probing olfactory receptors with sequence-specific antibodies

Eur. J. Biochem. 219, 829-835 (1994) 0 FEBS 1994

Probing olfactory receptors with sequence-specific antibodies Jurgen KRIEGER', Sabine SCHLEICHER', Jorg STROTMAN", h a WANNER', Ingrid BOEKHOFF', Klaus RAMING' Pieter DE GEUSZ and Heinz BREER' ' University Stuttgart-Hohenheim, Institute of Zoophysiology, Germany

Unilever Research Laboratory, Department of Biochemistry and Immunochemistry, Netherlands

(Received September 3, 1993) - EJB 93 1342/1

Molecular cloning has revealed the structure of several putative odorant receptors. Chemically synthesized peptides, that correspond to a predicted extracellular domain of the encoded proteins, were employed to generate receptor-specific antibodies. Immunohistological approaches as well as Westem-blot analysis confirmed the specificity of the antipeptide sera. Furthermore, deglycosylation experiments explained the observed discrepancy between the molecular mass of odorant receptors, as determined by SDSPAGE and Western-blot analysis of ciliary proteins (Mr 50000), and the predicted protein size based on the deduced primary structure from cloned receptor genes (Mr 30000-35 000). Receptor proteins become phosphorylated upon odorant stimulation of olfactory cilia preparations ; this was demonstrated by immunoprecipitation experiments employing the sequence-directed, receptor-specific antibodies. Functional assays revealed that the receptor-specific antibodies significantly attenuate second messenger signalling elicited by inositol 1,4,5-trisphosphate-inducing odorants, whereas activation of the CAMP cascade by appropriate odorants was not affected. These observation indicate that the sequence-specific antibodies not only recognize odorant receptors, but also discriminate between receptor subtypes coupling to different second-messenger pathways.

The olfactory system is capable of discriminating between a vast number and variety of different odorous molecules, some at concentrations as low as a few parts per tril- lion [ l , 21. The molecular basis of the perception and discrimination of odorants has long been an intriguing puz- zle. Several lines of evidence accumulated over the last de- cade support the concept that odorants are recognized by and bind to distinct receptor entities, most probably seven transmembrane domain receptor proteins which couple via G-proteins (guanyl-nucleotide-binding proteins) to second-messenger pathways. Although numerous approaches have been employed in the past to identify and isolate odorant receptor proteins, positive evidence for the proposed receptor molecules has remained elusive. A significant breakthrough was recently achieved by employing molecular cloning tech- niques leading to the identification of a large multigene family encoding candidate odorant receptors exclusively expressed in the olfactory epithelium [3,4]. It has recently been demonstrated by in situ hybridisation that a defined receptor subtype is only expressed in a distinct subpopulation of ol-

factory neurons [5 - 71. Meanwhile, several receptor-encoding cDNAs have been expressed in surrogate cells; for one of the receptor subtypes, it has been demonstrated that upon stimulation with certain odorants second-messenger signals are elicited in the host cells [8].

The deciphering of odorant receptor primary structure permits investigation of the structure and function of native receptor proteins in olfactory neurons using immunological approaches. Antibodies generated against chemically synthesized peptides based on sequence information derived from molecular cloning, have been found to react specifically with native molecules [9]. Site-directed antipeptide sera have suc- cessfully been employed as specific tools to explore subunit structure [lo], topology [ll, 121, tissue distribution [13] and function [ 141 of transmembrane signalling proteins.

In this study, sequence-specific antibodies have been employed for the identification of native odorant receptor protein and its in situ localization. In addition, the function of odorant-receptor subtypes has been probed using the site- directed antibodies.

Correspondence to H. Breer, Universitat Stuttgart-Hohenheim, Institut fur Zoophysiologie, Garbenstr. 30, D-70599 Stuttgart, Ger- many

Fax: +49 711 459 3726. Abbreviations. InsP,, inositol 1,4,5-trisphosphate ; Fmoc, 9-

fluorenylmethoxycarbonyl; G-protein, guanyl-nucleotide-binding protein; citralva, 3,7-dimethyl-2,6-octadienenitrile; lilial, 4-( 1,l- dimethylethyl)-a-methylbenzenepropanol ; ethylvanillin, 3-ethoxy-4- hydroxybenzaldehyde ; lyral, 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxyaldehyde; hedione, 3-0x0-2-pentylcyclopentane- acetic acid methyl ester; eugenol, 2-methoxy-4-(2-propenyl) phenol.

Enzyme. N-glycosidase F or glycopeptidase F (EC 2.2.2.18).

MATERIALS AND METHODS Materials

Sprague-Dawley rats were obtained from the Zentralinsti- tut fur Versuchstierzucht, Hannover, Germany. Citralva (3,7- dimethyl-2,6-octadienenitrile) was supplied from Intema- tional Flavors and Fragrances. Lilial [4-(1, 1-dimethylethy1)- a-methylbenzenepropanol], ethylvanillin (3-ethoxy-4-hydroxybenzaldehyde), lyral [4-(4-hydroxy-4-methylpentyl)-3- cyclohexene- 1 -carboxyaldehyde] , hedione (3-0x0-2-pentyl-

830

cyclopentaneacetic acid methyl ester) and eugenol [2-methoxy-4-(2-propenyl)phenol] were provided by W. Steiner, Baierbrunn, Germany. [y3*P]ATP was supplied by Du Pont. Radioligand assay kits for CAMP and inositol 1,4,5-trisphosphate (InsP,) were purchased from Amersham. Goat-anti- {rabbit IgG), labeled with horseradish peroxidase, was obtained from BioRad, labeled with alkaline phosphatase from Promega. Agarose-conjugated goat-anti-(rabbit IgG) and N- glycosidase F were supplied by Sigma. The purity of all chemicals used was >99%.

Methods Generation of sequence-specific antibodies

The peptide E464, with 14 amino acid residues (DMSALLKLACSDTR) corresponding to the second extracellular loop between hydrophobic domains 4 and 5 of the odorant receptor OR5 [8], was synthesized by the Fmoc (9- fluorenylmethoxycarbony1)-polyamide method using an au- tomated solid-phase peptide synthesizer (Applied Biosys- terns). The synthetic peptides were purified by reverse-phase HPLC and freeze dried. For immunization, the peptide was dispersed in 20 mM sodium phosphate, pH 7, at a concentration of 10 mg/ml for 4 h at 20°C. In this way, the material was cross-linked by the cysteine residues forming disulphide bridges. The resulting dispersion was diluted with 1.4 mM KH2P04, 8 mM Na,HPO,, 145 mM NaCl, pH 7.4 (NaCL'P,), to 2.5 mg/ml, mixed with one volume of Freund's complete adjuvant and injected into rabbits (subcutaneous and intra- muscular injections). At 6 - 8 weeks after immunization, titers had developed satisfactorily, and the immunoglobulin fraction was obtained by plasmaphoresis.

Isolation of olfactory cilia

Partially purified preparations of chemosensory cilia from rat olfactory epithelia were produced according to [15]. Briefly, rat olfactory epithelia, dissected from rat nasal sep- tum, and ethmoid turbinates were collected in Ringer's solution (120 mM NaCl, 5 mM KC1, 1.6 mM K2HP04, 1.2 mM MgS04, 25 mM NaHC03, 7.5 mM glucose, pH 7.4). This procedure and all subsequent operations were performed at 4°C. Cilia were detached from the epithelia, by raising the CaCl, concentration in the Ringer solution to 1OmM (calcium-shock procedure). After incubation for 20 min on an end-over-end shaker, the deciliated epithelia were separated by centrifugation for 10 min at 7700 g. The supernatant was collected and the pelleted epithelia were subjected to the same procedure. The supernatants were combined and centrifuged at 27000 g for 15 min. The pelleted cilia were suspended in 10 mM Tris/HCl, pH 7.4, 3 mM MgCl,, 2 mM EGTA and stored at -70°C.

ELISA assays

Microwell plates were coated with various preparations (200ng proteidwell) in 50mM NaHCO,, pH9.5, at 4°C overnight. Unbound material was removed by washing with 10 mM TrisMCl, pH 7.6, 150 mM NaCl (buffer A), containing 0.1% gelatin. After an incubation with buffer A, 0.1% gelatin containing 1 % bovine serum albumin antibody solutions (1 : 1000 dilution in buffer A with 1% bovine serum albumin) were added and samples were incubated for 1 h at 37 "C ; horseradish-conjugated goat anti-(rabbit IgG) (1 : 7500

dilution in buffer A) was subsequently added. Each step was followed by three buffer A washes. Antibody binding was visualized using a substrate solution containing 0.005% 3,5,3',5'-tetramethyLbenzidine and 0.003 % H,O, in 100 mM sodium acetate, 100 mM sodium citrate, pH 6.0; the enzyme reaction was stopped after 10min by applying 1 M H,S04. The absorbance was measured at 450nm with a Dynatech microplate reader (MR 700).

SDS/PAGE and Western-blot analysis

Protein samples (30 pg) in 16 pl water were mixed with 4 pl5X sample buffer (625 mM Tris/HCl, pH 6.8,50% glycerol, 5% SDS, 7.5 mM dithiothreitol, 0.05% bromophenol blue), boiled for 3 min and subjected to SDSPAGE on 12.5% gels using the Laemmli buffer system [16]. Proteins were transferred onto nitrocellulose according to [17]. Non- specific binding sites were blocked by incubation with 1 % bovine serum albumin in 10 mM Tris/HCl, pH 8.0, 150 mM NaC1, 0.05% Tween 20 (buffer B). Incubation with antibody solutions (1 : 1000 dilution in buffer B with 1 % bovine serum albumin) for 1 h was followed by three washes with buffer B for 15 min each. Blots were subsequently incubated with alkaline-phosphatase-conjugated goat anti-(rabbit IgG) (1 : 7500 dilution) and washed as described above. Bound antibodies were visualized employing a substrate solution containing 0.015 % nitro-blue tetrazolium and 0.007% 5-bromo- 4-chloro-3-indolylphosphate in 100 mM TrisBCl, pH 9.5, 100 mM NaC1, 5 mM MgC1,. The enzymic reaction was stopped by adding 5 mM EDTA, 20 mM TrisBCl, pH 8.0.

Deglycosylation

Isolated cilia from rat olfactory epithelia were prepared according to [18] and resolved in 150 mM NaCl, 10 mM Tris/HCl, pH 7.4, to a concentration of 0.5 pg proteidpl. 100 p1 (50 pg protein) of this preparation was centrifuged at 7800 g for 10 min. The pellet was resolved in 16 pl water with antiproteases [19] and mixed with 4 pl five-times concentrated sample buffer. After boiling the samples for 3 min and subsequent cooling to 20"C, 10 pl aqueous N-glycosidase F (1 U) was added, followed by an incubation at 37°C for 7 h. In control samples, N-glycosidase F was replaced by lop1 water. Afterwards, the proteins of each sample were separated by SDSPAGE and tested for immunoreactivity by Western-blot analysis.

fmmunoprecipitation

Membrane proteins were solubilized by incubating isolated olfactory cilia in 50 mM Tris/HCl, 150 mM NaC1, 1 mM EDTA, 1% Triton X-100, pH 7.5, for 30 min at 4°C and separated from unsoluble cytoskeletal elements by centrifugation at 27000 g for 15 min. Detergent was eliminated by a chromatographic step using PD-10 columns (Phar- macia). The eluate was concentrated and 100-p1 samples containing 50 pg protein were mixed with 2 pl E464 antiserum followed by an incubation for 2 h at 4°C. Subsequently, 25 pl agarose-coupled goat anti-(rabbit IgG) was added and the incubation was continued for 2 h. Thereafter, samples were centrifuged at 4800 g for 5 min. The pelleted antigedanti- body-agarose complex was treated with sample buffer for 30 min at 20°C and the solubilized proteins were analysed by SDSPAGE. After Coomassie-blue staining and destain-

831

ing, the gel was dried and exposed to a X-ray film for two weeks.

lmmunohistochemistry Immunohistochemical analysis was performed using

cryostat sections of nasal cavities from adult rats. Tissue samples were fixed at 4°C for 3 h in 0.8% p-formaldehyde- lysine-periodate fixative and, after three rinses in NaCVP,, were incubated for 24 h in ice-cold 30% sucrose in NaCVP,. Fixed and cryoprotected tissues were embedded in Tissue Tec (Miles) and rapidly frozen at -70°C. Sections (10-pm thickness) were cut using a Reichert and Jung cryostat model 2800 E instrument, thawed and mounted on chromalum gelatin-coated slides. After air drying for 30 min, the slices were treated with 0.1% Triton X-100 in NaCW, for 2min; any additional binding sites were blocked with 1 % bovine serum albumin in NaCVP, for 30 min. Sections were subsequently incubated for 1 h with E464 antiserum diluted 1 : 500 in NaCVP, with 1 % bovine serum albumin at 20°C followed by three NaCVP, washes for 5 min each; control sections were exposed to preimmune serum. As the second antibody, horseradish-peroxidase-conjugated goat anti-(rabbit Ig) (1 : 2000 dilution in NaClP, with 1 % bovine serum albumin) was applied for 1 h at 20°C. After three NaClP, washes, peroxidase activity was visualized by an incubation with 0.1% 3,3'-diaminobenzidine and 0.01% H,O, in 10 rnM Trid HC1, pH7.9, for 10min; the reaction was stopped by a S - min wash in NaClP,. Sections were mounted in (NaCVP,)/ glycerol (4 : 1, by vol.) and examined under a Zeiss Axiophot equipped with Nomarsky phase-contrast optics.

Determination of odor-induced second-messenger responses

Odorant-induced changes in second-messenger concentrations were monitored as described previously [20]. Briefly, odorants dissolved in dimethylsulfoxide were added to the stimulation buffer (200 mM NaC1, 10 mM EGTA, SO mM Mops, 2.5 mM MgCl,, 1 mM dithiothreitol, 0.05% sodium cholate, 1 mM ATP, 1 pM GTP and CaC1, to give a free Ca" concentration of 0.02 pM at pH 7.4). The odorant solutions were thoroughly mixed in an ultrasonic water bath and used immediately. The reaction was started by rapidly mixing cilia and odorants and was stopped by injecting 7% perchloric acid. The quenched samples were collected, cooled on ice and analysed for second-messenger concentrations.

Phosphorylation procedure Quantification by in vitro phosphorylation experiments

was performed according to the procedure described in [21]. The phosphorylation reaction was started by adding 20 p1 cilia preparation (20 pg protein) to 80 p1 previously warmed (25 "C) stimulation buffer containing odorants at the appropriate concentration and 1 pCi [y-32P]ATP. After incubation for 1 s, the reaction was stopped by adding 300 pl ice-cold 25% trichloroacetic acid, 5 mM phosphoric acid. The samples were placed on ice for 30 min followed by a thorough mixing of the precipitated protein. Aliquots (100 pl) of each sample were spotted onto Whatman 3 MM filter paper (4 cmX4 cm) and immersed in 10% trichloroacetic acid, 5 mM phosphoric acid. After thorough washing for 30 min, the filters were dried at 20°C for 2 h. The radioactivity ad- sorbed onto the filters was quantified by scintillation count- ing.

Cilia Bulb Cortex Muscle

Fig. 1. Tissue distribution of immunoreactivity for sequence-specific E464 antibodies. ELISA assays were performed using antipeptide sera (B) and preimmune serum (R), both at 1 : 1000 dilution, and assaying isolated olfactory cilia as well as membrane preparations from the olfactory bulb, cerebral cortex and skeletal muscle. Specific reactivity was only detected in cilia preparations. The data are the mean of three experiments ? SD. The absorbance was measured at 450 nm.

RESULTS

Since the antibodies generated against the synthetic peptide were required for analysing odorant receptors in situ, they must be able to recognize the native conformation of the protein. Therefore, an amino acid region which is supposed to be accessible on the protein surface was chosen for antibody production. Based on the prediction that the seven hydrophobic stretches of the odorant receptor sequences represent membrane-spanning domains, a peptide sequence corresponding to the second extracellular loop was synthesized. The 14-residue peptide was prepared by Fmoc-polyamide solid-phase chemistry; it was purified and cross-linked prior to rabbit immunization. The specificity of the antisera to the peptide was determined in ELISA assays giving a half maximal response at a dilution of approximately 1 : 10000. In order to identify those antisera that recognize specifically the native protein, the tissue specificity of the immunoreactivity was determined. As demonstrated in Fig. 1, the antipeptide serum designated E464 showed specific reactivity exclusively with preparations from the olfactory epithelium ; immunoreactivity was not detectable in membrane preparations from the olfactory bulb, cerebral cortex or muscle. The specific localization of the antigens recognized by the E464 antibodies was confirmed in immunocytochemical experiments; Fig. 2 shows cross-sections of the rat olfactory epithelium incubated with either preimmune serum or E464 antiserum. Only the uppermost layer of the epithelium containing the olfactory cilia is specifically labeled; no reaction with the preimmune serum was detected. Subsequently, isolated olfactory cilia as well as membrane preparations from the olfactory epithelium and the cerebral cortex were assessed for immunoreactive polypeptides by Western-blot analysis. In an olfactory cilia preparation, a single polypeptide band corresponding to M, 50000 is strongly labeled by the E464 antibodies (Fig. 3A). A faint band can also be observed in the

832

A B

Fig. 2. Topochemid localization of E464 immunoreactivity in the olfactory epithelium. Cryostat sections of the olfactory epithelium were incubated either with preimmune serum (B) or with E464 antibodies (A). The specifically bound antibodies were visualized using horseradish-peroxidase-coupled second antibodies.

A M,X 10-3

94 --

B ~ ~ ~ 1 0 - 3

94 -- 67 - 67 --

43- 43 - 30- 30 -

a b e d i II

Fig.3. Western-blot analysis using E464 antibodies. (A) Proteins from isolated olfactory cilia (lanes a, b) as well as membrane preparations from total olfactory epithelium (lane c) and cerebral cortex (lane d) were assayed employing preimmune serum (a) and sequence- specific antibodies (b-d). (B) Proteins from isolated olfactory cilia were incubated for 7 h with N-glycosidase F prior to SDSPAGE and and Western-blot analysis (lane I). Lane I1 shows the immunological response of control preparations (without N-glycosidase F) for comparison. The indicated molecular mass markers are as follows: phosphorylase b, M, 94000; bovine serum albumin, M, 67000; oval- bumin, M, 43000; carbonic anhydrase, M. 30000.

olfactory epithelium preparation ; no immunoreactivity was observed in the brain tissue.

The relative molecular mass of the immunoreactive protein (Mr 50000) is higher than the predicted size of odorant receptors based on their primary structure deduced from cloned genes (M. 30000-35000) [3, 81. Discrepancies between the molecular mass of proteins determined by SDS/ PAGE and the values calculated from the deduced primary structure are often due to glycosylation of proteins. To ana- lyse if the proteins detected by the E464 antibodies are glycosylated, isolated olfactory cilia preparations were treated with N-glycosidase F prior to SDSPAGE and Westem-blot analysis. As shown in Fig. 3B, N-glycosidase-F treatment of olfactory cilia proteins causes a disappearence of the immunoreactive M, 50000 band. Instead, a reactive polypeptide band of much lower molecular mass (Mr approximately 30000) is labeled. The size of this immunoreactive protein is in agreement with the relative molecular mass predicted on the basis of sequence data. These results further support the notion that E464 antibodies recognize glycosylated odorant receptor proteins in olfactory tissue.

It has previously been shown that stimulation of olfactory cilia in the presence of [y-’*P]ATP leads to labeling of a single polypeptide band which is supposed to represent receptor

proteins [22]. To verify this prediction, the receptor-specific E464 antibodies have been employed in immunoprecipitation experiments. Isolated olfactory cilia incubated with [Y-~~P]- ATP were stimulated with a mixture of odorants and subsequently treated with detergents. The solubilized proteins were subjected to immunoprecipitation using E464 antiserum. The immunoprecipitate was separated by electrophoresis and analysed for phosphorylated proteins by autoradiography. Only a single labeled band (Mr 50000) was detectable (Fig. 4); no labeling was found in preparations not stimulated with odorants. This result confirms that olfactory receptor proteins are phosphorylated upon odorant stimulation.

To approach the question if the receptor/antibody interaction may affect the receptor function, isolated cilia preparations were treated with preimmune serum and antiserum E464, before assaying for odor-induced second messenger response. Upon stimulation with a mixture of citralva, hedione and eugenole (1 pM each), the CAMP signal elicited in treated and untreated cilia preparations was quite similar (Fig. 5A); in contrast, the InsP, signal elicited by a mixture of lyral, lilial and ethylvanillin (1 pM each) was significantly attenuated in olfactory cilia previously treated with the E464 antiserum (Fig. 5B). Dosehesponse experiments indicate that the odorant-induced InsP,-response is almost completely

833

-

M,XIO-~

- U

.- 5 6 0 - > .- c

94 -- 67 -- 43 -- 30-

ants, was unaffected by the antibodies, whereas protein phosphorylation induced by InsP,-producing odorants was significantly reduced. These data emphasize that the sequence-specific I364 antibodies apparently discriminate between two sets of receptors ; they recognize those coupled to phospholipase C and do not interact with those coupled to adenylate cyclase.

A B DISCUSSION Fig. 4. Immunoprecipitation of olfactory diary proteins phosphorylated upon odorant stimulation. Isolated olfactory cilia were incubated with [y-32P]ATP and stimulated with a mixture of odorants (lyral, lilial, ethylvanillin) for 1 s. Ciliary membranes were treated with detergent and the solubilized proteins were subjected to immunoprecipitation using EM64 antibodies. Immunoprecipitated polypeptides from unstimulated (A) and stimulated (B) samples were separated on SDS/polyacrylamide gels. Dried gels were exposed to X-ray films for visualizing 32P-labeled polypeptides.

blocked at high antibody concentrations (Fig. 5C). The specificity of this antibody blockade was confirmed in experiments employing the synthetic peptides; a prior incubation of cilia with antibodies and the specific peptide simultaneously abolished the inhibitory effect. These observations suggest that the EX64 antibodies severely affect the function of odorant receptors coupled to the phosphatidylinositol pathway whereas receptors coupled to the cyclic nucleotide pathway are not affected. This notion was coIlfirmed in experiments assaying the odorant-induced phosphorylation of olfactory ciliary proteins. As demonstrated in Fig. 6, the incorporation of 32P-labeled phosphate, induced by CAMP-producing odor-

Sequence-specific antibodies have been employed to identify and characterize native odorant receptor proteins in the olfactory epithelium of rat. The immunoreactive molecules were found to reside preferentially in the ciliary layer of the olfactory epithelium. The localization of odorant receptors in the cilia provides the molecular basis for the elec- trophysiological findings that olfactory receptor cells respond strongly if odors are applied to the apical zone of the cells [23]. It is unclear why the immunreactivity could not be detected in the cell bodies; either the concentration of receptor proteins may be very low or the specific epitope for the antipeptide sera may not be accessible during synthesis and pro- cessing of the receptor proteins.

The relative molecular mass of the immunoreactive membrane protein (Mr 50000) as determined by Western- blot analysis (Fig. 3) and SDSPAGE of immunoprecipitated proteins (Fig. 4) is in the range of other G-protein coupled receptors [19, 241. However the relative molecular mass is significantly larger than the size of the protein predicted from the primary structure deduced from odorant receptor genes 13, 81. This discrepancy is solved by the deglycosylation experiments (Fig. 3B), demonstrating that the remaining pro-

A

1501

100-

8 h z Y

P Y

4 p"

50 -

W. Od. + PWSWum + E M W.

T

Od.

Fig. 5. Selective blockade of odorant-induced second-messenger signalling in olfactory cilia. Isolated olfactory cilia were treated with preimmune serum or antipeptide sera E464 for 15 min (1 : 500 dilutions in A and B). Subsequently, samples were stimulated with odorants and the second-messenger responses were determined after 50 ms. The basal levels were 84 -t 23 prnol cAMP/mg protein and 139 ? 64 pmol InsPJmg protein. The stimuli responses (100%) were 522 -t 127 pmol cAMP/mg protein; 568 ? 95 pmol InsP,/mg protein. (A) Stimulation with odorants inducing CAMP formation (citralva, hedione, eugenol ; 1 pM each). (B) Stimulation with odorants eliciting InsP,-responses (lyral, lilial, ethylvanillin, 1 pM each). Data are the mean of three experiments 2 SD. (C) Dose/response curve for the effect of the sequence- specific antibodies on the odorant-induced InsP, response of rat olfactory cilia preparations. The data are the mean of three experiments 2 SD.

834

A

- k 23

‘5 .- 800 z I

I

6

p 400

1200

h E - 2 .- b z 800 P

d .- 0

400

”M

T

PM

6

1200

* Pmswum .~404 + Pr08.rum 6 PIOSWUDI rE404 f Pmmrum rE404

Fig. 6. Sequence-specific antibodies specifically attenuate odor-induced phosphorylation of olfactory ciliary proteins. Isolated olfactory cilia were previously treated for 15 min on ice with preimmune serum or E464 antibodies (1 : 500 dilutions) and subsequently incubated with [y-”P]ATP and stimulated with odorants. (A) A previous treatment with peptide-specific antibodies neither affected the basal level of phosphorylation nor the incorporation of ’*P induced by citralva, hedione and eugenol. (B) In contrast, phosphorylation of ciliary proteins induced by lyral, lilial and ethylvanillin was significantly reduced. The data are the mean of three experiments L S. D.

tein core corresponds to the predicted molecular mass. Furthermore, this result is direct evidence that receptor proteins for odorants are in fact glycosylated, like many other membrane proteins involved in signal recognition and cell- cell interaction and is in agreement with the recent observation that certain lectins interfere with odorant-induced responses of the olfactory epithelium and isolated cilia [25, 261.

Recent experiments using specific inhibitors of protein kinases have provided evidence that the rapid termination of odorant-activated second-messenger cascades is mediated by phosphorylation of molecular components of the transduction apparatus [20, 27, 281. The results of immunoprecipitation experiments using the sequence-specific antibodies (Fig. 4) clearly demonstrate that the most likely candidates for regu- lation, the receptor proteins, are phosphorylated upon stimulation with the appropriate odorants. Thus, the principle mechanism for a rapid termination of second-messenger signalling in olfactory neurons is in line with the reactions that mediate desensitization of hormone and neurotransmitter receptors which have been explored in great detail 1241.

A major finding of this study is the observation that site- directed antipeptide sera generated against a predicted extracellular domain prevent the odorant-induced second-messenger responses (Fig. 5). The utility of antibodies in determining the specificity of receptor function in broken cell systems is well documented. Manifestations of agonist-mediated signalling that can be selectively disrupted with antibodies in- clude adenylate-cyclase [29] and phospholipase-C stimulation [30]. In most of these studies, where receptor/(;-protein interactions have been explored, it was concluded that the antigenic site of the receptor protein may contribute to the coupling domain. How the E464 antibodies prevent receptor activation by odorants is presently unclear. The notion that the second extracellular loop may be part of the binding site for odorous ligands contradicts the idea that odorous molecules interact with sites formed by the transmembrane do-

mains [3]. However, it is quite possible that immunoglobulin binding to the extracellular domain may sterically hinder odorous ligands from reaching their binding sites. Alterna- tively, immunoglobulin binding may block ligand-induced conformational changes of the receptor protein. In this context it is interesting to note that all antipeptide sera to the eleven hydrophilic domains of the P-adrenergic receptor failed to alter radio-ligand binding properties of the receptor [3 11. The observation that only the odor-induced InsP,-signal is attenuated by E464 antibodies, whereas the cAMP response to the appropriate odorants is not affected, indicates that the antibodies discriminate between the two families of odorant receptors. It can be concluded from these results that odorant receptor subtypes coupled to the cAMP or to the InsP, pathway not only differ in their cytoplasmic domains determining the G-protein coupling but also in extracellular epitopes.

A strategy combining a large panel of site-directed antipeptide sera in combination with various experimental approaches, including indirect immunofluorescence, immunoprecipitation and functional assays, will contribute to eluci- dating the topographical disposition of specific domains, like binding pockets, glycosylation and phosphorylation sites, with greater confidence.

This work was supported by the Deutsche Forschungsgernein- schaft, grant no. Br 712110-2.

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