Báo cáo Y học: Expression pattern in the antennae of a newly isolated lepidopteran Gq protein a subunit cDNA potx

Expression pattern in the antennae of a newly isolated lepidopteran Gq protein a subunit cDNA Emmanuelle Jacquin-Joly 1 , Marie-Christine Franc¸ois 1 , Michael Burnet 2 , Philippe Lucas 1 , Franck Bourrat 3 and Rosario Maida 4 1 INRA, Unite ´ de Phytopharmacie et Me ´ diateurs Chimiques, Route de Saint-Cyr, Versailles cedex, France; 2 Sympore GmbH, Reutlingen, Germany; 3 UPR 2197 DEPSN, Institut de Neurosciences A. Fessard, CNRS, Gif-sur-Yvette, France; 4 Max-Planck-Institut fu ¨ r Verhaltensphysiologie, Seewiesen, Germany From the antennae of the moth Mamestra brassicae, we have identified a lepidopteran G protein a subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino-acid sequence identity with vertebrate (80% with mouse or human Gqa) and invertebrate subunits (varying between 60 and 87% for Gqa from organisms as diverse as sponge and Drosophila). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this Gproteina subunit may be involved in the olfaction trans- duction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway. Keywords: G protein; a subunit; olfaction; Lepidoptera; in situ hybridization. For insects, olfaction plays an essential role in processing chemical signals from the environment, leading to the detection of food, reproductive partners, oviposition sites, hosts, prey or predators. In particular, pheromone percep- tion in moths has become a model for a growing number of studies on the mechanisms of olfactory reception and transduction. Although invertebrate chemosensory systems show a great diversity across phyla, there are strong similarities at the cellular level. The pheromone sensing system of moths is morphologically very close to olfactory systems from organisms as diverse as flies, nematodes or lobsters. In moths, pheromone receptor cells are localized in specialized sensory organs, the sensilla trichodea, distributed on the antennae. Pheromone molecules, usually emitted by the female, enter the sensilla of the male antennae and are bound by specialized soluble proteins that traffic through the extracellular lymph to the dendrite membrane where they are recognized by specific olfactory receptors. The transduction events following binding of the receptor have been recently clarified by the discovery of the first putative invertebrate odorant receptor genes in Drosophila [1–3]. The receptor proteins appear to belong to the seven-transmembrane G protein coupled receptor multigene family that also include vertebrate odorant receptor molecules [4]. These receptors relay signals from cell surface to intracellular effectors through guanine nucleotide-binding proteins: the G proteins. G proteins play a central role in a wide variety of signal transduction pathways, mediating the perception of environmental cues in all higher eukaryotic organisms. In particular, G pro- teins have been implicated in signal-transduction events underlying olfaction and vision (reviewed in [5]). They have been classified into different subtypes depending on which second messenger they predominantly control. Although these distinctions are not absolute, Gs frequently activates adenylate cyclase whereas G i inhibits it, Gq mediates the stimulation of phospholipase C and hence phosphoinosi- tide turnover, and G 12 regulates Na + /K + exchanges [6]. All G proteins consist of three subunits, a, b and c,with the nucleotide-binding and hydrolyzing a subunit defining the protein’s identity. The a subunit is believed to confer receptor and effector specificity on the heterotrimer. After its activation, different secondary pathways can occur: adenylate cyclase catalyses the formation of cAMP, whereas phospholipase C hydrolyses membrane phospha- tidylinositol, liberating inositol 1,4,5-triphosphate (InsP 3 ) and diacylglycerol. Although cAMP and InsP 3 cascades appear to be active as two alternative pathways in vertebrate olfaction [7], mechanisms of olfactory signal transduction in insects seem to involve the InsP 3 pathway. Experiments on the rapid kinetics of second messengers in antennal homogenates of insects demonstrated an elevation of InsP 3 upon stimulation with pheromones [8–10] and nonpheromonal compounds [11] and it has been shown that G proteins are functionally active in signal transduc- tion of different sensory systems of invertebrates [12]. Additionally, a phospholipase C b and a protein kinase C were recently identified in pheromone receptor neurons of the moth Antheraea polyphemus [13]. Correspondence to E. Jacquin-Joly, Phytopharmacie, INRA, Route de Saint-Cyr, 78026 Versailles cedex, France. Fax: + 33 1 30 83 31 19, Tel.: + 33 1 30 83 32 12, E-mail: jacquin@versailles.inra.fr Abbreviations:InsP 3 , inositol 1,4,5-triphosphate. (Received 28 November 2001, revised 28 February 2002, accepted 4 March 2002) Eur. J. Biochem. 269, 2133–2142 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02863.x G proteins from different families have been studied in several invertebrate species including locust Go [14], Dro- sophila Gq [15], the Lymnaea stagnalis Gq [16] or lobsters Gq [17–19] and Gs [20]. The presence of different G proteins was reported in lepidopteran antennae in toxin sensitivity studies [8,21,22]. However, using antibodies raised against different G proteins, Laue et al. [23] could detect positive stain only with an antiserum raised against the asubunit of a G protein belonging to the Gq/11 G protein family. So far, no G a subunit sequence is available in Lepidop- tera, except a Go a cloned in the moth Manduca sexta [24]. In order to develop a better understanding of all the elements of the olfactory signaling pathway in insects, we report here characterization, molecular cloning and expres- sion localization in the antennae of the first lepidopteran G protein a subunit belonging to the Gq family. MATERIALS AND METHODS Insects Animals were reared in Domaine du Magneraud (INRA, France) on a semiartificial diet [25] at 20 °C, 60% relative humidity, exposed to a 16-h/8-h light/dark photoperiod and sexed as pupae. Antennae from 3-day-old adults were dissected and stored at )80 °C until use. Preparation of extracts, gel electrophoresis and immunoblotting Two hundred whole antennae from either male and female adults were homogenized in 1 mL of 20 m M Tris/HCl, pH 7.3, with a home-made moto-driven homogenizer, and centrifuged at 10 000 g for 30 min The supernatant, con- taining soluble proteins and membrane vesicles, was used in the further experiments. PAGE was performed at a concentration of 10% of polyacrylamide in the presence of 5% SDS, according to the procedure of Laemmli [26]. Protein bands were detected with Coomassie Brilliant Blue R-250 (Serva). After electropho- retic separation, proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Germany) and were treated with 2.5% BSA, 2.5% gelatin, 1% goat serum and 0.05% Tween 20 in NaCl/P i for2hinorderto prevent unspecific binding and incubated overnight with a Gq/11 a antiserum (Calbiochem), at a dilution of 1 : 1000. Bound antibodies were detected with goat anti-rabbit 1 Ig conjugated with alkaline phosphatase (dilution 1 : 10 000), using 5-bromo-4-chloroindolyl phosphate/nitroblue tetra- zolium as substrate. The affinity purified Gq/11 a antiserum was raised against a synthetic decapeptide corresponding to the C-terminal of a G protein a subunit and cross-reacts with the a subunits of Gq and G 11 (Calbiochem). Primary cultures of antennal neurons Cultures were prepared as previously described [27]. Briefly, antennal flagella from 3-day-old male pupae were dissected in 3 + 2 medium (three parts of Leibovitz’s L15 medium and two parts of Grace’s medium supplemented with lactalbumine hydrolysate and yeastolate). Flagella were disrupted by incubation in L -cysteine-activated papain (1 mgÆmL )1 ) followed by trituration with a fire-polished Pasteur pipette. The resulting cell suspension was then plated onto uncoated Falcon Petri dishes. Two hours after plating the cells, the culture medium was replaced by a 3 + 2 medium supplemented with 5% fetal bovine serum. The cultures were then inverted to form a Ôhanging columnÕ and were maintained for 2–4 weeks at 22 °C in humid atmosphere. Antennal cells were grown in culture for 2–3 weeks prior to harvesting in 20 m M Tris/HCl, pH 7.3 buffer, and extracting into Laemmli sample buffer. RNA extraction and cDNA synthesis Total RNA was extracted from 200 antennae with the Tri-Reagent (Euromedex). Single stranded cDNA was synthesized from 1 lg of total RNA with M-MLV (USB), using buffer and protocol supplied with the enzyme. The reaction mixture contained dNTP mix (Pharmacia), Rnasin (Promega), oligodT 18 with an anchor: CATGCATGCGGC CGCAAGCT 18 VN (synthesized by Isoprim, Toulouse, France), sterile water and template RNA to a final volume of 50 lL. The mix was heated at 68 °C for 5 min and chilled on ice before adding the M-MLV (600 U), then incubated 1 hat37°C and finally the reverse transcriptase was inactivated at 95 °C for 5 min. For the 3¢ RACE, reverse transcription was performed on 1 lg of total RNA accord- ing to the manufacturer’s instructions (3¢-AmpliFIND- ER TM RACE Kit, Clontech), using a 20-lLreaction mixture. For the 5¢ RACE, cDNA was synthesized from 1 lg of male antennae total RNA at 42 °Cfor1.5husing the SMART TM RACE cDNA Amplification Kit (Clontech) with 200 U of Superscript II (Gibco BRL), 5¢ CDS-primer and SMART II oligonucleotide, according to the manufac- turer’s instructions. Internal amplification Two degenerate primers were designed according to consensus regions of several G protein a subunit sequences from different species, including the mollusk Lymnaea stagnalis sequence [16]. The nucleotide sequence of the sense primer is based on the amino-acid motif FIKQMR (5¢-CG C GAATTCNTTYATHAARCARATGMG-3¢)andthe antisense primer is based on the amino-acid sequence ATDTENL (5¢-TGT GGATCCTTITTYTCIGTRTCIG TNGC-3¢). EcoRI and BamHI restriction sites (indicated by underlining), respectively, have been included to facilitate subcloning. Approximately 1 ng of cDNA was used for polymerase chain reaction carried out with Taq polymerase (1 U) (Promega) in 10 m M Tris/HCl, pH 9.0, 50 m M KCl, 0.1% Triton X-100, 1.5 m M MgCl 2 ,0.2m M of each dNTP. A 900-bp PCR product was generated after 40 cycles consisting of 1 min at 94 °C, 1 min at 44 °Cand1minat 72 °C in a Hybaid thermocycler. Subcloning in pZERO (Invitrogen) using EcoRI and BamHI resulted in loss of a part of the amplified product due to a BamHI internal site. So, cloning was then performed using a TA vector from Invitrogen, pCR TM II, using the TOPO cloning kit. 3¢ RACE-PCR For the 3¢ RACE-PCR, two amplifications were con- ducted as described in the manufacturer’s instructions 2134 E. Jacquin-Joly et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (3¢-AmpliFINDER TM RACE Kit, Clontech). The first one was conducted on 1 lLofthe3¢ reverse transcription reaction, with a primary sense gene-specific primer deduced from the sequence obtained after the internal amplification (5¢-GCATTATAGAATACCCATTTGACCTG-3¢) and with an antisense Anchor Primer (furnished in the kit). It consisted of 30 cycles of 1 min at 94 °C, 1 min at 55 °Cand 1minat 72°C. The second amplification consisted of a nested PCR and was carried out on 1 lL of the first amplified product, using a second sense gene-specific primer (5¢-GACCTGGAAGAAATACGATTTAGAATGG-3¢) and the Anchor Primer from the kit, and consisted of 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C. A 600-bp amplification product was obtained. 5¢ RACE-PCR Amplification was performed on 2.5 lLof5¢-RACE-ready cDNA using Universal Primer Mix (Clontech) as a sense primer and an antisense gene-specific primer, designed according to the cDNA sequence obtained from the internal amplification (5¢-TCGCCTGCCGTCGTAGCAC TCCTG -3¢). The 50-lL amplification mix was prepared according to the SMART TM RACE cDNA Amplification kit instructions using the Advantage 2 Polymerase mix (Clontech). Touchdown PCR was performed using hot- start as follows: after 1 min at 94 °C,5cyclesof30sat 94 °C and 3 min at 72 °C, then 5 cycles of 30 s at 94 °C, 30 s at 70 °C and 3 min at 72 °C, then 25 cycles of 30 s at 94 °C, 30 s at 68 °C and 3 min at 72 °C, then 5 min at 72 °C. Cloning and sequencing The amplified cDNAs were ligated into the plasmid pCR TM -II using the TOPO cloning kit from Invitrogen (the Netherlands). Recombinant plasmids were isolated using Plasmid Midi kit from Qiagen and both strands were subjected to automated sequencing by ESGS (Evry, France). Database searches were performed with the BLAST program (NCBI) and sequence alignment with the CLUSTALW (NPS @IBCP). In situ hybridization RNA sense and antisense probes (900 bp long) were in vitro transcribed from linearized pCRII-cDNA plasmid, result- ing from the cloning of the internal amplification, using T7 and SP6 RNA polymerase (Promega) following recom- mended protocol and in the presence of 1.5 U of Rnasin (Promega). Probe quality was confirmed under denaturing conditions by formaldehyde agarose gel electrophoresis and the probes stored at )80 °C until use. For hybridization, antennae were removed from adult head, cut into pieces and fixed overnight at 4 °Cin4% paraformaldehyde in NaCl/P i . Fixed tissues were dehydra- ted in 100% methanol and stored at )20 °C. The hybrid- ization protocol was performed on whole-mount pieces of antennae as previously described [28]. Hybridization was detected using alkaline-phosphatase-conjugated anti- digoxygenin Ig (1 : 4000) and stained with Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (Boehringer Mannheim). After sufficient staining, specimens were washed in NaCl/P i and fixed in 4% paraformaldehyde for 20 min, then dehydrated through a graded series of ethanol and wax-embeded. Six-micro- meter longitudinal sections were cut and counter-stained with acridine orange. Sections were photographed, then pictures were digitized and processed using ADOBE PHOTO- SHOP 5.0. RESULTS Immunodetection of the Gq/11 a subunit. Proteins extracted from male and female antennae and from primary antennal cell culture of M. brassicae were separated by SDS/PAGE and analysed by Western-blot using a Gq/11 a antiserum (Fig. 1). Crude homogenates of male and female antennae contained an immunoreactive band with an apparent molecular mass of 40 kDa (Fig. 1, left, A,B). In the sample of primary cell culture of M. brassicae, a band with the same apparent molecular weight was labeled by the antiserum, indicating that the protein is also present in the in vitro cell cultures (Fig. 1, left, C). Cloning and cDNA sequencing A 900-bp cDNA product was amplified with RT-PCR using degenerate oligonucleotide primers. After cloning and sequencing, this product was translated and the deduce amino-acid sequence was compared with sequences in the GenBank database. This product appeared to be very similar to a subunits from G proteins belonging to the Gq family. It was then extended to the 5¢ and the 3¢ untranslated regions by 5¢ and 3¢ RACE, respectively. This allowed us to obtain the sequence of a full length Fig. 1. Biochemical detection of Gq/11 a. M, molecular markers. Left (A,B,C) Coomassie stain after 10% SDS/PAGE of antennal and cell culture homogenates. (A) Male M. brassicae (4 antennae equivalent), (B) female M. brassicae (6 antennae equivalent), (C) primary cell cul- tures of M. brassicae male antennae (15 Petri dishes equivalent to 15 antennae). Right (A,B,C) Western-blot after SDS/PAGE of antennal and cell culture homogenates using Gq/11 a antiserum (dilution 1 : 1000). The antiserum cross-reacted only with a single band of about 40 kDa in both male (A) and female (B) antennal extracts as well as in the primary cell culture extracts (C). Ó FEBS 2002 A lepidopteran Gq protein a subunit (Eur. J. Biochem. 269) 2135 cDNA of 1541 bp (Fig. 2). This sequence has been deposited in the GenBank database with accession number AF448447. Nucleotide sequence analysis revealed that the cDNA contains a putative coding region of 1059 bp, encoding a 353 amino-acid protein with a theoretical molecular mass of 41 400 Da and an isoelectric point of 5.35, as determined using MWCALC (Infobiogen) (Fig. 2). There are several upstream ATG codons but soon followed by stop codons. The ATG at position 199 has a favorable sequence context for translation initiation [29] and could be proposed to be the start of the protein coding domain. Sequence analysis of the 3¢ end cDNA revealed that there is a polyadenylation signal upstream of the poly(A). Analysis of the primary structure of M. brassicae Gqa The putative protein product encoded by the cloned cDNA was aligned with different G proteins from invertebrates and vertebrates retrieved from blast search (Fig. 3). This putative protein showed a high degree of identity to other known Gqa proteins from invertebrates (Drosophila, 87%; Limulus, 83%; lobster, 85%) but also from vertebrates (mouse, 80%; human, 80%), and is less similar to other Ga types (47.5% with Go of the Lepidoptera M. sexta,for example) (Table 1). Furthermore, the M. brassicae Gqa subunit exhibits important characteristics of other Gqa proteins, namely: the amino-acid sequence G40TGESGKST FI typical of the Fig. 2. cDNA and deduced amino-acid sequence of the M. brassicae Gq a subunit (GenBank accession number no. AF448447). The suggested start ATG and stop TGA codons are in bold italics. Positions of the primers for the internal amplification are underlined (solid line), as are gene specific primers and nested primer for the 3¢ RACE amplification (dashed lines) and the gene specific primer used for the 5¢RACE amplifi- cation (dotted line). Palmitoylation sites C3C4, G40TGES box and putative cholera toxin site Arg177 are in boxes. 2136 E. Jacquin-Joly et al. (Eur. J. Biochem. 269) Ó FEBS 2002 A domain, with the characteristic residues underlined [30], a N-terminal cysteine doublet (Cys3, Cys4) in a MXCC motif that represent putative sites for palmitoylation [31], a putative cholera toxin ADP-ribosylation site (Arg177) and a G40TGES ÔGAG boxÕ sequence that is present in the GTP- binding domain of other Gqa proteins (Fig. 2). Expression pattern in male antennae In situ hybridization experiments were performed using digoxigenin incorporated antisense and sense RNA probes against adult male antennae. The M. brassicae antenna is filiform,  1 cm long and comprises about 72 segments [32]. Each segment exhibits the same general organization: the dorsal side is covered with two rows of scales and the olfactory hairs (the sensilla) are located on the ventral side as can be seen using scanning electron microscopy (Fig. 4A). In males, the olfactory hairs are distributed in two classes according to their length. The long ones (long sensilla trichodea) are located on the lateral part of the ventral area and are arrayed in four to five parallel rows [32] (Fig. 4A, white arrows). Short sensilla trichodea are located medio-ventrally and are not arranged in rows. Sense strand controls gave no signals (not shown) whereas antisense probe hybridization is restricted to the sensilla (ventral) side of the antennae (Fig. 4B,E). Close examination revealed hybridization in cells at the bases of the sensilla hairs (Fig. 4C,D) and sometimes two labeled somata can be seen at the base of one sensillum (Fig. 4E). On longitudinal sections through the antennae, it is difficult to distinguish between long and short sensilla as only parts of the sensilla are visible (Fig. 4B,E). However, sections through the cuticle permitted the observation of labeled spots distributed in the ventro-lateral region with a row pattern consistent with the distribution of the long sensilla trichodea (Fig. 4F, white arrows). Typical structures of Fig. 3. Alignment using CLUSTAL W of G protein a subunit of the q family from different species, including invertebrates and vertebrates. Amino-acid identities are in bold. Sequences compared to M. brassicae Gqa sequence are from Drosophila melanogaster (GenBank accession numbers M58016; M30152; U31092), Homarus americanus (U89139), Panulirus argus (AF201328), the mouse Mus musculus (P21279), the dog Canis familiaris (Q28294) and human (P50148). Several motifs indicative of this Ga family are conserved: N-terminal cysteines, arginine177, and a GAG box. Ó FEBS 2002 A lepidopteran Gq protein a subunit (Eur. J. Biochem. 269) 2137 sensilla coeloconica, that resemble flowers, can be observed on sections through the cuticle, without any associated labeling (Fig. 4G, black arrows), whereas on the same section other sensilla without any particular distribution are labeled, that correspond to short sensilla trichodea. DISCUSSION Several studies have previously suggested that G-protein- mediated signal transduction pathway may occur in pheromone-sensitive receptor cells in insects. For example, it has been shown that nerve impulse activity of phero- mone receptor cells increased significantly after G protein- activating natrium fluoride application to their outer dendrite in single sensilla trichodea of the moth Bombyx mori [23]. Additionally, using a Gq/11 antiserum, the same authors revealed the presence of a protein that is likely to belong to the Gq family in antennae of both B. mori and Antheraea pernyi. In this context, we report here the immunodetection of a protein with the same characteristics, the molecular cloning of the corresponding cDNA to get the total amino-acid sequence of the protein, and the expression pattern of the corresponding mRNA as a first step to clarify the role of G protein a subunit in olfaction. Immunodetection of Gq/11 a subunit in antennae homogenate and in neuron primary culture The molecular mass of the immunoreactive band observed is consistent with the molecular mass of other G protein a subunits, and may represent the a subunit(s) of one or several proteins belonging to the Gq/11 family. The visualization of such proteins in our olfactory cell culture is consistent with the occurrence of a Gq protein in antennal primary cell culture already observed in lobster [17], which mediates excitatory odor transduction in olfac- tory receptor neurons in this species. Molecular cloning of a cDNA coding for a Gqa subunit in male antennae Because of the strong conservation of the G protein a subunit throughout evolution, we decided to use the PCR technique to identify a cDNA encoding M. brass- icae Gqa-like protein. We then identified a putative transcript encoding a G protein a subunit homologous to invertebrate and vertebrate Gqa, suggesting the presence of a specific Gqa gene in M. brassicae. The molecular mass of the predicted protein is consistent with that determined by Western blot after SDS/PAGE (Fig. 1). Table 1. G protein a subunit characterized in insects, including the Gq of M. brassicae described here, and in some other invertebrate groups. Databank accession numbers, references and expression pattern/putative role are also given. ORN, olfactory receptor neurons. Species G protein a subunit class Accession no. Ref. % identity with Gq of M. brassicae Possible function Insecta Lepidoptera Mamestra brassicae Gq AF448447 This paper 100 Expression in ORN Manduca sexta Go Z49080 [24] 47.5 Developmental role in embryonic neurons Diptera Drosophila melanogaster DGq1 M58016 [54] 81 retinal expression DGq2 M30152 [55] 81 Expression in nervous system and ovaries DGq3 G U31092 M23094 [15] [56] 87 48.6 Expression in chemosensory cells and central nervous system Expression in embryos and pupae Calliphora vicina G AJ250443 [57] 81.5 Visual protein of the compound eyes Orthoptera Locusta migratoria Go A61035 [14] 49 Expressed in nervous tissues Crustacea Panulirus argus Gq/11 AF201328 [19] 85 Expression in ORN Homarus americanus Gq U89139 [18] 85 Expression in neurons of olfactory organs and brain Chelicerata Limulus polyphemus Gq U88586 [58] 83 Expression in eyes Mollusca Patinopecten yessoensis Gq AB006456 [59] 80 Expression in visual cells Lymnaea stagnalis Gq Z23106 [16] 78 Expression in neurons Octopus vulgaris Gq AB025782 [60] 76 Expression in photoreceptor cells Loligo forbesi Gq L10289 [61] 75 Visual G protein a subunit Echinodermata Asterina pectinifera G(I) X66378 [62] 59 ? Demospongiae Geodia cydonium Gq Y14248 [63] 50.1 Oocyte maturation 2138 E. Jacquin-Joly et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The antibody used for the Western-blot is directed to the decapeptide QSALKEFNLA that corresponds to a defined C-terminal sequence found in both Gqa and G 11 a (Calbiochem). The deduced amino-acid sequence shares high C-terminal identity with this sequence (QLNLKEYNLV) and thus should have been detected with such antibodies. Although proteins of this class are highly conserved in sequence and molecular mass, the observation of only a single band on the Western blot, combined with the molecular cloning data, suggests that the cloned cDNA probably encodes the protein detected using commercial antibodies. The predicted protein we obtained shares high identities with other already known Gq protein a subunits and therefore can be placed within this family. Indeed, M. brassicae Gqa possesses all the characteristics observed in Gqa-like proteins. In particular, the cysteine doublet at the N-terminal part probably serves as a site for post- translational attachment of a palmitoyl group (16-carbon, saturated fatty acid) through a labile, reversible thioester linkage [33,34], and which could serve as a membrane anchor. It is noteworthy that proteins from Gq family are highly conserved throughout evolution: the putative M. brassicae Gq sequence is 80% identical to mouse, dog and human Gq; however, it shares only 47% identity with a Go sequence from M. sexta, another lepidopteran (Table 1). In particular, the M. brassicae G subunit is 87% identical to the dGqa-3 of Drosophila [15]. It differs only in two domains: 70–130 and the C-terminal region that is important for receptor interac- tions [35,36]. Expression pattern in the adult male antennae In situ hybridization revealed that this G protein subunit is expressed in both long and short sensilla trichodea (Fig. 4F,G) in cells that could be neurons because of several observations. On Fig. 4C, for instance, the labeled cell is located at the base of the cuticular hair and protrusions emanating from the soma that could corres- pond to the dendrite are seen entering the base of sensillum hair (Fig. 4C). Such protrusions have already been observed after in situ hybridization in labeled neurons of M. sexta antennae [37]. Furthermore, two somata can be seen that are labeled at the base of the same sensilla (Fig. 4E), possibly corresponding to the two receptor neurons observed in all sensilla. The shape, size and position of the stained cells also suggest their identity as olfactory neurons. The Gqa appeared to be associated only with sensilla trichodea, devoted to pheromone reception [32], with no expression in sensilla coeloconica. These latter structures have been shown to be involved in plant-related volatile detection, at least in B. mori [38]. We can then suppose that although both sensilla types are implicated in olfaction, they do not express same G protein a subunits, maybe according to the ligands they are tuned to. Such a phenomenon has already been observed in the vertebrate vomeronasal organ, an organ responsible for detecting pheromones. Two G protein subtypes are selectively activated by different classes of compounds [39]: some neurons express receptors encoded by one multigene family and the G protein a subunit a i , whereas some others express receptors encoded by another multigene family and the G protein a subunit a o . Fig. 4. Expression pattern of M. brassicae G protein a subunit revealed by in situ hybridiza- tion to mRNA in longitudinal sections of male antennae. (A) Scanning electron microscopy of a male antennae. The ventral surface is cov- ered by short and long sensilla, the last being arranged in parallel rows (white arrows). (B,E) expression of G protein a subunit on the sen- silla side of the antennae. (C,D) sensilla trichodea at higher magnification. (F) Section through the cuticule in the ventro-lateral region of the antennae showing G protein a subunit expression in row pattern (white arrows) consistent with the distribution of the long sensilla trichodea devoted to pheromone reception. (G) Sensilla coeloconica are not labeled (black arrows) whereas the surrounded sensilla (short sensilla) are labeled. Scale: (A,B,E,F) 50 lm; (C,D,G) 10 lm. Ó FEBS 2002 A lepidopteran Gq protein a subunit (Eur. J. Biochem. 269) 2139 Recently, the a subunit of a G protein of the Gq family has been immunolocalized in olfactory sensilla preparations of the silkmoth Antherea pernyi [23]. Using immunocytol- ogy, the authors were able to show that all types of olfactory sensilla are labeled. However, labeling is not restricted to sensillar cells and can be observed in auxiliary cells, epidermal cells and subcuticular extracellular space. This is not in contradiction with our observations given that the tools and the organisms used in the two studies are different: using antibodies they visualized the localization of the protein whereas by using in situ hybridization to mRNA we revealed only the expressing cells that are likely to be olfactory neurons. The immunological study [23] suggests that Gq plays a role in olfactory signal transduction as long as the protein predominates in the dendrites of olfactory receptor cells. Implication of Gq in lepidoptera olfaction Gqa have been frequently presumed to play a role in olfaction in invertebrates. For example, the protein dGqa-3 of Drosophila was detected in the third antennal segment, maxillary palps, the tip of the proboscis and in the brain [15]. Some of the immunoreactive cells have been identified as antennal olfactory neurons, non-neuronal accessory cells, or gustatory neurons, suggesting that this protein is involved in olfactory and gustatory responses in Drosophila. Several studies on lobsters support Gq involvement in odor transduction in olfactory receptor neurons. An anti-Gq/11 Ig has been shown to selectively block odor-evoked inward current in voltage-clamped cultured neurons and immuno- labeled a band of  45-kDa in Western-blot analyses [17]. Similarly, a Gqa protein has been cloned in two lobster species, Homarus americanus [18] and Panulirus argus [19], that is expressed in olfactory receptor neurons, suggesting that one function of Gqa is to mediate olfactory transduc- tion. In our study, expression of proteins in olfactory sensilla trichodea, apparently in neurons, leads us to hypothesize that, in M. brassicae, this G protein subunit is involved in pheromone reception. In addition, our data demonstrated that the olfactory organ of this species expresses a gene that is critical for the phosphoinositide signaling pathway: the fact that this protein belongs to the Gqa subunits suggests that a phospholipase C second messenger pathway may be implicated in transduction of olfactory signals in lepidoptera. Such a hypothesis has already been proposed for insects in a variety of species using kinetics based methodology (reviewed in [40]) and immunological detection of Gq/11a subunits in antennae [23]. Additionally, a phospholipase C b and a protein kinase C, two enzymes involved in the InsP 3 transduction pathway, were identified by using specific antibodies directed against molecules involved in intracellular olfactory signalling [13]. The two enzymes were detected after Western blot with homogenates of isolated pheromone-sensitive sensilla trichodea, containing no other cellular elements than the outer dendrites of pheromone receptor neurons. In lobsters, several recent studies showed that phospholipase C b mediates olfactory transduction as well [41]. Molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons has been provided [19]: a G protein a subunit of the Gq familyandanInsP 3 -gated channel or an InsP 3 receptor. In addition, the authors showed that the InsP 3 receptor is associated with the plasma membrane, suggesting a novel mechanism for regulating intracellular ions within restricted cellular compartments of neurons [19]. Interestingly, InsP 3 receptors have also been immunolocalized within the dendritic membrane of olfactory sensilla of moths [42]. Elevation of InsP 3 and InsP 3 -gated-Ca 2+ influx in phero- mone-stimulated cell cultured olfactory neurons has also been shown [43]. Here, we provide molecular evidence that support the previous findings and the first lepidopteran sequence of a Gqa subunit. The Caenorhabditis elegans genome project has revealed 20 genes encoding a-subunits of G proteins, 14 of which are expressed almost exclusively in subsets of chemosensory neurons [44,45]. Then it seems likely that this nematode uses multiple Ga subunits per cell, leading us to hypothesize that neurons mediating more than one sensory modality can do so via distinct intracellular pathways [46], each mediating a particular response to a specific class of chemical stimuli [47]. However, C. elegans expresses multiple chemosensory receptors per olfactory neurons, which is not the case in Drosophila where neurons are likely to express only a single olfactory receptor gene, although sometimes along with a broadly expressed receptor of unknown function [48]. In moths, the lack of any information on putative olfactory receptors does not permit such considerations. However, it cannot be excluded that different types of Ga subunits may be involved in olfactory transduction in Lepidoptera. Different G proteins are found in specialized tissue and they have there different functions, although they all share structural properties such as the heterotrimeric composition with a, b, c subunits. However, a subunits are distinct whereas b subunits are quite similar [49]. The similarity of our a subunit sequence with others implicated in olfactory transduction further supports our hypothesis that this subunit is involved in odor transduction cascade in moth antennae. Our identification of a Gqa subunit expressed in olfactory sensilla supports the hypothesis that G-protein-coupled olfactory receptors are functional in insects. In insects, seven transmembrane domain proteins coupled to G-protein- mediated second messenger cascades have been found to date only in Drosophila [1–3] and Anopheles gambie [50] and attempts to find similar receptor proteins in other insects have failed. An olfactory-specific protein (SNMP for sensory neuron membrane protein) of two transmembrane domains uniquely expressed in olfactory receptor neurons has been characterized in the silkmoth A. polyphemus [51,52] as well as in the moths B. mori, Heliothis virescens and M. sexta [37]. In this latter species, a second SNMP homologue was also identified [37,53]. These proteins are homologous with the CD36 receptor family, which pre- dominately recognizes proteinaceous ligands. One could then not exclude a possible role as olfactory receptor, considering the probable interaction with odorant binding proteins carrying the odorant molecule to the receptor. Although no olfactory receptor has been identified in Lepidoptera, the discovery of a Gqa subunit expressed in olfactory neurons and sharing high identities with the olfactory/gustatory Drosophila Gqa subunits suggests that seven transmembrane domain receptor proteins should exist in moth antennae and are involved in olfaction. 2140 E. 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