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A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes. The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide. This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

2. Grow CHO-K1 empty cells (without plasmids) (ATCC, Manassas, VA, USA) or other desired cell line in a T-25 flask (BD Falcon) at 37°C in a 5% CO 2 humidified incubator (Holmes et al., 2003). All further incubations should be done under these conditions unless specified otherwise. Maintain the empty cells in growth medium (F12K medium with 10% fetal bovine serum) with 1X Antibiotic-Antimycotic (Invitrogen,CA). 3. To split cells, warm up all solutions to 37 °C. Remove old medium in the T-25 flask and rinse with 5 ml PBS and then remove PBS. To trypsinize cells, add 2 ml PBS-Trypsin-EDTA (34 ml PBS, 2 ml 7.5% sodium bicarbonate, 4 ml 10X trypsin-EDTA) for 2 min. Add 3 ml medium and aspirate medium up and down to mix cells. Transfer medium with cells into a conical tube and centrifuge for 2 min at ˜200-300 g (1,000 rpm). Discard supernatant and re-suspend cells in 5 ml medium. Dilute the resuspended cells to a concentration of 1:5 or 1:10 with fresh medium and transfer 5 ml in a new T-25 flask. 4. After the cells are growing healthily (around 2-3 days), seed the CHO-K1 cells into T-25 tissue culture flasks and grow them overnight in growth medium without antibiotics until they are about 30% confluent (approximately 18 hours). The degree of confluency can be determined by observing cells under inverted fluorescent microscopy. 5. Prepare the following mixtures for each sample: 1. Combine 1-2 μg DNA (example: use 4μl of 265μg/μl pcDNA3.1/Aedae-KR) with 100 μl Opti-MEM I Reduced Serum Medium (Invitrogen). 2. Mix 6 μl Lipofectin Reagent (InvitrogenTM) into 100 μl F12K serum free medium, making a 1:1 ratio of Lipofectin to DNA. Incubate at room temperature for 30-45 min.
Gently mix the solutions from 1.5.1 with 1.5.2 in a drop-wise fashion (total volume = 200 μL). Let stand at room temperature for 10-15 min. 6. Remove the old growth medium from the cells and wash the cells with 5 ml F12K serum free medium and then remove the F12K serum free medium. In a 15 ml tube, gently mix the transfection mixture into 1.8 ml of fresh F12K medium in a drop-wise fashion. Then, washed the cells from step 1.5 with PBS and add this new transfection solution to the cells. Incubate for 18 hours. 7. Change the medium to F12K medium plus 10% fetal bovine serum without antibiotics and incubate overnight. Split the cells into two T-25 flasks with F12K medium plus 10% fetal bovine serum without antibiotics for another 18 hours (for splitting cells please see step 1.3). 8. Replace the medium with selective medium (F12K medium plus 10% fetal bovine serum with 800μg/ml GENETICIN, Invitrogen). Culture the cells using the selective medium for 3-4 weeks. Over time this will select for cells that have stably incorporated the plasmid into their genomic DNA. Continue maintaining the cells using maintenance medium (F12K medium plus 10% fetal bovine serum with 400μg/ml GENETICIN). Periodically freeze cell lines with 1:1 ratio of freezing media (selective medium with 20% DMSO) to prevent loses in case of unexpected contamination. 9. Selecting clonal cell lines: as a first step, trypsinize and centrifuge the cells from step 1.8 with maintenance medium as in step 1.3. Resuspend cells in 5 ml maintenance medium, take 0.5 ml of the cell suspend and add 4.5 ml of fresh maintenance medium to make a 10x dilution. Transfer the 10x dilution into 12 wells of a 96 well plate, adding 100 μl to each well to select for single cells. 10. Continue to make 10x serial dilutions of these cells for a theoretical final suspension of one cell per 100 μl (normally the final dilution will be in the range of 10 -11 to 10 -19 ; the total number of 10x serial dilutions is ˜1 9). Immediately following each dilution, transfer 100 μl of the dilution into 12 wells of the 96 well plate. After 18 hours incubation, observe 96 well plates under an inverted light or fluorescence microscope and mark wells that appear to only contain one single cell or contain two cells that obviously divided from one single cell. Keep observing every day and change medium every three days. 11. When the wells are 80% confluent (about a week), rinse marked wells with 200 μl PBS and trypsinize them with 100 μl of PBS-trypsin-EDTA solution. Transfer cells from each marked well into one well of a 6-well plate with 1 ml maintenance medium. Grow those cells for 3 days then transfer cells into individual T25 flask. Test these cells using the calcium bioluminescence assay with agonist peptides (please see section 2). 12. Select 1 cell line from step 1.11 with the highest response in the calcium bioluminescence plate assay and perform for a second time the single cell selection following steps 1.9-1.11. Periodically freeze cell lines. 13. From the secondary selection described in step 1.12, choose 2-3 cell lines with the highest response in the calcium bioluminescence plate assay and maintain them in culture for further calcium bioluminescence plate assays. Keep track of passage numbers. From time to time, freeze cell lines from early passages so that you can always go back to them if the cell lines with more passages stop performing consistently. . Incubate for another 24 hours until about 80% cell confluency. This is the optimal concentration of cells for the bioluminescence assay. 5. Prepare 90μl/well of a calcium-free DMEM media (Invitrogen) containing 5 μM coelenterazine (Invitrogen) in the dark (coelenterazine is light sensitive). Take the plate from 2.4, remove old media and add this 90μl into each well. Incubate plates for 3 hours in the dark at 37°C and 5% CO 2 , after which the cells in the plate are ready to be tested.
We also used this assay to determine which kinin residues are critical for the kinin peptide-receptor interaction. Insect kinin peptides share a Cterminal pentapeptide that represents the minimal sequence required for biological activity, also known as core. In Table 1, the kinin peptide core analogs were synthesized as an Alanine replacement series of the core kinin pentapeptide FFSWGa and tested by a calcium bioluminescence plate assay (Taneja-Bageshwar et al., 2006). We found that the amino acids Phe 1 and Trp 4 were essential for activity of the insect kinins for both receptors.
The assay also can be used to test peptides designed for enhanced biostability. Table 2 shows the designed kinin analogs containing amino isobutyric acid (Aib) tested on the tick recombinant kinin receptor and Figure 2 shows the activity comparison of six alpha-amino isobutyric acid analogs on the tick kinin receptor expressing CHO-K1 cell line by a calcium bioluminescence plate assay ( Taneja Figure 2).

Discussion
We were able to perform the functional characterization of the first neuropeptide receptor discovered from the Arachnida (ticks, mites and spiders), the tick kinin receptor, using this protocol. This method has three primary applications. First, the technique can be applied for receptor deorphanization through ligand activity measurements. Second, the assay can resolve ligand-receptor structure-activity relationships (SAR). Third, the methods can be used in drug discovery. Furthermore, this protocol can be used to study the activity of agonists or antagonists on almost any GPCR. We are beginning to adapt this protocol for screening of small libraries. The cell line we utilized does not express the ubiquitous G protein G 16 . We did not need it because arthropod kinin receptors signal through the Gq protein and the intracellular calcium cascade and they conserve this signaling properties in mammalian cells as shown here.

Disclosures
No conflicts of interest declared.