr Ben-Zion Levi, Technion, Haifa, Israel.
DNA sequencing and analysis Sequencing of the olp4 cDNA was performed on both strands using Tmq DNA polymerase and dye terminators and an automatic DNA \equencer (Applied Biosystem, model 373A) with universal vector primers and internal primers. The sequence was aligned, composed and analyzed using the programs Inherit version 1.1.0, Seqed version 1.0.3 (both by ABI) and the Genetic Computer Group software (Wisconsin University, USA).
Probe synthesis The olfactory-receptor-specificdegenerate oligonucleotide primers, OR5A and OR3B [I91 were used to amplify 500-bp PCR products from rat genomic olfactory epithelium DNA as previously described [ 191. Three olfactory receptor clones, which we selected to represent three different subfamilies (FS, F12 and 18) [ l ] were grown, plasmid DNA prepared and inserts isolated from agarose gels, using the Geneclean11 kit (BIO 101 Inc.). The different DNAs were combined and radiolabeled to a specific activity of 10'- 10' cpml pg, using an Amersham Megaprime DNA-labelling kit and [a-32P]dCTPat ~ 3 0 0 Ci/mmol. 0
Library screening A rat olfactory epithelium Lgtll cDNA library [26] was screened using the above complex probe. Hybridization to filters was carried out overnight in 6 X NaCVCit (1X NaCl/ Cit = 8.8 g/l NaCI, 4.4 g/l sodium citrate, pH 7.0), 10 mM sodium phosphate, pH 6.8, 1 mM EDTA, S X Denhardt's solution, 0.1 % SDS, 100 nig/ml sonicated salmon sperm DNA at 55°C. The filters were washed in 0.SX NaCKit, 0.1% SDS at SS-60°C for 60 min and exposed to Kodak XAR S X-ray film at -80°C. The positively hybridizing phage clones were analyzed by PCR using the olfactory-receptorspecific primers OR5A and OR3B. Clones that produced bands of the appropriate size were purified, phage DNA was prepared and inserts released using EcoRI, all according to standard procedures [27, 281.
Plasmid constructs The olp4 cDNA insert was ligated into the EcoRI site of the vector pBluescript (Stratagene). The resulting plasmid, pBlue-olp4 was used as a template for a PCR reaction, in which the olp4 open reading frame from the third amino acid glycine to the last amino acid (307-amino-acid long), was amplified with the following primers, which contain BamHI sites (underlined) at their 5' ends : olp4-N, TAGGATCCCGGCACTGGGAATCATTCTGC sense ; olp4-C, TAGGATCCTTAACTCCTGACTGAG antisense (see also Fig. 1A). PCR amplification was carried out on a PTC-100 (MJ Research Inc.) instrument in a buffer containing SO mM KC1, 10 mM Tris/HCl, pH 8.3, 0.01 % gelatine, 0.2 mM each deoxyribonucleotide, 1 mM each primer and 2.5 U vent (exo+) polymerase (NEB)/I 00 pl, as follows : 30 cycles of denaturation at 94"C, annealing at 55°C and extension at 72°C; each step for 1 min. The first step of denaturation and the last step of extension were 4 min long. The 933-bp amplification product was digested with BamHI and cloned into the BamHI site, 3' of the Flag epitope tag of the vector pET8c-FlagI. The resulting sense-orientation vector PET-Flag-olp4 (see Fig. 2A) and antisense-orientation vector PET-Flag-olp4-rev were used for in vitro transcription. The N-terminal part of the olp4 open reading frame containing the Flag sequence (473 bp), isolated from PET-Flag-olp4, digested with NcoI and XbaI, was ligated together with the 3' part of the olp4 cDNA (836 bp) isolated from Blue-olp4 cleaved by XbaI and HindIII, into the vector Bluescript to produce Blue-Flagolp4. The Flag-olp4 fragment (1325 bp) was purified from Blue-Flag-olp4 digested by SmaI and EcoRV and ligated into the eukaryotic expression vector pcDNAI (Invitrogen) cut with EcoRV. Clones which are sense oriented (pcDNAI-Flagolp4) and antisense oriented (pcDNAI-Flag-olp4-rev) to the vector T7 promoter were identified by restriction analysis. The FS cDNA open reading frame (from the first methionine which was changed to leucine) and 3' non-coding sequence (1073 bp), was amplified by PCR as before from pBlue-FS, using a BamHI-site-containing the sense-strand primer, FS-N (CGGGATCCTCCTGAGCAGCACCAACC) and a Xbal-site-containing antisense primer, FS-3' ( G C n AGAGGCACGAGATTTGAGAG). The product was digested and inserted in-frame 3' to the Flag sequence of the vector pcDNAI-Flag-olp4, of which the olp4 sequence was removed by cleavage with BglII and XbaI, to create pcDNAIFlag-FS (Fig. 2B). The baculovirus transfer vector SG1-Flagolp4 was constructed by ligating the Flag-olp4 insert isolated from the vector pcDNAI-Flag-olp4-rev NotI- EcoRV cut
1159 into the vector pAcSGl (Pharmingen), cleaved with Not1 and SmaI (Fig. 2C). The validity of all the described constructions was confirmed by restriction analysis and DNA sequencing.
In vitro transcription and translation The following plasmids were linearized with the restriction enzymes indicated in brackets : pBlue-olp4 (XhoI), pBlue-F5 (Xbal), PET-Flag-olp4 (CZaI), PET-Flag-olp4-rev (ClaI) and pcDNAI-Flag-FS (XbaI), transcribed with T7 RNA polymerase and their mRNAs purified by phenoUchloroform extraction and LiCl precipitation, as described [29]. For translocation and glycosylation experiments, mRNA of yeast a-factor (Promega) was included as a control. About 0.5 pg of each transcript was translated in vitro using a rabbit reticulocyte lysate kit (Promega). Incubation was for 1 h at 22°C in a volume of 50 pl in the presence of 60 pCi ["Slmethionine (1000 Ci/mmol). 1-2 pl translation mix was added to buffer A (62 mM Tris/HCl, pH 6.8,4% SDS, 2% 2mercaptoethanol, 20% glycerol) and subjected to 12% SDS/ PAGE as described [30]. The gel was fixed, soaked in Amplify solution (Amersham) for 15 min, dried and exposed to Fuji RX-ray film at -80°C for 1-7 days.
Glycosylation and membrane association For the glycosylation and membrane-translocation assays, canine pancreatic microsomes (Promega) were added to the translation mixture (4%, by vol.). For deglycosylation, 10 p1 translation mix was incubated with 1000 U PNGase F (NEB) in 0.5% SDS, 1% 2-mercaptoethanol, 1% Nonidet P40 and SO mM Na,HPO,, pH 7.5, for 1 h at 37°C. Translocation was monitored either at pH 7.5 in the presence of 20 mM EDTA, which dissociates the ribosomes and weakly bound proteins from the membranes [31], or the pH was adjusted to 11.5 with 100 mM NaOH, [32]. After 10-min incubation on ice, the neutral pH samples were overlayed on 300 p1 0.5 M sucrose, 50 mM triethanolamine, pH 7.5, 20 mM EDTA, while the NaOH-treated samples were overlayed on 300p1 0.2M sucrose, 60mM Hepes, pH 11.5, 50mM KOAc, 2.5 mM Mg(OAc), and 1 mM dithiothreitol. The samples were centrifuged in a Beckman ultracentrifuge at 120000 gav.The supernatant and the sucrose cushion were removed and the translation products collected from this fraction by precipitation with equal volume of 20% trichloroacetic acid for 10 min on ice, followed by centrifugation in a microfuge for 10 min and methanol washing. The membranes and trichloroacetic-acid-precipitated pellets were dissolved in sample buffer and subjected to SDSPAGE, followed by autography.
Antibody production Four peptides from different segments of the olp4 sequence were synthesized: the N-terminal peptides NT1 (positions 1-12) and NT2 (positions 16-29), the cytoplasmic loop-3 peptide CL3.2 (positions 220-238) and the C-termihus peptide CT2 (positions 292-308), except for the N-terminal serine, which was changed to cysteine. The peptides were coupled to keyhole limpet hemocyanin and used to immunize three-month-old New Zealand White rabbits with 1 mg coupled peptide in complete Freund's adjuvant as described [33]. The rabbits were boosted twice at two-week
intervals, and the sera were tested for reactivity with their antigenic peptides by ELISA [33].
Production of recombinant Flag-olp4-containing baculovirus The transfer vector SG1-Flag-olp4 ( 5 pg) was cotransfected with BaculoGold DNA (0.5 pg, Pharmingen) as recommended by the manufacturer and the supernatant was collected after 5 days by centrifugating the cell medium. The transfection supernatant was used to infect fresh Sf9 cells and this amplification step was repeated once until a high titer virus (10' plaque-forming units/ml) was obtained. The titer of the viral supernatants was determined by end-point dilution as described [24]. Samples of the supernatants were treated with detergents (Nonidet P-40 and Tween 20) and proteinase K (Boehringer Mannheim) for lysis of the virions and the release of viral DNA as described [34]. PCR was then performed using olp4-specific primers (sense, positions 346-365 ; antisense, positions 1035 - 1053) as described above. Sf9 cells were infected with SG1-Flag-olp4 recombinant virus at a multiplicity of infection of 10. Control transfections were performed with wild-type virus and a recombinant virus containing the P-galactosidase gene. 40 h after infection, about 4X106 cells were centrifuged and suspended in TNM-FH medium without methionine for 1 h, followed by the addition of 500 pCi [35S]methionine(1000 Ci/mmol) for 4 h. The labelled Sf9 cells were washed in NaCIP,, phosphate-buffered saline (8 g/l NaCl, 0.2 g/l KCl, 1.09 g/l KH,PO, 0.1 g/l CaCl, 0.1 g/l MgC1, pH7.3) at 4°C and lysed for 1 h in 1 ml buffer B on ice (1% Triton X-100, 10mM Tris/HCI, 130mM NaC1, 10mM NaF, 1OmM sodium phosphate, 10 mM sodium pyrophosphate, pH 7.5) in the presence of a protease-inhibitor cocktail containing 1 mM phenylmethylsulfonyl fluoride, 16 mg/ml benzamidine/HCl and 10 mg/ml phenantroline, aprotinin, leupeptin and pepstatin A (all from Sigma). For determination of [35S]methionine incorporation, 10-p1 aliquots were subjected to trichloroacetic acid precipitation as described [28].
Immunoprecipitation Anti-CT2 serum (10 pl) was incubated with 15 pl protein-G-Sepharose suspension (Sigma) in 0.5 ml buffer C (10 mM Tris/HCl, pH 7.4, 150 mM NaCl) for 1 h at room temperature. The protein-G- Sepharose-antibody complex or anti-Flag M2 serum coupled to agarose gel (M2-ge1, International Biotechnologies 1nc.-Kodak) were washed twice in 1 ml buffer C, suspended in 1 ml buffer D (1% Triton X100, 25 mM Tris/HCI, pH 7.4, 300 mM NaC1, 1 mM CaCl,) and 2-5 pl translation mixture were added as well as 10 pg peptide when competition experiments were carried out. Following incubation for 2 h at room temperature, the immune complexes were washed 5 times with 1 ml buffer and once with buffer C. The immunoprecipitated proteins were released with buffer A (10 min, 65°C) and subjected to SDS/ PAGE analysis as above. N-terminal Flag fusion bacterial alkaline phosphatase (BAP-Flag, IBI-Kodak) was iodinated to a specific activity of 3 Ci/pmol using iodo-beads (Pierce) and lZ5I (Amersham). About 800 cpm were included in the immunoprecipitation mix as an internal control. For Sf9 cells, the cell lysates were centrifuged for 15 min in a microfuge at 10000 rpm, and protein-G-Sepharose-
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Fig. 1. The cDNA and protein sequence of olp4. (A) The full coding sequence of olp4 is shown in upper-case letters and the noncoding sequences in lower-case letters. The seven transmembrane domains are underlined in the protein sequence and marked in latin numerals, and the N-terminus (NT), cytoplasmic loops (CL), extracellular loops (EL) and the C-terminus (CT) are marked. PCR primers used for the screening of the cDNA library are also underlined in the nucleotide sequence and marked. The two possible N-glycosylation sites are marked by @. Serine residues, which could be sites for phosphorylation by protein kinases, are tagged by #. Small open reading frames in the 5' non-coding region are marked and numbered (start codons underlined and stop codons in bold). Possible adenylation signals are underlined in the 3' noncoding region and RNA destabilization signals are marked in bold. (B) Alignment of the deduced protein sequences of full-length olfactory receptors from four species, representing different subfamilies. Transmembrane domains are marked by lines with roman numerals. Dark boxes represent amino acid identity, while different shades of gray indicating two different degrees of conservative substitutions. Positions of identity among all the sequences typical to many seven-transmembrane-domain receptors are marked by a full circle, while positions that are specific to olfactory receptors are marked by a full square. The putative glycosylation sites are marked by '3 for the general N-terminal site and by (0) for specific sites.
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anti-CT2 or M2-eel were added to the supernatants. Immunoprecipitation was carried out as described above, but incubation was for 12 h at 4°C and washing was in buffer B. For deglycosylation, the antigen dissociated from the immune complexes was treated with 2000 U PNGase F for 2 h at 37 "C, followed by electrophoresis.
Immunohistochemistry Cells were stained by standard indirect immunofluorescence techniques. In brief, lo6 cells were washed twice in
1% fetal calf serum in NaCI/P,, and were then either permeabilized with ethanol for 1 h at 4"C, following by rehidration with 1% fetal calf serum overnight at the same temperature or were immediately incubated with 3 pg anti-(Flag M2) for 30 min. After incubation with first antibodies, the cells were washed three times with 1% fetal calf serum and stained either with 5 pg fluorescein-isothiocyanate (F1TC)-conjugated goat-(anti-mouse) IgG Fab' fragment (Sigma) or with rhodamine-conjugated goat-(anti-mouse) IgG (Sigma) for 30min at room temperature, followed by four washes. All samples were finally fixed with 1% paraformaldehyde and
1162 the cells were observed using a fluorescence microscope at excitation wavelengths of 450-490 nm and 510-560 nm.
A.
Flag-olp4
Fluorescence-activated cell-sorter (FACS) analysis Cells prepared as described above were analyzed on a FACScan (Becton-Dickinson, CA, USA) instrument, and fluorescence was monitored at 530 nm for FITC. Approximately 5000 events were scoredsample (containing about 10‘ cells). Forward and side scattering as well as FITC l-luorescence were collected. Histograms were generated using LYSYS I1 software (Becton-Dickinson).
Partial purification of Flag-olp4 protein from Sf9 cells Sf9 cells (1.6XlO’) infected with Flag-olp4 virus were lysed for 1 h at 4°C with 1 ml 10 mM Tris/HCl, pH7.4, 130 mM NaC1, 1% Triton X-100, followed by centrifugation at 10000 rpm for 15 min at 4°C in a microfuge. The cleared lysate was loaded onto 0.5 ml M2-Gel column, and the flowthrough was twice reloaded to ensure maximal recovery. The column was washed with 10 ml of the above buffer and Flag-olp4 protcin was eluted with 1.5 ml buffer contained 200 pg Flag peptide (125 mM; IBI-Kodak) in 300-pl aliqots collected at 5-min intervals. Eluted proteins were precipitated by nine vol. methanol overnight at -20°C. The precipitates were dissolved in buffer A and subjected to SDSPAGE, followed by silver staining [28]. Densitometry of the gel was performed on Computing Densitometer, model 300A (Molecular Dynamics).
RESULTS Cloning of a new member of the olfactory receptor superfamily A rat Rgtll olfactory epithelial cDNA library (8X10s phage clones) [26] was screened using a probe containing a complex PCR product amplified from rat genomic DNA, together with three previously published olfactory receptor cDNA clone inserts representing different olfactory receptor subfamilies 111. Ten strongly hybridizing plaques out of several hundred positive plaques were tested for positivity in PCR amplification with the ORSA and OR3B oligonucleotides, spanning transmembrane domains 3 and 7. Six clones showing the expected 500-bp product were purified and their phage DNA was prepared. The cDNA inserts were released by restriction with EcoRI and one of the cDNAs, about 1400bp long, termed olp4, was subcloned into the vector pBluescript (pBlue-olp4) and sequenced. The olp4 cDNA sequence, as well as the deduced protein sequence, is shown i n Fig. 1A. The 309-amino-acid sequence (Fig. 1B) shows 4060% identity to other members of the olfactory receptor superfamily [2, 191, and is therefore a novel member of this superfamily.
In vitro transcription and translation The pBlue-olp4 construct was linearized and transcribed in vitro using T7 RNA polymerase. The RNA was purified and translated zn vitro using rabbit reticulocyte lysate in the presence of [35S]methionine.SDSPAGE showed a major labelled band of 27 kDa (Fig. 3A). An anti-peptide rabbit polyclonal antibody (anti-CT2) was produced against the 17 Cterminal refidues of the deduced olp4 sequence. This anti-
...
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Flag 995Da
Olp4 1442Da
34174 Da
Fig. 2. Flag constructs used in this study. The Flag-olp4 (A) and Flag-FS (B) constructs used for in vitro translation are depicted, as well as the Flag-olp4 construct used for baculovirus expression (C). The amino acid sequence of the Flag and polylinker sequences at the N-termini are enlarged. The molecular masses of the different components are also indicated. Restriction sites used for the construction of these vectors or for their linearization are shown, as are the promoters used for expression (full arrows).
body immunoprecipitated a band of similar molecular mass from the in vitro translation mix (Fig. 3A), confirming the identity and full-length nature of the polypeptide. Three additional peptides related to the olp4 sequence, including the 12 N-terminal residues, did not produce reactive antibodies. Thus, we employed, in parallel, the Flag octapeptide epitope tag fused to the N-terminus, together with corresponding commercially available monoclonal antibodies [35].Bases 209-1441 of the olp4 cDNA were amplified by vent polymerase (to minimize the error rate), using primers spanning the olp4 open reading frame (olp4-N and olp4-C). The resulting PCR fragment was inserted 3’ to the Flag sequence in a pET8c-Flag1 vector (Fig. 2A). Subclones representing the sense orientation (in frame, PET-Flag-olp4) as well as the reverse orientation (PET-Flag-olp4-rev) were isolated. When PET-Flag-olp4 was transcribed and translated in vitro, it produced a band of a higher molecular mass (29 kDa; Fig. 3A), as expected for an open reading frame with an additional 19 residues (Fig. 2). Immunoprecipitation with the anti-Flag monoclonal antibody M2 also showed a 29-kDa band for PET-Flag-olp4, but not for the original pBlue-olp4 which has no epitope tag. Also, no product was seen when the reverse-oriented PET-Flag-olp4-rev was similarly analysed (Fig. 3B). The anti-CT2 also immunoprecipitated the Flag-olp4 29-kDa product (Fig. 3A), indicating that the same polypeptide is recognized by both antibodies. The antigenic peptides abolished the immunoprecipitation of the 29-kDa polypeptide by each of the corresponding antibodies (Fig. 3A), while the heterologous peptides had no inhibitory
1163 anti-Flag
n 5
TP
a
A
3
6 94 6-
304---
+Bap-Flag +
-21.5-
14-
2 17514I
Peptide :
Flag
CT2
Fig. 3. In vitro translation and immunoprecipitation. (A) Olp4 and Flag-olp4 RNAs were translated in vitro in the presence of [35S]methionine in rabbit reticulocyte lysate. Aliquots of the translation mixes were applied directly to a 12% polyacrylamide gel (two left lanes) or the mixes were subjected to immunoprecipitation, using the anti-(Flag M2) IgG (anti-Flag, three middle lanes) or the anti-CT2 (anti-CT2, three right lanes), in the presence or absence of the corresponding peptides. Arrows indicate the positions of the major bands of olp4 at 27 kDa (bottom) and Flag-olp4 at 29 kDa (top). The positions and sizes, in kilodaltons, of the molecular-mass markers are shown. The absence (-) or presence of the peptides is indicated below the immunoprecipitation lanes. (B) Flag-olp4 and Flag-olp4-rev translation mixes were subjected to immunoprecipitation, as described in (A), using the anti-(Flag M2) IgG. The arrows indicate the position of the iodinated BAP-Flag immunoprecipitation control, which migrates as a 55-kDa band (top), and of the Flag-olp4 band (bottom).
effect (not shown), demonstrating the specificity of the antibodies recognition. To examine the generality of the above results, we studied similarly another olfactory receptor gene ; the rat F5 cDNA, published in the first group of olfactory receptor genes discovered [l]. F5 belongs to a different olfactory receptor family and subfamily [2, 191, with only 46% amino acid identity to olp4, and is thus a good candidate for comparison. The F5 cDNA was amplified with oligonucleotides F5-N and F5-3' spanning from the second codon to a position 129-bases 3' to the termination codon. This fragment was subcloned in-frame in the sense orientation into a pcDNAI vector containing the flag sequence (Fig. 2B). In vitro transcriptiodtranslation, followed by immunoprecipitation with the anti-Flag M2 monoclonal antibody showed a polypeptide product of 29 kDa, the same as for olp4. The signal disappeared upon inhibition with the Flag peptide (not shown). The olp4-specific C-terminal antibodies (anti-CT2) did not, however, inmunoprecipitate the F5 product, probably because the terminal half of the respective sequences bears only marginal mutual similarity (Fig. 1 B).
Membrane association and glycosylation Since olfactory receptors have an inferred topology of integral membrane proteins which span the lipid bilayer seven times, the in-vitro translation products are expected to be incorporated into membranes. To examine this, in vitro translations of the olfactory receptors and of a control secreted yeast a-factor protein, were performed in the presence of pancreatic canine microsomes. The samples were then subjected to ultracentrifugation through a sucrose cushion and the presence of the radiolabelled proteins was examined in the supernatants and in the membrane pellets. When, after translation, the reaction mixture was exposed to pH 11.5,which disrupts biological membranes to open sheets
and strips off all peripheral proteins [32], the labelled olfactory receptor polypeptide products were largely found in the microsomal pellet fraction (Fig. 4A), demonstrating that they are integrated in the membrane. In contrast, the soluble a factor appeared in the supernatant after treatment at pH 11.5, while in intact microsomes (at neutral pH), it appeared largely in the pellet (Fig. 4A). That the association was not due to protein-protein interaction and aggregation was proved by the fact that practically no olfactory receptor polypeptide products were found in the pellet if the microsomal membranes were added after the translation had been completed (not shown). Both the olp4 and F5 protein sequences contain consensus sequences for N-glycosylation at expected extracellular domains: olp4 at the N-terminus as well as at the second extracellular loop, while F5 contains consensus sequences at the N-terminus and at the third extracellular loop (Fig. 1B). A first indication for such a potential post-translational modification is seen in the lower electrophoretic mobility (3 1 kDa) of the microsome-associated translation product of F5 (Fig. 4A). An additional band at 18 kDa may be a degradation product or a translation product at an internal initiation site. To further test whether both olfactory receptor proteins are indeed glycosylated, the Flag-olp4 and Flag-FS RNAs were translated in vitro in the presence of pancreatic canine microsomes and immunoprecipitated with anti-Flag serum. As shown in Fig. 4B, the Flag-F5 product showed a clear shift in molecular mass from 29 kDa to 31 kDa. The 18-kDa band is not immunoprecipitated, suggesting that it does not contain the epitope tag and hence is likely to be truncated at its N-terminal region. A similar shift in size was detected with the 27-kDa labelled F5 (without flag) polypeptide produced by cell-free translation in the presence of microsomes (not shown). Treatment of the Flag-F5 product with PNGase F, which removes N-linked carbohydrates from glycosylated
1164
-21.5-
-1
I
4-
S P S P S P S P S P S P y H = 7.5
pH = 11.5
Fig. 4. Translocation and glycosylation of olfactory receptors. (A) The Flag-olp4 and Flag-FS RNAs, as well as control yeast n-factor RNA were translated in v i m in the presence of microsomes, followed by incubation at either pH 7.5 (left) or at pH 11.5 (right). S, trichloroacetic-acid-precipitatedsupernatants ; P, microsomal pellets. (B) Flag-FS was translated in vitro in the absence or presence of canine pancreatic microsonies ( +mic) and immunoprecipitated with anti-(Flag M2). In the rightmost lane, the FS translation mix was treated prior to immunoprecipitation with PNGase F.
proteins, show5 a return to the original 29-kDa size, suggesting that, indeed, the FS polypeptide is glycosylated (Fig. 4B). The Flag-olp4 product did not show a detectable change of molecular mass, and may not be glycosylated significantly under the conditions employed (not shown).
Expression in the baculovirus system A Flag-olp4 transfer vector construct (SG1-Flag-olp4) was generated in a three-step procedure, in which the sequence immediately preceding the initiation codon was made highly compatible with the preferred consensus for translation [36]. The construct included the olp4 open reading frame from residue 3 to the end, as well as a 310-bp 3' non-coding region of the original olp4 cDNA (Fig. 2C). The SGl-Flagolp4 vector was co-transfected with the Baculogold DNA into Autogvuphn cal$irnicu Sf9 cells and a high titer recombinant viral supernatant was prepared (Flag-olp4 virus). PCR performed on this supernatant using olp4 open-readingframe-specific primers showed one band affirming that the majority of the viral population does not contain deletions (not shown). The Sf9 cells were infected with Flag-olp4 virus and, after 40 h, were labelled with [%'3]methionine. The cells were solubilized in 1 % Triton X-100 and subjected to immunoprecipitation with anti-CT2 and anti-Flag. In both immunoprecipitates, a major band appears at about 29 kDa (Fig. 5A), which comigrates with the cell-free translation product of Flag-olp4 (Fig. 5B). These immunoreactivities were inhibited by the respective free peptides (not shown). A second, weaker band appears in the Flag-olp4 virus-infected Sf9 cell immunoprecipitates, which is 4 kDa heavier. This band appears to partly convert to lower molecular-mass forms upon treatment with PNGase-F (not shown), suggesting that it is a glycosylated form of the polypeptide. An additional minor band appears at approximately 70 kDa, which may be an SDS-resistant dimer, similar to that observed for the other seven-transmembrane-domain receptor proteins [37]. All three bands are specific, as they do not appear in non-in-
fected Sf9 cells, in cells infected with wild-type virus or with a virus containing a /I-galactosidase gene (Fig. SA) [25].
Affinity purification of the baculovirus-expressed Flag-olp4 protein To assess the amount of olp4 protein expressed in Sf9 cells and to confirm the identity of the immunoprecipitated polypeptide, we subjected the expressed Flag-olp4 protein to partial purification. 1.6X 10' Flag-olp4-expressing Sf9 cells (containing 3.6 mg protein) were grown and solubilized in 1 % Triton X-100. The extract was passed over an anti-Flag affinity column. The absorbed protein was eluted with the Flag peptide. The whole cell extract and the elution fraction are shown in Fig. 5 C. This fraction contains a major band at approximately 30 kDa, the same as the position of the major Flag-olp4 immunoprecipitated polypeptide. A densitometric analysis of the eluate indicates that it contains about 30 ng Flag-olp4. This translates to about 40000 copies of the proteidcell, on average.
Immunofluorescence studies on Flag-olp4 virus-infected Sf9 cells In order to study the extent and patterns of expression of the Flag-olp4 polypeptide in Sf9 cell, we used immunohistochemistry and FACS analysis. Cells were infected with Flagolp4 virus, ethanol permeabilized and labelled with anti-(flag M2) IgC followed by FITC or rhodamine conjugated second antibodies (Fig. 6). Fluorescence microscopy showed numerous intenscly labelled cells and the staining appeared to be concentrated at the cell perimeter, characteristic of a cellsurface epitope. A control of the same preparation in the presence of the Flag peptide showed only residual staining (not shown). The staining pattern was not dependent on the permeabilization of the cells (Fig. 6). Flag-olp4 virus-infected unpermeabilized cells were subjected to FACS analysis with anti-M2. A broad peak of fluorescent cells appeared at fluorescence intensities 3 - 100-
1165
Fig. 5. Immunoprecipitation and partial purification of the Flag-olp4 product expressed in Sf9 cells. (A) Sf9, uninfected; /I-gal, infected with /I-gal-containing virus ; Flag-olp4, infected with Flag-olp4 virus ; wild-type, infected with wild-type A. californica nuclear polyhedrosis virus. The arrows indicate the position of the Flag-olp4-specific band (bottom) and its putative glycosylated form (top). (B) The Flag-olp4 products of the in vivo (Sf9) system (two left lanes) are compared to the product of the in vitro (RRL) system (right lane). The two bottom arrows indicate the bands, as described in (A); the top arrow indicates the putative dimer form. (C) Flag-olp4-virusinfected Sf9 cells were lysed and applied to an M2 gel-affinity column. The silver-stained SDSPAGE patterns of the chromatography are shown. 1, whole cell lysate; 2, Flag-peptide elution fraction.
Fig. 6. Immunohistochemistry.Flag-olp4 virus-infected cells, permeabilized with ethanol, were fluorescently labeled with anti-(Flag M2), followed by rhodamine-conjugated anti-mouse IgG antibodies. (Inset) unpermeabilized cells stained similarly.
times higher than the mean background fluorescence (no antibody; Fig. 7A). Such specific labelling was abolished by incubation with the Flag peptide. Cells infected with wildtype virus or P-galactosidase-containing virus and similarly labelled, showed a fluorescence peak indistinguishable from the background intensity. When unpermeabilized Flag-olp4 virus-infected cells were labelled with anti-CT2, only a slight enhancement of fluorescence was observed (mean fluorescence twice as high as the mean background fluorescence (Fig. 7C). In contrast, when cells were permeabilized with ethanol and similarly stained, the mean fluorescence increased six times over the background level, suggesting an enhanced exposure of the antigen (Fig. 7D). Practically no such dependence on permeabilization was observed for the anti-Flag N-terminal antibodies (Fig. 7B).
DISCUSSION In the last few years, considerable knowledge has accumulated on olfactory receptor genes and cDNAs. However, relatively little is currently known about their protein receptor products. In order to obtain such information, we studied one novel rat olfactory receptor gene product, olp4, and examined its properties in detail. Olp4 is a banafide member of the olfactory receptor gene superfamily. It belongs to the same gene family (3) with several other rodent olfactory receptor genes, as well as with a large subfamily of human olfactory receptor genes coded within an olfactory receptor gene cluster on chromosome 17 [19]. The olp4 deduced amino acid sequence includes 30 highly conserved residues unique to olfactory receptors, e.g. the motif FXLXG at the N-terminus, the motif PMY at the second transmembrane
1166 Anti-Flag M2
Anti CT.2
C
A
fluorescence
D m
fl uorrscmcr
fluorrsccncc
Fig.7. FACS analysis. Flag-olp4 virus-infected Sf9 cells were either left intact (A, C) or ethanol permeabilized (B, D). Cells were either sorted without immunoiabeling (open peaks) or after labeling (shaded) with anti-(Flag M2) (A, B) or anti-CT2 (C, D).
helix, the motif VAXC at the second cytoplasmic loop, the motif CXXL at the fourth transmembrane helix, the motif LXFC at the second extracellular loop, the motif KXXXTCXSH at the sixth transmembrane helix and the motifs PM and LR at the seventh transmembrane helix. Olp4 thus fully qualifies to serve as a model olfactory receptor protein. Olp4 also has the structural features common to all the seven-transmembrane-domain G-coupled receptors; the seven deduced hydrophobic transmembrane helices, the potential disulfide bond between the highly conserved cysteins in extracellular loops 1 and 2, a potential glycosylation site in the N-terminus and several potential phosphorylation sites in intracellular regions. The later include potential sites for CAMP-dependant protein kinase e.g. (RSS at positions 230232, RFS at positions 304-306), protein kinase C (RFSVR at positions 304- 308) and calmodulin-dependant protein kinase I1 (RIRS at positions 228-231) which are found at the third cytoplasmic loop (positions 219-236) and at the Cterminus (positions 294-309) domains of the receptor. Olp4 also shares 27 highly conserved residues common to all seven transmembrane-domain receptors, including the motifs TXXGNL in the first transmembrane helix, FLXXL in the second transmembrane helix, DR in the third transmembrane helix, VXXF in the sixth transmembrane helix and NPXIY in the seventh transmembrane helix. Examination of the untranslated regions of the olp4 cDNA reveals that the 5' untranslated region contains many start and stop codons, which produce several small open reading frames, such as those found in some other genes of the seven-transmembrane-domain receptor family [38]. A 5' small open reading frame peptide product has recently been shown to regulate the translation of the P-adrenergic receptor 1391, a mechanism to be studied in the future for olfactory receptor proteins. The 3' non-coding region of the olp4 cDNA interestingly contains three mRNA destabilization sequences (ATTTA), which are known to cause degradation of regulatory cellular mRNAs of oncogenes, cytokines, lympho-
kines and transcriptional activators [40,41]. These sequences probably also play a role in the regulation of the gene expression. The expression of the novel olp4 cDNA, as well as that of an additional olfactory receptor cDNA (F5) identified previously [l],is studied here by two parallel methods, in vitro transcriptiodtranslation using a mammalian cell-free reticulocyte lysate system and intact insect cells, using the baculovirus expression system. An expressed polypeptide of 27 kDa (29 kDa with the 2568-Da epitope tag, including polylinker addition) is seen for both cDNAs in both expression systems, lending credence to the observed molecular mass. A 29-kDa polypeptide is immunoprecipitated by the two different antibodies raised against an olp4 C-terminal peptide sequence and against a Flag N-terminal epitope tag. This provides additional confirmation for the identity of the observed protein. An identical electrophoretic mobility (slightly below the 30-kDa carbonic anhydrase standard) has been reported for the rat OR5 olfactory receptor protein in native ciliary membranes after deglycosylation, when visualized by immunoblotting with an anti-peptide IgG [23]. The calculated molecular mass of the olfactory receptor proteins is 34451 Da for olp4 and 34679 Da for F5. The discrepancy between the calculated molecular mass and the apparent molecular mass in SDS/PAGE is accountable by the abnormally high electrophoretic mobility observed for other seven-transmembrane-domain receptors, such as the turkey erythrocytes /3-adrenergic receptor, whose predicted molecular mass is 54 kDa and which migrates as a 45-kDa band [42]. Another abnormal electrophoretic behavior is the formation of dimers, observed here for the Sf9-expressed olp4, and previously reported for other seven-transmembrane-domain receptors [37, 431. Both the olp4 and FS sequences have two potential Nglycosylation sites. One is at a site common to most olfactory-receptor sequences, at the N-terminus, while the other is in the second extracellular loop for olp4 and in the third extracellular loop for F5. Only F5 appears to undergo glyco-
1167 sylation in the cell-free expression system, as seen by the appearance of a 31-kDa band following the addition of microsomes, and the decreased molecular mass (back to 29 kDa) upon PNGase F treatment. The reason for the difference between the two receptors may be related to the effect of the different locations of their glycosylation sites. The Olp4 protein is, however, translocated into the microsomal membranes in vivo and does show evidence for glycosylation in the baculovirus expression system. The nominal expected increase in molecular mass is 2-3 kDdglycosylation site, consistent with the modification of only one site in vitro (F5), and 1 or 2 sites in the baculovirus expression system (olp4). In contrast, the OR5 protein [23], which also has two nominal glycosylation sites, has been reported to undergo a 20kDa change upon glycosylation, possibly due to a difference in the glycosylation machinery in native olfactory cilia. An important question asked in the present study is whether the expressed olfactory receptor protein is membrane associated. Furthermore, we asked whether the olfactory receptor protein behaves as a class-IIIb polypeptide [44], i.e. extracellular N-terminus without cleaveable signal peptide, several transmembrane domains and intracellular C-terminus. The in vitro transcriptiordtranslation in the presence of microsomal membranes shows that both the Flag-olp4 and Flag-F5 proteins are translocated into the membrane vesicle bilayer without N-terminal proteolysis, and are incorporated into membranes in a manner characteristic of integral membrane proteins. The immunofluorescence and FACS analysis of the Sf9expressed olp4 protein show that a considerable fraction of the protein is on the cell surface. More importantly, the increased labelling upon cell permeabilization observed for the C-terminal antibody, but practically not for the N-terminal antibody, suggests that the protein is in the proper classIIIb orientation. An antibody such as the C-terminally directed CT2 could be used in the future for localization in permeabilized olfactory epithelial tissue sections, as previously performed with other antibodies to olfactory receptors [15, 231. The large olfactory receptor protein superfamily has a highly variable N-terminal sequence. We, therefore, chosen to insert a Flag epitope tag at the N-terminus of the olp4 and F5 cDNAs, an approach successfully employed for other members of the seven-transmembrane-domain receptor family without considerable interference with function [45, 461. The high specificity and affinity of the anti-Flag M2 monoclonal antibody allows reproducible immunolabelling, immunoprecipitation and affinity purification. In the future, additional olfactory receptor sequences can easily be fused to the Flag sequence using the same vector. This approach complements the production of antibodies against inferred intracellular or extracellular olfactory receptor peptide sequences, as used here (for the CT2 peptide) as well as in other studies [15, 21, 231. We have performed partial purification of the Flag-olp4 protein from the infected Sf9 cells using mild elution with Flag peptide. Such methodology is favorable for future functional reconstitution studies, as it is likely to produce relatively large amounts of an undenatured protein in the detergent of choice. This is particularly important for a seventransmembrane-domain receptor probed for structure/function relationships in ligand binding, coupling to G-proteins, and ligand-dependent protein phosphorylation. Partial purification allows the first visualization of a silver-stained olfactory receptor protein, confirming the molec-
ular mass of 29 kDa determined by other methodologies. The amount of the protein was found to be 0.2 pmol/mg protein, corresponding to approximately 40000 copieskell on average. Fluorescent-microscopy and cell-sorting analyses show a heterogeneity of expression levels, possibly related to the efficiency of infection in different cells. The cells with highest expression levels (fluorescence values above 100 U in Fig. 7A) constitute 15% of the total population, and are calculated to have about 10 times more protein than the average, i.e. roughly 400000 protein moleculeskell, comparable to the high expression observed in isolated clones of other baculovirus-expressed seven-transmembrane-domain receptors [37]. This research was supported by grants from the US National Institutes of Health (DC00305), the US-Israel Binational Science Foundation, the Human Frontier Science Program and the Forschheimer Center for Molecular Genetics, the Minerva Foundation (Munich, Germany), the Mordoh Mijan de Salonique Foundation and a Wolfson Research Award of the Israel Academy of Sciences. We thank Dr Ben-Zion Levi and Tzafra Cohen, Technion, and Dr Igor Met (this institute) for help in Sf9 cell culture and baculovirus expression, T. Mehlmann for automated sequencing and computer analysis, Andrei Matsaev for help in FACS analysis, Dr Alexander Bershadsky and Dr Tova Volberg for assistance in immunohistochemistry and photography.
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