Adult A. aegypti (Liverpool) were derived from an established laboratory-reared colony raised under conditions described previously (17).

Total RNA was isolated and purified from select A. aegypti tissues and organs, reverse transcribed into cDNA. GPA2 and GPB5 transcript abundance was quantified using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) (see SI Materials and Methods for details) and transcript abundance was normalized to transcript levels of two reference genes; ribosomal protein 49 (GenBank accession: AY539746) and 60S ribosomal protein S18 (GenBank accession: XM_001660270), following the ΔΔCt method as previously described (13). Experiments were repeated using a total of three technical replicates per sample and three biological replicates for each tissue/organ.

Using gene-specific primers (Table S1), A. aegypti GPA2 and GPB5 sequences were amplified from previously prepared constructs (13) that contained the GPA2 and GPB5 complete open reading frames (ORFs). Sense and antisense probes were then generated following a similar protocol as recently reported (19). Briefly, cDNA fragments were ligated to pGEM T Easy vector (Promega, Madison, WI, USA) and used to transform NEB 5-α competent Escherichia coli cells (New England Biolabs, Whitby, ON, Canada). After screening plasmid constructs for directionality using T7 promoter oligonucleotide and gene-specific primers (Table S1), template sense or anti-sense cDNA strands for GPA2 and GPB5 probe synthesis were created by PCR amplification and verified by Sanger sequencing for base accuracy (The Centre for Applied Genomics, Sick Kids Hospital, Toronto, ON, Canada). Digoxigenin (DIG)-labeled anti-sense and sense RNA probes corresponding to GPA2 and GPB5 subunits were synthesized using the HiScribe T7 High Yield RNA Synthesis kit (New England Biolabs, Whitby, ON, Canada). Fluorescence in situ hybridization (FISH) was then used to detect GPA2 and/or GPB5 transcript in the mosquito central nervous system using 4 ng μl⁻¹ (GPB5) and/or 6 ng μl⁻¹ (GPA2) RNA sense/antisense probes, following a previously established protocol (30). Preparations were analyzed with a Lumen Dynamics X-Cite™ 120Q Nikon fluorescence microscope (Nikon, Mississauga, ON, Canada), or a Yokogowa CSU-XI Zeiss Cell Observer Spinning Disk confocal microscope, and images were processed using Zeiss Zen and ImageJ software. All microscope settings were kept identical when acquiring images of control and experimental preparations.

GPB5 immunoreactivity in the abdominal ganglia of adult mosquitoes was examined in newly emerged and four-day old A. aegypti that were lightly CO₂ anesthetized, and dissected in PBS at RT. Tissues were fixed, permeabilized and incubated in a custom affinity-purified rabbit polyclonal GPB5 antibody (1 μg ml⁻¹; Genscript, Piscataway, NJ) designed against an antigen sequence (CDSNEISDWRFP) positioned at residues 69–80 of the deduced A. aegypti GPB5 protein sequence (13) for 48 h at 4°C rocking. Control treatments involved pre-incubated A. aegypti GPB5 primary antibody solution containing 100:1 peptide antigen:antibody (mol:mol). After several washes, tissues were incubated overnight at 4°C in Alexa Fluor 488-conjugated goat anti-rabbit Ab (1:200) secondary antibody (Life Technologies, Carlsbad, CA) in PBS containing 10% normal sheep serum. The next day, samples were washed and then mounted onto coverslips using mounting media containing 5 μg μl⁻¹ Diamidino-2-phenylindole dihydrochloride (DAPI), and analyzed using a Lumen Dynamics X-Cite™ 120Q Nikon fluorescence microscope (Nikon, Mississauga, ON, Canada), or optically sectioned using a Yokogowa CSU-XI Zeiss Cell Observer Spinning Disk confocal microscope. All images were processed using Zeiss Zen and ImageJ software. Further details concerning the wholemount immunohistochemical protocol were reported in an earlier study (17).

Plasmid expression constructs were designed to study A. aegypti and H. sapiens GPA2/GPB5 subunit dimerization patterns and receptor signaling. Using previously available hexa-histidine-tagged A. aegypti GPA2-His (13), as well as FLAG (DYKDDDDK)-tagged (-FLAG) H. sapiens GPB5-FLAG (Genscript, Clone OHu31847D) plasmid vectors as template, the full ORF of each (A. aegypti and H. sapiens) GPA2 and GPB5 subunit coding sequence, including a consensus Kozak translation initiation sequence, was amplified and a hexa-histidine or FLAG tag sequence was incorporated on the carboxyl-terminus of subunits to produce the following fusion proteins; A. aegypti GPA2-FLAG, H. sapiens GPB5-His (Table S2). A pcDNA3.1⁺ mammalian expression construct containing mCherry, which was a gift from Scott Gradia (Addgene plasmid # 30125), was utilized to verify cell transfection efficiency. Experiments also utilized previously prepared pcDNA3.1⁺ constructs with A. aegypti GPB5-His and A. aegypti LGR1 coding sequences and dual promoter vector pBudCE4.1 containing both A. aegypti GPA2-His and GPB5-His (13). Additionally, pcDNA 3.1⁺ mammalian expression vector construct containing FLAG tagged H. sapiens thyrotropin receptor (TSHR-FLAG) (Genscript USA Inc., Clone OHu18318D), H. sapiens GPA2-FLAG (Genscript USA Inc., Clone OHu31847D), H. sapiens GPB5-FLAG (Genscript USA Inc., Clone OHu55827D) and pGlosensor™−22F cyclic adenosine monophosphate (cAMP) biosensor plasmid (Promega Corp., Madison, WI), which were used for receptor activation and intracellular signaling assays.

The ORFs of A. aegypti GPA2 and GPB5 sequences were tethered together in order to promote heterodimer interactions for testing in receptor activity assays with mammalian cell lines. A hexa-histidine tagged artificial linker sequence involving three glycine-serine repeats was used to fuse the amino-terminus of A. aegypti GPA2 propeptide sequence to the carboxyl-terminus of A. aegypti GPB5 prepropeptide sequence, using multiple PCR amplifications with several primer sets (Table S2) as performed previously using lamprey and Amphioxus GPA2 and GPB5 sequences (31, 32) (see SI Materials and Methods for details).

Human embryonic kidney (HEK 293T) cells were grown in complete growth media (Dulbecco's modified eagles medium: nutrient F12 (DMEM) media, 10% heat inactivated fetal bovine serum (Wisent, St. Bruno, QC) and 1X antimycotic-antibiotic) and maintained in a water-jacketed incubator at 37°C, 5% CO₂. When cells reached ~80–90% confluency, they were transfected with mammalian expression plasmid constructs in 6-well tissue culture plates (Thermo Fisher Scientific, Burlington, ON) using Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA) with 3:1 (μL:μg) transfection reagent to DNA ratio. Before transfection, culture media was replaced with either serum-free medium (DMEM and 1X antimycotic-antibiotic) for experiments that collected secreted proteins, or fresh complete growth medium for experiments that dually-transfected cells with pGlosensor™−22F cAMP biosensor plasmid and either H. sapiens TSHR, A. aegypti LGR1, or mCherry.

At 48 h post-transfection, serum-free culture media containing secreted proteins were collected and concentrated in 0.5 mL 3-kDa molecular weight cut-off centrifugal filters (VWR North America). In some experiments, cells were dislodged with PBS containing 5 mM ethylenediaminetetraacetic acid (EDTA; Life Technologies) (PBS-EDTA) pH 8.0, pelleted at 400 × g for 5 min, resuspended in PBS and transferred to 1.5 mL centrifuge tubes for a subsequent centrifugation. Cell lysates were then prepared by resuspending and sonicating cells for 5 s in cell lysis buffer containing 37.5 mM Tris, pH = 7.5, 1.5 mM EDTA, pH 8.0, 3% sodium dodecyl sulfate, 1.5% protease inhibitor cocktail (v/v), and 1.5 mM dithiothreitol (DTT). For receptor activity assays, in order to prevent carry-over of lysis buffer, cell lysates were concentrated in 3-kDa molecular weight cut-off centrifugal filters and re-constituted back to initial volumes with serum-free media for a total of three repetitions. Proteins were then used for cross-linking analysis, deglycosylation, and immunoblotting or used as ligands for functional receptor activation using the cAMP signaling biosensor assays.

Dissuccinimidyl suberate (DSS), a chemical cross-linker that is primarily reactive toward amino groups providing stabilization of weak or transient protein intermolecular interactions, was employed to study GPA2 and GPB5 protein-protein interactions. A. aegypti and H. sapiens GPA2-FLAG and GPB5-His proteins were kept separate or combined together (GPA2-FLAG/GPB5-His) and then treated with DSS (Sigma Aldrich, Oakville, ON). In some experiments, A. aegypti GPA2-His/GPB5-His were co-expressed using a dual promoter plasmid and as such, media containing both His-tagged subunits were directly treated with DSS. To treat secreted concentrates of culture media containing H. sapiens GPA2 and GPB5 proteins, 0.68 mM DSS was used whereas both 0.68 mM (data not shown) and 2.04 mM DSS was used to test the dimerization characteristics of A. aegypti GPA2-His/GPB5-FLAG proteins. Cross-linking was performed for 30 min at RT and reactions were quenched with 50 mM Tris, pH 7.4 for 10 min under constant mixing. To remove N-linked sugars, protein samples were treated with peptide-N-Glycosidase F (PNGase) (New England Biolabs, Whitby, ON) following manufacturers guidelines, with the only modification being that protein samples were not heated to 100°C before enzymatic deglycosylation. Experiments aimed to determine the effects of protein glycosylation on cross-linking ability first treated A. aegypti GPA2-His/GPB5-His subunits with PNGase and subsequently cross-linked samples with DSS after deglycosylation.

Samples were prepared in 2x Laemmli buffer (Sigma Aldrich, Oakville, ON) containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH ~6.8, and resolved on 10 or 15% SDS-polyacrylamide gels under reducing conditions at 120 V for 90–110 min. Using a wet transfer system, proteins were then transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 75 min. For GPA2-FLAG/GPB5-His heterodimerization experiments, samples were run in duplicate on the same gel and following transfer, membranes were cut in half for separate primary antibody incubations. Membrane blots containing protein samples were blocked for 1 h in PBS containing 0.1% Tween-20 (Bioshop, Burlington, ON, Canada) and 5% skim milk powder (PBSTB) rocking at RT. After blocking, membranes were incubated overnight at 4°C on a rocking platform in PBSTB containing mouse monoclonal anti-His (1:500 dilution) or mouse monoclonal anti-FLAG (1:500) primary antibody solutions. The next day, membranes were washed three times with PBS containing 0.1% Tween-20 (PBST) for 15 min each wash and then were incubated in PBSTB containing anti-mouse HRP conjugated secondary antibody (1:2000–1:3000 dilution) for 1 h rocking at RT before washing again with PBST (3 × 15 min washes). Finally, blots were incubated with Clarity Western ECL substrate and images were developed using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Mississauga, ON) and molecular weight measurements and analysis were performed using Image Lab 5.0 software (Bio-Rad Laboratories, Mississauga, ON). Western blots used to study recombinant GPA2 and GPB5 dimerization were repeated at least three times.

HEK 293T cells were co-transfected to express (i) H. sapiens TSHR, A. aegypti LGR1, or mCherry along with (ii) pGlosensor™−22F cAMP biosensor plasmid (Promega Corp., Madison, WI), which encodes a modified form of firefly luciferase with a fused cAMP binding moiety providing a biosensor for the direct detection of cAMP signaling in live cells. At 48 h post transfection, recombinant cells were dislodged with PBS-EDTA, pelleted at 400 × g for 5 min, resuspended in assay media [DMEM:F12 media with 10% fetal bovine serum (v/v)] containing cAMP GloSensor reagent (2% v/v), and incubated for 3 h rocking at RT shielded from light. White 96-well luminescence plates (Greiner Bio-One, Germany) were loaded under low light with previously prepared secreted or cell lysate protein concentrates (described above), forskolin or assay media alone, and incubated at 37°C for 30 min prior to performing receptor activity assays. For stimulatory G-protein (Gs) pathway detection, recombinant cells expressing either H. sapiens TSHR, A. aegypti LGR1, or mCherry along with the cAMP biosensor were pre-treated with 0.25 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min at RT with rocking and shielded from light. Using an automatic injector unit (BioTek Instruments Inc., Winooski VT), co-transfected HEK 293T cells were then loaded into wells containing various treatments, including 250 nM forskolin as a positive control or concentrates of protein fractions from mCherry-transfected cells as a negative control. For inhibitory G-protein (Gi/o) pathway testing, A. aegypti GPA2 and/or GPB5 was tested for the ability to inhibit a forskolin-induced cAMP-mediated bioluminescent response. Using an automatic injector unit (BioTek Instruments Inc., Winooski VT), cells expressing LGR1 or mCherry (LGR1 activation and signaling negative control) were loaded into wells that contained various ligand treatments. Subsequently, 250 nM or 1 μM forskolin was added to wells immediately after the addition of cells, using a second automatic injector unit. For bioluminescent assays with tethered GPA2/GPB5 fusion proteins, recombinant cells expressing or not expressing LGR1 were equally divided to test Gs and Gi signaling pathways simultaneously using the same batch of cells per biological replicate. In all assays, luminescence was measured every 2 min for 20 min at 37°C using a Synergy 2 Multimode Microplate Reader (BioTek, Winooski, VT) and averaged over 4–8 technical replicates for each treatment. To calculate the relative luminescent response, data were normalized to luminescent values recorded from treatments with 250 nM or 1 μM forskolin alone. Assays were performed repeatedly and involved 3–6 independent biological replicates. Relative luminescent values were compiled on Excel and transferred into GraphPad Prism 8.0 for figure preparation and statistical analysis. Data were analyzed using one-way or two-way ANOVA with Tukey's multiple comparison post-test (p < 0.05).

To determine the distribution of GPA2 and GPB5 subunit expression in the central nervous system and peripheral tissues, adult mosquito organs were analyzed using RT-qPCR. GPA2 and GPB5 subunit transcript was detected in the central nervous system of adult male and female mosquitoes, with significantly enriched expression in the abdominal ganglia relative to other regions of the nervous system and peripheral tissues (Figures 1A,B). Fluorescence in situ hybridization was employed to localize cell-specific expression of the GPA2 and GPB5 transcripts in the abdominal ganglia. GPA2 and GPB5 anti-sense RNA probes identified two bilateral pairs of neuroendocrine cells (Figures 1C,D) within each of the first five abdominal ganglia in male and female mosquitoes whereas the control sense probes did not detect cells in the nervous system (Figures 1E,F). Within each of these first five abdominal ganglia, GPA2 transcript localized to similar laterally-positioned cells as the GPB5 transcript (Figures 1G,H). To determine if cells expressing GPA2 transcript were the same cells expressing GPB5 transcript, abdominal ganglia were simultaneously treated with both GPA2 and GPB5 anti-sense RNA probes. Using this dual probe approach, results confirmed the detection of only two bilateral pairs of cells (Figures 1I,J) that displayed a greater staining intensity compared to the intensity of cells detected when using either the GPA2 or GPB5 anti-sense probe alone (Figures 1C,D).

Using a custom antibody targeting A. aegypti GPB5, we next sought to immunolocalize GPB5 protein in the abdominal ganglia. GPB5 immunoreactivity localized to two bilateral pairs of cells (Figure 2A) within the first five ganglia, which were in similar positions to cells expressing GPA2 and GPB5 transcript (Figures 1C,D,G–J). Regardless of sex, in 30% of mosquitoes examined, three instead of two bilateral pairs of cells immunolocalized to each of the first five abdominal ganglia of the ventral nerve cord (Figure 2B), whereas no cells were ever detected in the sixth terminal ganglion (Figure 2C). For a given mosquito, there were no differences in the number of GPB5 immunoreactive cells detected between different ganglia. Along the lateral sides of each of the first five abdominal ganglia, GPB5 immunoreactive processes were observed to closely associate into a tract of axons that emanated through the lateral nerve (Figure 2B). Control treatments with GPB5 antibody preabsorbed with the GPB5 immunogenic antigen did not detect any cells in ganglia (Figure 2D).

A. aegypti GPA2 and GPB5 subunit protein interactions were studied using western blot analysis of recombinant proteins from HEK 293T cells expressing each subunit independently or co-expressing both subunits in the same cells using a dual promoter plasmid. Under control conditions, GPA2 protein is represented as two bands at 16 and 13 kDa, which correspond to the glycosylated and non-glycosylated forms of A. aegypti GPA2, respectively (Figure 3A). Following deglycosylation with PNGase, the higher molecular weight band of GPA2 is eliminated, and the non-glycosylated lower molecular weight band intensifies (Figure 3A). Interestingly, when the GPA2 subunit was tested to examine potential homodimerization, an additional strong band at ~32 kDa was detected, which migrates to ~30 kDa when cross-linked protein samples were deglycosylated using PNGase (Figure 3A). Under control conditions, GPB5 protein is represented as a band size at 24 kDa and the migration pattern is not affected by PNGase treatment (Figure 3B). Following treatment with DSS, a faint second band appears at 48 kDa, which does not change in molecular weight after treatment with PNGase (Figure 3B). Three independent band sizes at 24 kDa (GPB5), 16 kDa (glycosylated GPA2) and 13 kDa (non-glycosylated GPA2) were detected in lanes loaded with protein isolated from HEK 293T cells co-expressing GPA2 and GPB5 subunits (Figure 3C). After removal of N-linked sugars, the 24 kDa band is not affected but the 16 kDa band disappears and 13 kDa band intensifies (Figure 3C), as observed when assessing the GPA2 subunit independently (Figure 3A). Cross-linked samples show the addition of two higher molecular weight bands at ~48 and ~32 kDa, the latter of which migrates lower to ~30 kDa when subjected to PNGase treatment (Figure 3C). Given the resolution and intensity of the detected bands, the molecular weight band at ~45 kDa could indicate GPA2/ GPB5 heterodimeric interactions (13 or 16 kDa + 24 kDa = 37–40 kDa). Alternatively, these bands could reflect GPB5 (24 + 24kDa = 48 kDa) homodimers. As a result of this uncertainty, additional experiments were performed to clarify whether A. aegypti GPA2 and GPB5 subunits are heterodimeric partners.

Using yeast two-hybrid analyses and cross-linking experiments, it was previously shown that human GPA2 (hGPA2) and GPB5 (hGPB5) subunits are capable of heterodimerization (4). As a result, to verify whether A. aegypti GPA2 and GPB5 subunit proteins are heterodimeric candidates, experiments were performed alongside hGPA2/hGPB5 subunit proteins, using the latter as a positive experimental control for heterodimer detection.

Initially, single-promoter expression constructs were designed to incorporate a FLAG-tag and His-tag on the C-terminus of (human and mosquito) GPA2 and GPB5 subunits, respectively (i.e., GPA2-FLAG and GPB5-His). When probed with an anti-His antibody, no bands were detected in lanes containing only GPA2-FLAG (human and mosquito) protein (Figures 4A,B). Similarly, when an anti-FLAG antibody, no bands were detected in lanes containing only GPB5-His protein (human and mosquito) (Figures 4A,B).

Lanes loaded with cross-linked hGPB5-His protein revealed two bands at 18 kDa (monomer) and 36 kDa (homodimer) (Figure 4A). Similarly, in lanes loaded with cross-linked hGPA2-FLAG protein, two bands at 20 kDa (monomer) and 40 kDa (homodimer) were detected (Figure 4A). To determine subunit heterodimerization, hGPA2-FLAG protein was combined with hGPB5-His protein (i.e., produced separately in different cell batches) and subsequently crosslinked using DSS. Results showed an intense 38 kDa band size that correlated to the molecular weight of the hGPA2-FLAG/hGPB5-His heterodimers; detected with both anti-His and anti-FLAG primary antibody solutions (Figure 4A).

Identical experiments were conducted using A. aegypti GPA2-FLAG/GPB5-His protein but using three-fold higher concentration of DSS cross-linker to help improve detection of inter-subunit interactions. Results demonstrated that lanes loaded with DSS-treated GPB5-His protein resulted in a 24 kDa monomer band (Figure 4B), whereas lanes containing cross-linked GPA2-FLAG detected a 16 kDa (glycosylated monomer), 30 and 32 kDa (homodimers) band size (Figure 4B). As a result, unlike immunoblots containing hGPA2-FLAG and hGPB5-His protein (Figure 4A), lanes loaded with cross-linked A. aegypti GPA2-FLAG and GPB5-His failed to provide evidence of bands correlating to the predicted molecular weight of an A. aegypti GPA2/GPB5 heterodimer, which would be expected at 37–40 kDa (Figure 4B).

Bioluminescent assays were employed to confirm LGR1 interaction with mosquito GPA2 and/or GPB5 subunits and elucidate downstream signaling pathways upon receptor activation. Since human GPA2/GPB5 (hGPA2/hGPB5) has previously been shown to bind and activate human thyrotropin receptor (hTSHR) mediating a stimulatory G protein (Gs) signaling pathway (4), we first validated our experimental design using hGPA2/hGPB5 and hTSHR as a positive control. Recombinant hGPA2 and hGPB5 subunit proteins were produced in HEK 293T cells with single promoter expression constructs containing the hGPA2 or hGPB5 sequences. Conditioned culture media containing secreted proteins were subsequently concentrated, and crude extracts containing hGPA2, hGPB5 or a combination of hGPA2 and hGPB5 subunits were tested as ligands on HEK 293T cells expressing the hTSHR and a cAMP-sensitive firefly luciferase biosensor, which produces bioluminescence upon interacting with cAMP. Control treatments involved the incubation of hTSHR/ luciferase-expressing cells with concentrated media collected from mCherry-transfected cells (negative control), and data was normalized to treatments with 250 nM forskolin (positive control) (Figures 5A,B). As expected, the results indicate that incubation with extracts containing a combination of both hGPA2 and hGPB5 were required to stimulate a cAMP-mediated luminescent response from hTSHR-expressing cells, but not when incubated with extracts containing individual subunits (Figure 5A). We next performed identical experiments using A. aegypti GPA2/GPB5 and LGR1. Given the availability of a dual promoter plasmid (pBudCE4.1), an additional treatment was performed whereby GPA2/GPB5 subunits were co-expressed within the same cells and conditioned media was concentrated as described above. Unlike the results using hGPA2/hGPB5 subunit homologs on HEK 293T cells expressing the hTSHR (Figure 5A), no combination of mosquito GPA2 and/or GPB5 subunit proteins led to an increase in the cAMP-based luminescent response in HEK 293T cells expressing mosquito LGR1 (Figure 5B).

Using an in silico analysis to predict coupling specificity of A. aegypti LGR1 and human TSHR to different families of G-proteins (33), we determined that A. aegypti LGR1 is strongly predicted to couple to inhibitory (Gi/o) G proteins Table S3). As a result, to determine whether A. aegypti GPA2 and/or GPB5 activate a Gi/o signaling pathway, various combinations of GPA2 and GPB5 were tested for their ability to inhibit a 250 nM forskolin-induced rise in cAMP measured by changes in bioluminescence (Figures 5C,D). Results revealed that sole treatments of GPB5 proteins alone significantly inhibited a forskolin-induced luminescent response, relative to control treatments with mCherry proteins, when incubated with cells expressing LGR1 (Figure 5C). However, similar inhibitory effects of GPA2 and GPB5 proteins were also observed with cells lacking LGR1 expression (Figure 5D).

Activity on the hTSHR was only observed when both hGPA2 and hGPB5 subunits were coapplied for receptor activation (Figure 5A) and, unlike the heterodimerization of hGPA2/hGPB5 observed in our experiments, mosquito GPA2/GPB5 lacked evidence of heterodimerization (Figure 4). In light of these observations, we hypothesized that the activation of A. aegypti LGR1 also required subunit heterodimerization. To mimic GPA2/GPB5 heterodimers using the heterologous expression system, both GPA2 and GPB5 mosquito subunits were expressed as a tethered, single-chain polypeptide by fusing the C-terminus of the GPB5 prepropeptide sequence with the N-terminus of the GPA2 propeptide sequence, using a tagged linker sequence composed of twelve amino acids, involving three glycine-serine repeats and six histidine residues.

HEK 293T cells were transfected to transiently express a single promoter plasmid construct containing the tethered GPA2/GPB5 sequence, or the red fluorescent protein (mCherry) as a negative control. To verify expression of the construct, at 48 h post-transfection, cell lysates along with the conditioned culture media, the latter of which contains secreted proteins, were collected for immunoblot analysis. No bands were detected in lanes containing cell lysate or secreted protein fractions of mCherry transfected cells (Figure 6A). However, in the lysates of cells transfected to express tethered GPA2/GPB5, an intense band at 32 kDa, and less intense band at 37 kDa were detected; the latter of which corresponds to the predicted molecular weight of non-glycosylated GPA2 (13 kDa) plus GPB5 (24 kDa) (Figure 6A). In lanes containing secreted fractions of tethered GPA2/GPB5- transfected cells, a band at 37 kDa was again detected, as well as a stronger 40 kDa band size that correlates to the predicted molecular weight of glycosylated GPA2 (16 kDa) plus GPB5 (24 kDa) (Figures 6A,B). After secreted protein extracts containing tethered GPA2/GPB5 proteins were treated with PNGase, the higher 40 kDa molecular weight band disappears and the 37 kDa band size intensifies, which confirms the observed molecular weight shift results from removal of N-linked oligosaccharides (Figure 6B). Thus, the tethered GPA2/GPB5 undergoes similar post-translational processing as observed for the subunits expressed individually.

The activity of tethered GPA2/GPB5 proteins on LGR1 activation was examined. Cell lysate or secreted protein fractions collected from mCherry- or tethered GPA2/GPB5-transfected cells were incubated with HEK 293T cells co-expressing the cAMP luciferase biosensor and either A. aegypti LGR1 or mCherry (i.e., not expressing LGR1). Whether tethered GPA2/GPB5 proteins (cell lysate or secreted fractions) could elevate cAMP or inhibit a forskolin-induced rise in cAMP was assessed and compared to negative control treatments with proteins harvested from mCherry-transfected cells.

Without the addition of tethered GPA2/GPB5 proteins, the basal luminescent levels were higher in LGR1-transfected cells compared to mCherry-transfected cells (Figure 7), suggesting constitutive activity of A. aegypti LGR1 elevating cAMP levels. However, the application of neither secreted protein fractions nor cell lysates of tethered GPA2/GPB5-transfected cells elicited an increase in the cAMP-mediated luminescent response relative to control treatments with mCherry proteins (Figures 7A,B). Secreted protein fractions containing tethered GPA2/GPB5 protein had no effect on the forskolin-induced cAMP-dependent luminescence, compared to control treatments with mCherry secreted proteins in LGR1-expressing cells (Figure 7C). Notably, however, treatments of LGR1-expressing cells with cell lysates containing tethered GPA2/GPB5 significantly inhibited the luminescent response owing to the forskolin-induced rise in cAMP relative to treatments with mCherry-transfected cell lysates, which was not observed in cells lacking LGR1 expression (Figure 7D).

The central nervous system (CNS) of adult mosquitoes is comprised of a brain and a ventral nerve cord, consisting of the suboesophageal ganglion, three fused thoracic ganglia and six abdominal ganglia. GPA2 and GPB5 transcripts are significantly enriched in the abdominal ganglia of adult mosquitoes relative to peripheral tissues and other regions of the CNS. Although low levels of GPA2 and GPB5 transcripts were detected in the thoracic ganglia and brain using RT-qPCR, fluorescence in situ hybridization (FISH) techniques used to localize GPA2 and GPB5 transcripts, along with immunohistochemical detection of GPB5, did not identify specific cells in these regions of the nervous system. Instead, GPA2 and GPB5 transcripts, as well as GPB5 immunoreactivity was identified in 2–3 laterally-localized bilateral pairs of neuroendocrine cells within the first five abdominal ganglia. These findings are consistent with previous findings in the fruit fly D. melanogaster, where GPA2 and GPB5 subunit transcripts were localized to four bilateral pairs of neuroendocrine cells within the abdominal neuromeres in the fused ventral nerve cord, that were distinct from cells expressing other neuropeptides including leucokinin, bursicon, crustacean cardioactive peptide or calcitonin-like diuretic hormone (18).

Both FISH and immunohistochemical techniques revealed GPA2 and GPB5 expression within two bilateral pairs of neuroendocrine cells, that were positioned slightly posterior to where the lateral nerves emanate from these ganglia. It is possible that GPB5 immunoreactive axons follow similar projection patterns as ovary ecdysteroidogenic hormone (OEH I), a gonadotropin produced in lateral neurosecretory cells in the abdominal ganglia of mosquitoes (34). OEH I positive cells emanate through the lateral nerves, similar to our findings with GPB5 immunoreactive cells, and terminate on perivisceral organs that function as neurohaemal release sites (34).

In some abdominal ganglia preparations, a third bilateral pair of cells immunoreactive for GPB5 protein was detected; however these additional cells were not detected using FISH, which suggests GPB5 transcript may be differentially regulated between different bilateral pairs of cells. Given that the same number of cells were observed to express GPA2 transcript, and these cells localized to similar positions as GPB5 expressing cells, the glycoprotein hormone subunits are likely co-expressed in the same cells. To investigate cellular co-expression of GPA2/GPB5, abdominal ganglia were simultaneously treated with both GPA2- and GPB5-targeted anti-sense RNA probes. From this analysis, again only two bilateral pairs of cells were detected and these were more intensly stained compared to preparations treated with either probe alone, which confirms that GPA2 and GPB5 are indeed co-expressed within the same neurosecretory cells of the first five abdominal ganglia in adult mosquitoes. The cellular co-expression of GPA2 and GPB5 proteins implies that, upon a given stimulus, both subunits are regulated in a similar manner and are likely simultaneously released following the appropriate stimulus. Importantly, since co-expression and heterodimerization of the classic vertebrate glycoprotein hormone subunits takes place within the same cells (21, 22), these findings indicate that the mosquito GPA2/GPB5 subunits may be produced and released as heterodimers in vivo.

To study the interactions of A. aegypti GPA2 and GPB5 subunits in vitro, hexa-histidine tagged proteins secreted into the culture media of transfected HEK 293T cells were collected and analyzed under denaturing conditions after cross-linking treatments, which had been utilized previously to show GPA2/GPB5 heterodimerization in other organisms (4, 6, 29). Cross-linked protein samples were then deglycosylated to identify whether the removal of N-linked sugars affected dimerization. Treatment of mosquito GPA2 and GPB5 subunits individually with cross-linker resulted in the detection of bands with sizes corresponding to homodimers of GPA2 (~32 kDa) and GPB5 (48 kDa). GPA2 homodimer bands migrated lower to ~30 kDa following deglycosylation treatment with PNGase. However, experiments performed with cross-linked GPA2/GPB5 protein were not able to confirm heterodimerization since the detected band sizes could also reflect GPB5 homodimeric interactions. Similarly, previous studies that demonstrated GPA2/GPB5 heterodimerization in human (4, 6) and fruit fly (29) did not provide evidence on the interactions of each subunit alone to determine if homodimerization is possible. In these earlier studies, molecular weight band sizes that were identified as heterodimers could have been the result of homodimeric interactions (4, 6, 29). As a result, the findings herein with mosquito GPA2/GPB5 indicate additional experiments are required to confirm GPA2/GPB5 heterodimerization in these organisms.

To clarify whether A. aegypti (mosquito) GPA2/GPB5 heterodimerize, each subunit was differentially tagged (GPA2-FLAG and GPB5-His), and immunoblots containing various combinations of cross-linked subunits were probed with either anti-FLAG or anti-His antibody. As a positive control, experiments were first performed using H. sapiens (human) hGPA2/hGPB5 subunit proteins. Similar to mosquito GPA2 and GPB5 subunits, results showed hGPA2 and hGPB5 subunits are capable of homodimerization. To study heterodimeric interactions, GPA2 and GPB5 subunit proteins were expressed separately in HEK 293T cells. Upon combining and treating protein samples with DSS, a molecular weight band size at 38 kDa, corresponding to the molecular weight of hGPA2/hGPB5 heterodimers (hGPA2-FLAG and hGPB5-His tagged), was detected and migrated differently than bands corresponding to hGPA2 (40 kDa) and hGPB5 (36 kDa) homodimers. Taken together, our results confirm hGPA2 and hGPB5 subunits are indeed capable of heterodimerization in vitro. Further, an induction of cAMP was observed when both subunits were present for TSHR functional activation, whereas treatments with individual subunits failed to significantly increase cAMP-mediated luminescence. As a result, hGPA2/hGPB5 is capable of heterodimerization, and since a combination of both subunits were required to signal a TSHR-mediated elevation in cAMP, these heterodimers are required to functionally activate its cognate glycoprotein hormone receptor.

Analogous experiments using the same concentration of DSS cross-linker (data not shown) as well as three-fold higher concentrations were performed with mosquito GPA2-FLAG and GPB5-His subunit proteins. Irrespective of the DSS concentration used, no bands corresponding to the expected molecular weights of mosquito GPA2/GPB5 heterodimers (37–40 kDa) were observed, but rather, only GPA2 and GPB5 homodimers were detected. Moreover, mosquito GPA2/GPB5, either each subunit alone, mixed from different cell batches or co-expressed in the same cells using a dual promoter vector, was unable to stimulate a cAMP-mediated luminescent response in HEK 293T cells expressing LGR1. Since we identified A. aegypti LGR1 is predicted to couple a Gi/o signaling pathway (Table S3), we examined if mosquito GPA2/GPB5 could inhibit a forskolin-induced cAMP response. Sole treatments of GPA2 and GPB5 subunit proteins inhibited a forskolin-induced rise in cAMP, however these inhibitory actions were not owed to G protein signaling events related to A. aegypti LGR1, since inhibition was observed in control cell lines in the absence of LGR1. These results suggest mosquito GPA2 and GPB5 subunit proteins may non-specifically interact with other endogenously expressed proteins, like the orphan receptors LGR4 and LGR5 that are highly expressed in HEK 293T cells (35).

Activation of the human thyrotropin receptor was only observed when both hGPA2 and hGPB5 subunits were present which, unlike data obtained involving mosquito subunits, demonstrated hGPA2/hGPB5 subunit heterodimerization in vitro using the mammalian heterologous system. The inability of mosquito GPA2 and GPB5 subunits to successfully heterodimerize could result from improper protein folding of insect-derived secretory proteins in HEK 293T cells used for heterologous expression. As a result, future experiments should test the expression and heterodimerization of A. aegypti GPA2/GPB5 in other heterologous systems such as insect cell lines, which could provide a more appropriate environment for tertiary and quaternary protein structure formation. Nonetheless, given the observed co-localization of the subunits within bilateral pairs of cells in the first five abdominal ganglia in A. aegypti, which is comparable to cellular colocalization shown earlier in D. melanogaster (18), we proposed that A. aegypti GPA2/GPB5 heterodimers would be required to functionally activate LGR1 in vitro. To confirm this possibility, a tethered construct was designed linking the C-terminus of GPB5 to the N-terminus of GPA2 using a histidine tagged glycine/serine-rich linker sequence. Natural and synthetic linkers function as spacers that connect multidomain proteins, and are commonly used to study unstable or weak protein-protein interactions (36). The incorporation of a linker sequence between glycoprotein hormone subunits has been performed previously, and does not affect the assembly, secretion or bioactivity of human FSH (37), TSH (38), and CG (39). The conversion of two independent glycoprotein hormone subunits into a single polypeptide chain using a glycine-serine repeat linker sequence has also been performed recently with lamprey GPA2/GPB5 (31), which was shown to induce a cAMP response. Interestingly, similar proteins involving TSH alpha fused to TSH beta with carboxyl-terminal peptide (CTP) as a linker promoted a three-fold higher induction of cAMP compared to wild-type TSH, likely because the addition of a CTP linker increases protein stability and flexibility (40).

Thus, tethered A. aegypti GPA2/GPB5 was expressed in HEK 293T cells and secreted protein fractions along with cell lystaes were collected for expression studies. In cell lysates, immunoblot studies revealed the detection of two bands at 32 and 37 kDa which correlate to immature (incompletely processed) forms of tethered GPA2/GPB5 proteins and non-glycosylated GPA2 (13 kDa) plus GPB5 (24 kDa), respectively. In secreted fractions, a faint band corresponding to non-glycosylated GPA2/GPB5 as well as an intense band at 40 kDa, glycosylated GPA2 (16 kDa) plus GPB5 (24 kDa), was detected. Moreover, treatments with PNGase verified the higher molecular weight band size in secreted fractions at 40 kDa is glycosylated, as observed for GPA2 expressed independantly in earlier experiments herein and in previous studies (13). Bands in cell lysates indicate the retention of immature and non-glycosylated GPA2/GPB5 heterodimers. For this reason, cell lysates and secreted protein fractions of tethered GPA2/GPB5-expressing cells were separately tested for their ability to activate A. aegypti LGR1. Unlike secreted fractions that were more rich in glycosylated GPA2/GPB5 proteins, cell lysates that lacked glycosylated GPA2/GPB5 were able to activate LGR1, leading to a reduction in cAMP-mediated luminescence. Thus, our results suggest that post-translational modifications like glycosylation may impact ligand-receptor activity. Alternatively, it is possible that a lack of inhibitory activity of secreted fractions could be owed to smaller concentrations of tethered GPA2/GPB5 compared to lysate fractions, given that the amounts of secreted and cell lysate fractions were not equalized. Future experiments should aim to create novel constructs that improve expression and secretion of tethered GPA2/GPB5.

In humans, GPA2/GPB5-TSHR signaling stimulates adenylyl cyclase activity to increase intracellular cAMP via interaction with a Gs protein (4, 6, 11), and these results were confirmed in our studies. Comparatively, GPA2/GPB5 signaling was also shown to increase levels of cAMP upon binding LGR1 in D. melanogaster (29). Interestingly, our experiments demonstrate low level constitutive activity of adenylyl cyclase in LGR1 expressing cells since cAMP luminescent response was moderately greater in LGR1-transfected cells compared to cells not expressing LGR1. High basal constitutive activity of LGR1 has been observed previously in D. melanogaster (29). Moreover, constitutive activity of glycoprotein hormone receptors is well-known in vertebrates and has been demonstrated to be stronger for the thyrotropin receptor than for the LH/CG receptor (41). Given that GPA2/GPB5 receptor homologs in vertebrate and invertebrate model organisms have been shown previsously, and herein, to promote constitutive synthesis of cAMP, it may be suggestive that a common downstream effector and/or function exists for LGR1 and homologs in different organisms.

Surprisingly, however, our experiments indicate that incubations of LGR1-expressing cells with mosquito GPA2/GPB5 tethered protein triggers a switch from low level constitutive Gs coupling to Gi/o coupling for A. aegypti LGR1, given that tethered GPA2/GPB5 signficantly inhibited the forskolin-induced increase in cAMP in LGR1-expressing HEK 293T cells but not in control cells lacking LGR1 expression. This finding, while highly interesting, is not entirely unusual since promiscuous G protein coupling has been reported for glycoprotein hormone receptors like the TSH receptor (Gs and Gq) and LH/CG (Gi and Gs) (42–44).

To help stabilize heterodimerization, the beta subunit sequences of the classic glycoprotein hormones (FSH, LH, TSH, and CG) contain two additional cysteine residues that form an additional disulfide bridge which wraps around and “buckles” the alpha subunit (26). Though heterodimerization can occur with mutated forms of this “seatbelt” structure, there is a dramatic decrease in heterodimer stability (21, 22). GPB5 in vertebrates and invertebrates lack the seatbelt structure required to stabilize heterodimerization (26). Thus, the hypothesis that GPA2/GPB5 functions as a heterodimer in a physiological situation (i.e., without chemical cross-linking) is challenged. The dissociation constant (Kd) associated with heterodimerization of the classic glycoproteins hormone subunits and GPA2/GPB5 is 10⁻⁷ to 10⁻⁶ M, which indicates heterodimeric interactions are favored at these concentrations (45, 46). However, since the classic beta subunits contain an additional disulfide bridge that strengthens its association with the common alpha subunit, heterodimeric interactions are stabilized in circulation at physiological concentrations as low as 10⁻¹¹ to 10⁻⁹ M (21). Without this seatbelt structure, GPA2/GPB5 heterodimeric interactions are posssible only at micromolar concentrations, which are not typically observed in circulation (21, 26). Together, the limited evidence so far using heterolougous expression challenges the possibility of endocrine regulation by GPA2/GPB5 heterodimers. Nonetheless, it was argued in D. melanogaster that the large neurosecretory cells co-expressing the glycoprotein hormone subunits, along with their corresponding axonal projections that localized distinctly from organs that express the GPA2/GPB5 receptor (LGR1), did support that this system is indeed endocrine in nature (18). Alternatively, the subunits could function independently or regulate physiology as a heterodimer in a paracrine/autocrine fashion. In rats, GPA2/GPB5 is expressed in oocytes and may act as a paracrine regulator of TSHR-expressed granulosa cells in the ovary to regulate reproductive processes (6). Another possibility to consider is that additional endogenous co-factors may be involved, but remain unidentified, which help to strengthen interaction between the GPA2 and GPB5 subunits, since the tethered mosquito GPA2/GPB5 was indeed capable of activating LGR1 in vitro inducing a Gi/o signaling cascade.

Our results establish that human and mosquito GPA2 and GPB5 subunits can weakly and strongly, respectively, homodimerize. However, whether these homodimers have a physiological function in vivo is unknown. Treatments of either mosquito or human GPA2 and GPB5 subunits alone did not stimulate specific downstream signaling in LGR1- or TSHR-expressing cells, upholding that only GPA2/GPB5 heterodimers can activate their cognate glycoprotein hormone receptors. However, it is possible that GPA2 and GPB5 homodimers may target other unidentified receptors. In insects, the molting hormone bursicon is a heterodimer of two subunits called burs and pburs. Burs/pburs heterodimers act via a glycoprotein hormone receptor (i.e., LGR2) to regulate processes such as tanning and sclerotization of the insect cuticle as well as wing inflation after adult emergence (47). Recently, it was demonstrated that bursicon subunits can homodimerize (i.e., burs/burs and pburs/pburs) and these homodimers mediate actions independently of LGR2 to regulate immune responses in A. aegypti and D. melanogaster (48, 49).

In addition to the human and mosquito GPA2/GPB5 homodimers observed in our studies, human GPA2 was also shown to interact with the beta subunits of CG and FSH (4). Lastly, the expression patterns of GPA2 and GPB5 in a number of organisms do not always strictly co-localize, since GPA2 expression exhibits a much wider distribution and is expressed more abundantly than GPB5 in a number of vertebrate and invertebrate organisms (7, 12, 23, 24, 50, 51). Taken together, this raises the possibility that GPA2 and GPB5 subunits may interact with other unknown proteins that could activate different receptors or signaling pathways and elicit distinct functions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.