Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits.


Introduction
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are molecular switches that mediate many extracellular signaling processes by coupling cell surface receptor activation with the diverse signal transduction effector molecules that drive cellular responses. The heterotrimeric G protein complex is composed of an Į-subunit (GĮ) that functions in guanine nucleotide binding/hydrolysis and a heterodimer composed of a ȕ and Ȗ subunit (GȕȖ). In the absence of receptor stimulation, the three subunits are associated and GDP is bound to GĮ. Receptor activation triggers GDP exchange for GTP and dissociation of GȕȖ from the GĮ-GTP complex. The dissociated GĮ and GȕȖ subunits are then able to modulate the activity of various downstream effector proteins (ion channels, adenylyl cyclases, phospholipase Cȕ, etc.). The intrinsic GTPase activity of GĮ hydrolyzes GTP to GDP, which promotes reassociation of the heterotrimeric G protein complex and terminates the signal [1][2][3]. Based on this intermediary molecular role, heterotrimeric G proteins play pivotal roles in determining the specificity and duration of the cellular response to extracellular signals.
The GĮ subunits form a large multigene family composed of 39-52 kDa proteins that share 35%-95% sequence identity and have been grouped into four subfamilies (GĮs, GĮi/o, GĮq, and GĮ12) based on structural and functional similarities [1][2][3]. GĮs subfamily members couple receptors to adenylyl cyclase stimulation (i.e., increases in cAMP), whereas the GĮi/o subfamily has the opposite effect. The GĮq subfamily regulate the activity of phospholipase C ȕ isoforms (i.e., diacylglycerol and inositol triphosphate production) [1,2] and GĮ12 has been extensively characterized based on their ability to activate Rho-specific guanine nucleotide exchange factors [4,5].
The western tarnished plant bug (Lygus hesperus) is a polyphagous pest of numerous crops [37,38] that utilizes chemosensory signals to aid in identification of host plants and conspecific mates [39][40][41][42]. Despite the pest status of the Lygus spp. complex, transcriptional resources have only recently been developed [43][44][45][46], and our knowledge of chemosensory signal transduction is limited to odorant binding proteins [45,47] and the olfactory receptor co-receptor (Orco) [12] in L. lineolaris and L. hesperus. Furthermore, while G proteins have been studied in a number of insects with GĮ subunits cloned from Drosophila melanogaster [17,21,[48][49][50], Anopheles gambiae [20], Bombyx mori [19,51,52], Manduca sexta [53], Locusta migratoria [54], Lissorhoptrus oryzophilus [55], Helicoverpa assaulta [56], Mamestra brassicae [18], Bemisia tabaci [57], and Oncopeltus fasciatus [58], little progress has been made on the role of these genes in mediating chemosensory behaviors in plant bugs such as Lygus. In this study, we sought to begin to address this lack of knowledge by identifying the molecular sequences and expression profile of GĮ subunits in L. hesperus. Using homology-based PCR and transcriptome database mining methods, we cloned a group of cDNAs with high sequence homology to each of the GĮ subfamilies. In addition, we performed detailed sequence comparisons of the L. hesperus transcripts with those from other insects, profiled transcript expression levels, and examined the subcellular localization of a subset of recombinantly expressed L. hesperus GĮ proteins in cultured insect cells.

Insect Rearing
L. hesperus were obtained from an in-house stock colony (USDA-ARS Arid Land Agricultural Research Center, Maricopa, AZ, USA) periodically outbred with locally caught conspecifics. The colony is fed an artificial diet packaged in Parafilm M [59,60] and maintained under rearing conditions consisting of 27 °C, 40% humidity and a L14:D10 photoperiod. Experimental nymphs were generated from eggs deposited in oviposition packets and maintained as described previously [61].

Identification and Cloning of L. hesperus GĮ Subunits
To identify L. hesperus GĮ subunits (LhGĮ), we initially utilized a degenerate PCR approach similar to that reported previously in B. mori [51,52] using degenerate primers (Table 1) designed to conserved amino acid stretches identified in protein sequence alignments of known insect GĮ sequences. Total RNA was isolated from adult L. hesperus female heads and bodies using TRI Reagent RNA Isolation Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Isolated total RNA was quantified based on absorbance at 260 nm using a Take3 multi-volume plate on a Synergy H4 hybrid multi-mode microplate reader (BioTek Instruments, Winooski, VT, USA). First strand cDNA was synthesized from 1 ȝg of DNase I-treated total RNA in separate Thermoscript or SuperScript III (Life Technologies, Carlsbad, CA, USA) first-strand cDNA synthesis reactions with random hexamers. To minimize primer bias towards particular classes of GĮ proteins [62,63], multiple PCR amplifications were performed using ExTaq DNA polymerase (Takara Bio Inc./Clontech, Palo Alto, CA, USA) with 0.7 ȝL (35 ng) cDNA template and 2.5-3 ȝL (0.5-0.6 ȝM) of each primer and varying thermocycler conditions ( Figure 1). Nested PCR was performed as above but using a 1-ȝL aliquot of the previous reaction as the template. PCR products were electrophoresed on 1.7% agarose gels and stained with SYBR Safe (Life Technologies). Amplimers of the expected sizes were gel-excised using an EZNA Gel Extraction kit (Omega Bio-Tek Inc., Norcross, GA, USA), cloned into the pGEM T Easy-TA cloning vector (Promega, Madison, WI, USA) and sequenced at the Arizona State University DNA Core Lab (Tempe, AZ, USA).
The partial fragments amplified above were extended by RACE PCR using templates generated with a SMARTer RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) and 2 ȝg DNase I-treated RNA. Amplification was performed using ExTaq with 0.5 ȝL (50 ng) cDNA, primers corresponding to one of the Universal Primers supplied with the SMARTer RACE cDNA Amplification kit, a gene specific primer (Table 1), and touchdown thermocycler conditions ( Figure 1). PCR products were electrophoresed on 1.5% agarose gels with amplimers of the expected sizes gel excised and sequenced. Incorporating the resulting 5' and 3' RACE sequence data with the degenerate PCR derived sequences yielded sufficient data to design gene specific primers encompassing the putative start and stop codons ( Table 1). The respective L. hesperus GĮ open reading frames (ORFs) were amplified in multiple independent reactions using ExTaq DNA polymerase and sequence verified. The consensus nucleotide sequence data are available in the GenBank database under the accession numbers: AEK80438 (LhGĮi), AEK80436 (LhGĮs), and AEK80437 (LhGĮq1). To identify additional GĮ subunits and potential variants of the LhGĮ subunits identified above, L. hesperus transcriptomes [43,46], which became available after the initiation of the LhGĮ cloning project, were searched using BLASTx (E value 10 í10 ) with queries consisting of the consensus LhGĮ sequences and other insect GĮ subunits. Sequence hits were then re-evaluated against the NCBI nr (non-redundant) database and duplicates removed. This search identified two additional GĮ subunits (LhGĮo and LhGĮ12) and three potential LhGĮq variants. Primers were designed to the putative start and stop codons of LhGĮo and LhGĮ12 and to unique portions of the LhGĮq variants ( Table 1). The respective sequences were amplified from multiple independent reactions using Sapphire Amp Fast PCR Master Mix (Takara Bio Inc./Clontech), subcloned where possible into a pCR2.1 TOPO TA cloning vector (Life Technologies) and sequence verified. The nucleotide sequence data are available in the GenBank database under the accession numbers: KM610199-KM610202 (LhGĮq2-LhGĮq5), KM610203 (LhGĮ12), and KM610204 (LhGĮo).

Bioinformatic Analyses
LhGĮ sequences were evaluated against the NCBI nr database by BLASTx (E value 10 í5 ). Putative myristoylation sites were predicted using NMT-MYR Predictor (http://mendel.imp.ac.at/myristate/ SUPLpredictor.htm) and palmitoylation sites with CSS-PALM (http://csspalm.biocuckoo.org/index.php) [64]. x 5 x 35 x 35 x 10 x 10 x 20 nested PCR x 10 x 10 x 20 nested PCR x 5 x 5 x 30 x 5 x 5 x 30 x 35 x 35 To determine potential phylogenetic relationships, multiple sequence alignments of the putative LhGĮ subunits and other insect GĮ subunits (nine per subfamily) were constructed using default settings in MUSCLE [65,66]. Phylogenetic inferences were made using the maximum likelihood, minimum evolution, NJ, and UPGMA modules implemented in MEGA6.06 [67] with bootstrap analysis conducted of 1000 replicates. Data shown are for the maximum likelihood method based on the JTT matrix-based model [68]. Initial tree(s) for the heuristic search were obtained by applying the NJ method to a matrix of pairwise distances estimated using a JTT model. The analysis involved 53 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 350 positions in the final dataset.

Transcriptional Profiling of L. hesperus GĮ Subunits
The expression profiles of the respective LhGĮ transcripts were examined across L. hesperus development and within sex-specific adult body tissues. Developmental profiling consisted of eggs, pooled samples from each of the five nymphal instars, and mixed sex adults comprising equal numbers of males and females at 1, 10, and 20 days post-adult emergence. Adult tissue profiling was performed using cDNAs generated from pooled, sex specific virgin 7-day-old adult bodies, heads, midgut/hindgut, Malpighian tubules, antennae, probosci, and legs as well as pooled tissue sets of female ovaries and seminal depositories, and male medial/lateral accessory glands and testes. Samples were homogenized in TRI Reagent Solution (Ambion/Life Technologies) using a TissueLyser (Qiagen, Valencia, CA, USA) with total RNA extracted based on recommendations from the manufacturer. First-strand cDNAs were generated using a Superscript III first-strand cDNA synthesis kit (Life Technologies) with custom-made random pentadecamers (IDT, San Diego, CA, USA) and 500 ng of DNase I-treated total RNAs. End-point PCR amplification was done using Sapphire Amp Fast PCR Master Mix with 0.4 ȝL (10 ng) cDNA template, sequence-specific primers (Table 1) designed to amplify ~500-600 bp fragments of the LhGĮ transcripts, and thermocycler conditions described in Figure 1. Both developmental and adult tissue expression profiles were replicated at least three times using cDNA templates prepared from different biological replicates. Differing combinations of primer sets (see Table 1) designed from transcriptomic data were used to profile the LhGĮq1-4 variants: LhGĮq1 (LhGaq 468 F1/LhGaq 1036 R1), LhGĮq2 (LhGaq 468 F1/LhGaq 1035 R2), LhGĮq3 (LhGaq 474 F2/LhGaq 1036 R1), and LhGĮq4 (LhGaq 474 F2/LhGaq 1035 R2). PCR products were electrophoresed on 1.5% agarose gels and representative amplimers of the expected sizes were sub-cloned and sequence verified.

Immunocytochemical Localization of L. hesperus GĮ in Cultured Insect Cells
To examine the intracellular localization of select LhGĮ subunits, the respective coding sequences lacking endogenous stop codons were amplified from plasmid DNAs using KOD HotStart DNA polymerase (Toyobo/Novagen, EMD Biosciences, San Diego, CA, USA) and sub-cloned into a pIB/V5-His TOPO TA expression vector (Life Technologies) upstream of the plasmid-derived epitope tag such that the translated LhGĮ subunits contain a carboxyl terminal 6×-His tag. All resulting expression plasmids were sequence verified. Adherent Trichoplusia ni (Tni) cells (Orbigen Inc., San Diego, CA, USA) attached to 35-mm #1.5 glass bottom dishes (In Vitro Scientific, Sunnyvale, CA, USA) were transfected with 2 ȝg plasmid DNA using Insect Gene Juice transfection reagent (Novagen) for 5 h. Transfected cells were maintained in serum-free media for 48 h at 28 °C and then fixed for 15 min at 4 °C with 3.5% formalin/IPL-41. The cells were blocked and permeabilized for 1 h at 25 °C in PBS/10% fetal bovine serum/0.1% Triton X-100. The cells were then incubated for 2 h at 25 °C with 1:50 rabbit polyclonal anti-His antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; #SC-804), which recognizes the plasmid-derived His epitope tag. After washing, the cells were incubated with 1:100 goat anti-rabbit IgG-TRITC (Southern Biotechnology; Birmingham, AL, USA; #4030-03) for 2 h at 25 °C. Fluorescent imaging was performed on an Olympus FSX-100 fluorescence microscope with FSX-BSW imaging software (Olympus, Center Valley, PA, USA). Images were processed for publication with Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

Identification of L. hesperus GĮ Sequences
To identify GĮ proteins expressed in L. hesperus (LhGĮ), we initially utilized a homology-based approach with degenerate primers designed to conserved regions of GĮ proteins and both PCR and nested PCR conditions. Sequence analysis indicated amplimers of the expected sizes were partial fragments of proteins homologous with GĮs, GĮi, and GĮq proteins. Further extension of the partial sequences using conventional RACE PCR methods identified putative start and stop codons. Primers designed to those regions facilitated amplification of the respective open reading frames (ORFs). Based on sequence similarities with known GĮ subunits (  [3]. Because the degenerate primers used in the homology-based PCR approach have the potential to bias toward particular classes of GĮ proteins [62,63], we sought to use recently assembled L. hesperus transcriptomes [43,46] to more comprehensively evaluate LhGĮ expression. The respective databases were queried with the LhGĮi, LhGĮs, and LhGĮq sequences as well as GĮ subunits from other insects. All three LhGĮ transcripts are present in the databases with minimal (>99% nt identity) sequence variation. In addition, complete transcripts for GĮo and GĮ12 subunits were identified. The putative LhGĮo ORF encodes a 355 amino acid protein with highest sequence similarity to a GĮo subunit cloned from a migratory locust (Locusta migratoria) head cDNA library [54]. While the putative LhGĮ12 encodes a 368 amino acid protein that has significant sequence identity with genomic sequences annotated simply as GĮ subunit-like proteins (Table 2), it is 63% identical (E value = 3e í154 ) with the D. melanogaster GĮ12 homolog, concertina [50]. To confirm correct assembly of the transcriptomic data, the complete coding regions for both LhGĮo and LhGĮ12 were amplified from L. hesperus cDNAs in multiple independent reactions and sequenced. As before, the cloned sequences exhibited >99% nt sequence identity with the transcriptomic sequences.  Multiple sequence variants have been reported for GĮ subunits [20,48,51,52,69,70] with variants/isoforms also predicted in many insect genomes. Furthermore, high throughput sequencing methods, such as those used to construct the L. hesperus transcriptome databases, offer the possibility of identifying low representation and/or unique transcripts [71][72][73]. Consistent with previous findings, our transcriptome database search identified three additional LhGĮq variants, which we have designated LhGĮq2-4. Sequence identity among the four subunits varies from 89%-96% with all four variants identical through Leu155, at which point identity is maintained between LhGĮq1/2 and LhGĮq3/4 up to Pro292, with identical residues then shared between LhGĮq1/3 and LhGĮq2/4 throughout the rest of the protein (Figure 2A). This variation is consistent with the alternative exon splicing described in A. gambiae [20] and D. melanogaster [70] and is present in a number of species from disparate orders, suggesting that the putative splice sites have been evolutionarily conserved. While characterizing the respective LhGĮq variants, we cloned a partial sequence corresponding to a fifth variant (LhGĮq5) that is not represented in either of the transcriptomic databases and which lacks residues 291-326 ( Figure 2B). While this variant is also present in A. gambiae (AAW50316) and Diaphorina citri (XP_008479779) ( Figure 2B), we were unable to identify it from other insects, which suggests that the splice site is either not conserved or that it is a cryptic site [73]. The GĮq locus in A. gambiae spans 11 exons, three of which (identified as D/D*, G/G*, and H/H*) are homologous and undergo alternative splicing [20]. While the genomic structure of the L. hesperus GĮq locus has not been determined, we can surmise based on the transcript sequences that similar alternative splicing likely generates the five variants ( Figure 2C).
In their characterization of GĮ in A. gambiae, Rützler et al. [20] identified a sixth GĮq variant (AAW50317) characterized by inclusion of a 43 amino acid insertion that corresponds to two of the exons alternatively spliced in the other variants. Although this variant is a predicted product in a number of insect genomes, it was considered to be a premature transcript as inclusion of the second exon could potentially disrupt the catalytic pocket of the GTP hydrolysis domain. We were unable to detect this variant during characterization of the other LhGĮq subunits nor was it represented in the L. hesperus transcriptomes [43,46]. No other LhGĮ sequence variants were identified. The respective transcriptomes, however, may underrepresent the number of GĮ transcripts actively expressed in L. hesperus due to the exclusion of temporally or spatially restricted transcripts.
The conserved guanine nucleotide binding/hydrolysis motifs characteristic of GĮ subunits are present in the predicted LhGĮ proteins (Figure 4) including sequences critical for diphosphate binding (GXGESGKS), Mg 2+ binding (RXXTXGI and DXXG), and guanine ring-binding (NKXD and TCAT) [3]. Deviations from the canonical sequences, however, are present in the TCAT motif in LhGĮs (TCAV), LhGĮ12 (TTAV), and LhGĮq2/4 (TTAT). These deviations are not specific to the L. hesperus sequences as all of the GĮs and GĮ12 sequences used in the phylogenetic analysis had the same sequence changes and numerous GĮq sequences (e.g., NP_001128385, B. mori; ACJ06653, Spodoptera frugiperda; CAB76453, Calliphora vicina; XP_005180085, Musca domestica; XP_004526037, Ceratitis capitata) have a TTAT motif. Mutations to the TCAT motif in mammalian GĮ subunits mimic an activated receptor by enhancing GDP release [74,75]. Thus, activation of insect GĮ subunits with the modified TCAT motif may proceed more readily, which could account for the observed heterogeneity in receptor-G protein interactions and promiscuous activation of multiple GĮ subunits by some receptors [1]. Further analysis of the LhGĮ sequences indicated the presence of conserved modification sites for fatty acids and toxin-driven ADP-ribosylation ( Figure 4). Palmitoylation of GĮ amino terminal Cys residues and/or myristoylation of amino terminal Gly residues in GĮi/o subunits can influence cellular localization/membrane targeting, interactions with downstream effector proteins, and secondary structure [2,3,76]. ADP-ribosylation of a carboxyl terminal Cys by pertussis toxin uncouples GĮi/o subunits whereas similar modification of an internal Arg in GĮs subunits by cholera toxin abolishes GTP hydrolysis activity and leads to constitutive GĮs activation [1,3].
The last five residues of the GĮ carboxyl terminus are critical for receptor interactions, with minor modifications of this region altering receptor specificity and ADP-ribosylation uncoupling GĮi/o subunits from the respective receptor [2]. The identical carboxyl terminal ends shared by LhGĮq1/3 and LhGĮq2/4 raises questions regarding potentially overlapping functional roles. One possibility is that the respective subunits exhibit different expression profiles (see below), which would limit functional redundancy. A second possibility is that the sequence variations that differentiate the respective LhGĮq subunits also function to stabilize receptor interactions. Thus, despite identical carboxyl terminal ends the LhGĮq subunits interact with the receptors differently. Consequently, despite the critical role the carboxyl terminus plays, functional specificity is driven by the summation of receptor contact points.

End Point PCR-Based Transcriptional Expression Profiling
The tissue and/or developmental specificity of transcript expression can provide insights into gene functionality. To begin to assess the potential functional role of the LhGĮ subunits, we examined their transcriptional expression as ~500-600 bp fragments across L. hesperus development, from eggs through 5th instars and in 1-day-old, 10-day-old, and 20-day-old adults ( Figure 5A). While most LhGĮ subunits were ubiquitously expressed in all stages examined, the expression of LhGĮq2 and LhGĮq4 was more restrictive. The LhGĮq4 product was absent in eggs but was detected throughout nymphal development and in adults ( Figure 5A). Even though LhGĮq2 and LhGĮq4 share identical carboxyl terminal ends (see above), no amplimers were detected for LhGĮq2, suggesting little functional redundancy with respect to receptor specificity between the two variants. Despite overlap with the LhGĮq4 primer set (as demonstrated by the serendipitous cloning of LhGĮq5 while verifying the LhGĮq4 sequence), no LhGĮq5 amplimers, which would migrate as a lower molecular weight product (i.e., 476 bp vs. 584 bp for LhGĮq4) were detected, suggesting low transcript levels for this variant.
We also examined the expression profile of the LhGĮ subunit fragments in sex-specific adult tissues ( Figure 5B). A majority of the LhGĮ transcripts were amplified from all of the tissue sets from both sexes, albeit to varying degrees. Similarly wide tissue distribution profiles for GĮ subunits have been reported in B. mori [19], A. gambiae [20], D. melanogaster [21], B. tabaci [57], and L. oryzophilus [55] and likely reflect the critical role of G proteins in mediating the diverse signal transduction cascades that drive cellular processes. LhGĮq4 was the lone LhGĮ transcript to exhibit tissue specific expression with amplification limited to head-derived cDNAs ( Figure 5B). LhGĮq4 shares significant sequence identity with D. melanogaster GĮq1 (i.e., Gq-RD), the GĮ subunit involved in phototransduction [77,78], and the presumptive A. gambiae ortholog, Agq1 [20]. All three are derived from analogous alternative splice sites and are specifically expressed in adult heads and pre-adult stages with no detectable embryonic expression [20,21,77]. These similarities suggest that functionality may also be conserved, with LhGĮq4 likewise mediating phototransduction. This, however, remains to be experimentally verified. No amplimers corresponding to LhGĮq2 were detected in any of the tissues examined, which is consistent with the developmental expression profile ( Figure 5A). With the exception of LhGĮq2 and LhGĮq4, all of the LhGĮ subunits were amplified to varying degrees from chemosensory tissues (antenna, proboscis, and leg) indicating the absence of a chemosensory specific subunit ( Figure 5B). While variation in amplification across the chemosensory tissues was observed for LhGĮo (highest in antennae) and LhGĮq1 (highest in leg), more accurate determinations (e.g., quantitative real time-PCR) of transcript abundance are required to draw definitive conclusions regarding expression. GĮs has been reported to be more highly expressed in antennae than other GĮ subunits in A. gambiae, D. melanogaster, and B. mori [19][20][21]. The expression of GĮs in olfactory neurons coupled with abnormal olfactory behavior following disruption of the GĮs signal transduction cascade [25] has led some to postulate that GĮs functions in olfaction. However, elevated levels of GĮo and GĮq transcripts have been reported in antennae and olfactory neurons of a number of insects [17][18][19]54,55]. Furthermore, similar to the GĮs pathway, downstream effectors of GĮq such as Ca 2+ /calmodulin can also activate adenylyl cyclase [79] and RNAi-mediated knockdown of GĮq likewise reduces antennal responses [29]. In contrast, other studies have suggested that GĮ proteins have little role in insect olfaction [35]. Given the conflicting conclusions drawn by disparate groups and the critical role of GĮ proteins in normal cellular function, it is becoming increasingly clear that simple co-localization of GĮ transcripts within chemosensory tissues, while correlational, is not indicative in and of itself of an olfactory function.

Intracellular Localization of Transiently Expressed LhGĮ Subunits
Post-translational lipid modifications (i.e., myristolation/palmitoylation) facilitate targeting and subsequent anchoring of GĮ subunits to the inner surface of the plasma membrane [76,80]. To further characterize and confirm the sequence validity of the cloned LhGĮ transcripts, we sought to examine the intracellular localization of a subset of the LhGĮ proteins (LhGĮq1, LhGĮs, and LhGĮi) following transient expression in cultured insect cells. To facilitate detection, expression vectors were constructed in which a 6×-His tag was incorporated in frame with the carboxyl terminal ends of the respective LhGĮ sequences. Immunofluorescence analyses were performed in cultured Trichoplusia ni cells 48 h after transfection using a polyclonal anti-His antibody in conjunction with a TRITC-tagged anti-rabbit antibody. No fluorescence was observed in non-transfected cells ( Figure 6). In contrast, plasma membrane-associated fluorescence was clearly observed in cells transfected with the respective LhGĮ-His constructs ( Figure 6). These results are consistent with previous findings [12] and indicate that intracellular trafficking of the cloned LhGĮ sequences is as expected. In addition to the clear plasma membrane-associated signal, we also observed a diffuse red fluorescent signal throughout the cytosol of cells transfected with the respective LhGĮ subunits. The current model of G protein trafficking suggests that interactions between GĮ subunits and GȕȖ subunits are crucial for plasma membrane localization. Consequently, overexpression of one subunit (e.g., GĮ subunits) may disrupt the necessary stoichiometry and lead to inefficient localization [76]. Thus, the intracellular signal we observed might be "free" GĮ subunits that lack the apparent GȕȖ binding partners that facilitate plasma membrane localization. Alternatively, the signal may represent the normal trafficking profile of GĮ subunits as both cell membrane and discrete cytosolic localization for GĮ subunits have been reported in both native tissue and cell culture [81][82][83].

Conclusions
As part of our continuing efforts to further elucidate molecular mechanisms driving signal transduction in L. hesperus, we identified nine GĮ subunits. Expression analyses and sequence similarities strongly suggest that LhGĮq4 is orthologous to D. melanogaster Gq-RD, which functions in phototransduction. While the presence of multiple LhGĮ transcripts in chemosensory tissues is consistent with potential roles in olfaction and/or gustation, localization at the tissue level alone does not imply function in chemosensory-based signal transduction. To address that issue, the actual role of each of the LhGĮ subunits and variants in chemosensory functionality must be established, including demonstration of specific expression of LhGĮ within olfactory/gustatory receptor neurons and in vivo functional studies examining the biological effects of GĮ mutations, GĮ knockdown, and/or GĮ overexpression. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.