protocol-2732(1).pdf

Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2732|Page 1 of 6Video ArticleA Calcium Bioluminescence Assay for Functional Analysis of Mosquito(Aedes aegypti)and Tick(Rhipicephalus microplus)G Protein-coupledReceptorsHsiao-Ling Lu1,Cymon N.Kersch1,Suparna Taneja-Bageshwar1,2,Patricia V.Pietrantonio11Department of Entomology,Texas A&M University(TAMU)2Department of Molecular and Cellular Medicine,Texas A&M University(TAMU)Correspondence to:Patricia V.Pietrantonio at p-pietrantoniotamu.eduURL:http:/ 50,Aequorin calcium reporter,coelenterazine,G protein-coupled receptor(GPCR),CHO-K1 cells,mammalian cellculture,neuropeptide SAR studies(SAR=structure-activity relationships),receptor-neuropeptide interaction,bioluminescence,drug discovery,semi-throughput screening in platesDate Published:4/20/2011Citation:Lu,H.L.,Kersch,C.N.,Taneja-Bageshwar,S.,Pietrantonio,P.V.A Calcium Bioluminescence Assay for Functional Analysis of Mosquito(Aedes aegypti)and Tick(Rhipicephalus microplus)G Protein-coupled Receptors.J.Vis.Exp.(50),e2732,doi:10.3791/2732(2011).AbstractArthropod 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 kininreceptors 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 systemsis 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 stablyexpressed in the mammalian cell line CHO-K1.Functional analyses of these receptors were completed using a calcium bioluminescence plateassay 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 aequorinplasmid(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 calciumconcentration.As the kinin receptor signals through the release of intracellular calcium,the intensity of the signal is related to the potency of thepeptide.This protocol is a synthesis of several previously described protocols with modifications;it presents step-by-step instructions for the stableexpression of GPCRs in a mammalian cell line through functional plate assays(Staubly et al.,2002 and Stables et al.,1997).Using thismethodology,we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors,compare the potency of threemosquito kinins,identify critical amino acid positions for the ligand-receptor interaction,and perform semi-throughput screening of a peptidelibrary.Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases,they are severely limited in use astools for pest control or endocrinological studies.Therefore,we also tested kinin analogs containing amino isobutyric acid(Aib)to enhance theirpotency and biostability.This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs andmay aid in the development of neuropeptide-based arthropod control strategies.Video LinkThe video component of this article can be found at http:/ of stable cell lines1.Clone your GPCR of interest and insert it into an expression vector incorporating a 5 Kozak consensus sequence(GCCA/GCCATGG)around the start codon for optimal ribosomal binding in a mammalian system(Kozak,1986).Here we use the plasmid pcDNA3.1/Bm-KR forthe tick kinin receptor,and the plasmid pcDNA3.1/Aedae-KR for the mosquito kinin receptor(Holmes et al.,2003;Pietrantonio et al.,2005).The pcDNA3.1(-)vector encodes ampicillin resistance for selection in bacteria and neomycin(G418)resistance for selection in mammaliancells.Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2732|Page 2 of 62.Grow CHO-K1 empty cells(without plasmids)(ATCC,Manassas,VA,USA)or other desired cell line in a T-25 flask(BD Falcon)at 37C in a5%CO2 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.Totrypsinize 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 mediumand 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(1,000rpm).Discard supernatant and re-suspend cells in 5 ml medium.Dilute the resuspended cells to a concentration of 1:5 or 1:10 with freshmedium 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 ingrowth medium without antibiotics until they are about 30%confluent(approximately 18 hours).The degree of confluency can be determinedby observing cells under inverted fluorescent microscopy.5.Prepare the following mixtures for each sample:1.Combine 1-2 g DNA(example:use 4l of 265g/l pcDNA3.1/Aedae-KR)with 100 l Opti-MEM I Reduced Serum Medium(Invitrogen).2.Mix 6 l Lipofectin Reagent(InvitrogenTM)into 100 l F12K serum free medium,making a 1:1 ratio of Lipofectin to DNA.Incubate atroom 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 L).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 freemedium.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 cellsfrom 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-25flasks 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 800g/ml GENETICIN,Invitrogen).Culture thecells using the selective medium for 3-4 weeks.Over time this will select for cells that have stably incorporated the plasmid into their genomicDNA.Continue maintaining the cells using maintenance medium(F12K medium plus 10%fetal bovine serum with 400g/ml GENETICIN).Periodically freeze cell lines with 1:1 ratio of freezing media(selective medium with 20%DMSO)to prevent loses in case of unexpectedcontamination.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.Re-suspend 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 10 xdilution.Transfer the 10 x dilution into 12 wells of a 96 well plate,adding 100 l to each well to select for single cells.10.Continue to make 10 x serial dilutions of these cells for a theoretical final suspension of one cell per 100 l(normally the final dilution will bein the range of 10-11 to 10-19;the total number of 10 x serial dilutions is 19).Immediately following each dilution,transfer 100 l of the dilutioninto 12 wells of the 96 well plate.After 18 hours incubation,observe 96 well plates under an inverted light or fluorescence microscope andmark wells that appear to only contain one single cell or contain two cells that obviously divided from one single cell.Keep observing everyday and change medium every three days.11.When the wells are 80%confluent(about a week),rinse marked wells with 200 l PBS and trypsinize them with 100 l of PBS-trypsin-EDTAsolution.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 thentransfer 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 thesingle 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 bioluminescenceplate assay and maintain them in culture for further calcium bioluminescence plate assays.Keep track of passage numbers.From timeto time,freeze cell lines from early passages so that you can always go back to them if the cell lines with more passages stop performingconsistently.2.The calcium bioluminescence plate assay1.Ligate the reporter gene of interest into an expression vector.Here we used aequorin plasmid mtAEQ/pcDNA1(a gift from Drs.C.J.P.Grimmelikhuijzen and Michael Williamson,University of Copenhagen,Denmark).Transform the plasmid in E.coli cells MC1061/PS(Invitrogen)and purify them using a QIAprep spin miniprep kit(Qiagen Inc.).In the final step elute the plasmid with Tris buffer without EDTA,not water.2.Grow cell lines from step 1.13 expressing the desired receptor in maintenance medium.When the cells are 90%confluent,trypsinize,centrifuge and then re-suspend the cells in 5 ml maintenance medium as in step 1.3.Dilute cells(about 10 x with maintenance medium)andcount cell number with cell counter(Bright-Line Hemacytometer)under microscopy.Adjust the cell number to approximately 2 x 105 cells/ml(average 20 cells in one of the 9 squares showed in the Hemacytometer).Seed 2 ml diluted cells in media into each well of a six well plate.Incubate for 24 hours(the cells should reach about 60%confluency after incubation).3.Change media in the 6 wells plate to OPTI-MEM medium.For each well,mix 96 l OPTI-MEM with 4 l FuGENE 6 Transfection reagent(Roche Biochemicals)in a microfuge tube and let sit at room temperature for 5 min.Add 1 g of aequorin/pcDNA 1 plasmid DNA into eachtube and then gently shake the sample for 1 min,incubate at room temperature for 15-20 min.Add each mixture into each well in a dropwisefashion while gently shaking the well plate.Incubate the plates for 6 hours and change the medium to F12K medium containing 10%fetalbovine serum without antibiotic.4.After incubating the cells in six well plate for 24 hours,trypsinize,centrifuge and re-suspend the cells as step 1.3.Count the cell number to400,000 cells/ml as step 2.1 and transfer 100 l(total 40,000 cells/100 l)into each well of a 96-well white thin bottom microtitre plate(Costar3610).Incubate for another 24 hours until about 80%cell confluency.This is the optimal concentration of cells for the bioluminescence assay.5.Prepare 90l/well of a calcium-free DMEM media(Invitrogen)containing 5 M coelenterazine(Invitrogen)in the dark(coelenterazine is lightsensitive).Take the plate from 2.4,remove old media and add this 90l into each well.Incubate plates for 3 hours in the dark at 37C and 5%CO2,after which the cells in the plate are ready to be tested.Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2732|Page 3 of 63.Instrument operation and data analysis1.Each bioluminescence plate reader is different.We perform our assay using a NOVOstar(BMG Labtechnologies)plate reader inbioluminensence mode.If you use a different instrument,you must adapt the protocol.2.Purge the plate reader pumps(or PRIME PUMPS)before use.Turn off the light in the room before putting the plate on the plate holder.Oncethe plate holder has closed,turn the lights on.3.Solubilize peptides(in a 1.5 ml Eppendorf tube)in the calcium-free DMEM media.Set the Aspirate Depth and Position Determination ofthe peptide solution before use.Challenge the cells with 10 l(10 x)of varying concentrations of the peptides(FFFSWG-NH2,Aedes-K1-3,orother desired peptide)and immediately begin recording the light emission.We have set the instrument to record the light emission(465 nm)for each well every 2 seconds for a total time of 50 seconds.4.Make sure to include a positive control such as an active analog(analog FFFSWGa has been used,Taneja-Bageshwar et al.,2009)anda negative control such as vector only transfected cells.The negative control will be necessary during data analysis to set the baselinethreshold(see representative results).An unrelated,inactive peptide can also be added as a negative control.5.After the run is complete,wash the instrument pump(or PRIME PUMPS)then place your next peptide sample.Save your data and wash thepumps again.6.Data handling:Transfer the data of light emission from each well into a Microsoft Excel data sheet.7.Paste the data from Excel to Prism software 4.0 from GraphPad Software Inc.(San Diego,CA,USA).The various peptide concentrations isthe X-axis and bioluminescence units is the Y-axis.To normalize the data,plot a log-response curve.Select a nonlinear regression curve fitanalysis(sigmoidal dose-response equation with variable slope)to obtain concentration-response curves for each peptide.The program plotsthe values in the end and gives the EC50.8.Each experiment should be repeated three times for data analysis.4.Representative Results:When expressed in CHO-K1 cells,the mosquito Aedes aegypti kinin receptor behaved as a multiligand receptor and functionally responded toconcentrations as low as 1 nM of the three endogenours Aedes kinins,Aedae kinins 1-3,tested singly using the calcium bioluminescence plateassay.Figure 1 shows that the rank order of potency obtained was Aedae-K-3 Aedae-K-2 Aedae-K-1,based on the respective EC50 valuesof Aedae-K-3,16.04 nM;Aedae-K-2,26.6 nM and Aedae-K-1,48.85 nM,which were statistically significantly different(P 0.05)(Pietrantonio etal.,2005).We also used this assay to determine which kinin residues are critical for the kinin peptide-receptor interaction.Insect kinin peptides share a C-terminal pentapeptide that represents the minimal sequence required for biological activity,also known as core.In Table 1,the