Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 1 pp. 65ñ70, 2008 ISSN 0001-6837 Polish Pharmaceutical Society Adenosine plays important role not only intra- cellulary, but also as extracellular signal compound. In the central neural system, adenosine modulates sleep, locomotion, nociception, drug addiction and other vitally important processes. Adenosine medi- ates its actions through specific G protein-coupled receptors, which are classified into four subtypes: A 1 , A 2A , A 2B and A 3 receptors (1). A 1 and A 2A recep- tors bind adenosine with high affinity (k d 0.1 ñ 1.0 µM), whereas A 2B and A 3 receptors have lower affin- ity for adenosine (k d 10 µM) (2). Adenosine A 2A receptors are localized in the central nervous sys- tem, specially in striatum, where they modulate glu- tamatergic neurotransmission via the GABAergic enkephalinergic neurons (3). At the presynaptic side, they are localized in active zone, forming het- eromeric complexes mostly with adenosine A 1 , but also with glutamatergic mGlu 5 and dopaminergic D 2 receptors. At the postsynaptic side they are localized at the perisynaptic ring adjacent to the postsynaptic density, forming heteromeric complexes with gluta- matergic mGlu 5 and dopaminergic D 2 receptors. As G s , G olf proteins-coupled receptors, adenosine A 2A receptors transmit signal intracellulary, through adenyl cyclase, protein kinase A cascade (4). A 2A and D 2 receptors regulate the function of the GABAergic enkephalinergic neurons by antagonis- tic interactions (5, 6). Consequently, by blocking adenosine A 2A receptors, dopaminergic transmission can be increased. This effect can be used in the new, alternative therapies of some neurodegenerative dis- orders, specially Parkinsonís disease (7). To date, there is no drug with adenosine A 2A receptor antag- onistic properties in Parkinsonís disease available at the European market, but some compounds are being evaluated in clinical trials and one (Istra- defylline) may reach the US market this year (8). Green Fluorescent Protein (GFP) (9) and its variants have become one of the most widely used tools to study cellular distribution and trafficking of proteins of interest. Genetic engineering of fluores- cent proteins leads to different spectral properties, increased brightness and folding efficiences, as compared with the wild types (10). Attachment of such proteins to the G protein-coupled receptors has become widely applied method to study receptors in living cells (11). Moreover, interactions between receptors and other elements can be investigated in FLOW CYTOMETRY APPLICATION FOR STUDIES ON ADENOSINE A 2A RECEPTORS EXPRESSION TOMASZ W”JCIK 1 *, MICHA£ BERETA 2 , AGATA FARON-G”RECKA 3 , MARTA DZIEDZICKA-WASYLEWSKA 3 and KATARZYNA KIE∆-KONONOWICZ 1 1 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Str., 30-688, KrakÛw, Poland 2 Department of Immunology, Jagiellonian University Medical College, 18 Czysta Str., 31-121 KrakÛw, Poland 3 Institute of Pharmacology, Polish Academy of Sciences, 12 SmÍtna Str., 31-343 KrakÛw, Poland Abstract: Adenosine A 2A receptors belong to the heptaspanning membrane receptors family A, also known as G protein-coupled receptors. In human brain they are highly expressed in striatum, where they co-exist and co- function with adenosine A 1 , glutamate mGlu 5 and dopamine D 2 receptors. As glutaminergic neurotransmission modulators in GABAergic enkephalinergic neurons, adenosine A 2A receptors are attractive targets for new, alternative therapies of neurodegenerative disorders, like Parkinsonís disease and Huntingtonís disease. The aim of the research was to obtained fluorescently tagged adenosine A 2A receptors. Gene encoding human adeno- sine A 2A receptor was inserted into plasmid pEYFP-N1, bearing enhanced yellow fluorescent protein (EYFP). The construct was expressed in HEK 293 cells. Fluorescence was observed by flow cytometry and epifluores- cence microscopy. Functional ligand binding properties were investigated by saturation binding analysis of adenosine A 2A receptors specific agonist [ 3 H] CGS 21680. Keywords: flow cytometry, adenosine A 2A receptors, fusion proteins, radioligand binding assays, fluorescence microscopy 65 * Corresponding author: [email protected]; tel./fax +4812 657 04 88
Transcript
Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 1 pp. 65ñ70, 2008 ISSN 0001-6837Polish Pharmaceutical Society
Adenosine plays important role not only intra-cellulary, but also as extracellular signal compound.In the central neural system, adenosine modulatessleep, locomotion, nociception, drug addiction andother vitally important processes. Adenosine medi-ates its actions through specific G protein-coupledreceptors, which are classified into four subtypes:A1, A2A, A2B and A3 receptors (1). A1 and A2A recep-tors bind adenosine with high affinity (kd 0.1 ñ 1.0µM), whereas A2B and A3 receptors have lower affin-ity for adenosine (kd 10 µM) (2). Adenosine A2A
receptors are localized in the central nervous sys-tem, specially in striatum, where they modulate glu-tamatergic neurotransmission via the GABAergicenkephalinergic neurons (3). At the presynapticside, they are localized in active zone, forming het-eromeric complexes mostly with adenosine A1, butalso with glutamatergic mGlu5 and dopaminergic D2
receptors. At the postsynaptic side they are localizedat the perisynaptic ring adjacent to the postsynapticdensity, forming heteromeric complexes with gluta-matergic mGlu5 and dopaminergic D2 receptors. AsGs, Golf proteins-coupled receptors, adenosine A2A
receptors transmit signal intracellulary, through
adenyl cyclase, protein kinase A cascade (4). A2A
and D2 receptors regulate the function of theGABAergic enkephalinergic neurons by antagonis-tic interactions (5, 6). Consequently, by blockingadenosine A2A receptors, dopaminergic transmissioncan be increased. This effect can be used in the new,alternative therapies of some neurodegenerative dis-orders, specially Parkinsonís disease (7). To date,there is no drug with adenosine A2A receptor antag-onistic properties in Parkinsonís disease available atthe European market, but some compounds arebeing evaluated in clinical trials and one (Istra-defylline) may reach the US market this year (8).
Green Fluorescent Protein (GFP) (9) and itsvariants have become one of the most widely usedtools to study cellular distribution and trafficking ofproteins of interest. Genetic engineering of fluores-cent proteins leads to different spectral properties,increased brightness and folding efficiences, ascompared with the wild types (10). Attachment ofsuch proteins to the G protein-coupled receptors hasbecome widely applied method to study receptors inliving cells (11). Moreover, interactions betweenreceptors and other elements can be investigated in
FLOW CYTOMETRY APPLICATION FOR STUDIES ON ADENOSINE A2A RECEPTORS EXPRESSION
TOMASZ W”JCIK1*, MICHA£ BERETA2, AGATA FARON-G”RECKA3, MARTA DZIEDZICKA-WASYLEWSKA3 and KATARZYNA KIE∆-KONONOWICZ1
1 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Str., 30-688, KrakÛw, Poland
2 Department of Immunology, Jagiellonian University Medical College, 18 Czysta Str., 31-121 KrakÛw, Poland
3 Institute of Pharmacology, Polish Academy of Sciences, 12 SmÍtna Str., 31-343 KrakÛw, Poland
Abstract: Adenosine A2A receptors belong to the heptaspanning membrane receptors family A, also known asG protein-coupled receptors. In human brain they are highly expressed in striatum, where they co-exist and co-function with adenosine A1, glutamate mGlu5 and dopamine D2 receptors. As glutaminergic neurotransmissionmodulators in GABAergic enkephalinergic neurons, adenosine A2A receptors are attractive targets for new,alternative therapies of neurodegenerative disorders, like Parkinsonís disease and Huntingtonís disease. Theaim of the research was to obtained fluorescently tagged adenosine A2A receptors. Gene encoding human adeno-sine A2A receptor was inserted into plasmid pEYFP-N1, bearing enhanced yellow fluorescent protein (EYFP).The construct was expressed in HEK 293 cells. Fluorescence was observed by flow cytometry and epifluores-cence microscopy. Functional ligand binding properties were investigated by saturation binding analysis ofadenosine A2A receptors specific agonist [3H] CGS 21680.
detail. For example, interaction with ligands withagonistic/antagonistic properties (12), as well aswith G proteins can be visualized, as well as inter-action with β-arrestines (13). Another area of inter-ests, which has been widely investigated in recentyears is cross-talk between receptors of the sametype (homodimerization) or different types (het-erodimerization) (14). The best established ways tomeasure this type of protein-protein interactions areresonance energy transfer and protein complemen-tation assays. In both approaches both partnersrequire the attachment of the reporter proteins. Inresonance energy transfer assays one reporter is
donor fluorophore or bioluminescent protein andthe other reporter is longer wavelength acceptor flu-orophore. The efficiency of non-radiative transferbetween donor and acceptor protein is determinatedby the distance between them (15). In protein com-plementation assays the reporters are halves of aprotein, which can only function when the two frag-ments are brought together and fold around eachother.
In the area of instrumentation, a great varietyof tools is presently offered. Steady-state fluores-cence spectroscopy can be applied to get fluores-cence emission spectra from cells population in
Figure 1. Flow cytometric profile of non-transfected HEK293 cells.
Flow cytometry application for studies on adenosine A2A receptors expression 67
solution. Flow cytometry offers fast and exactmeasurements of each cell. Such parameters likenumber, shape, viability, mobility, together withfluorescent properties can be measured.Fluorescent resonance energy transfer can be alsomeasured by flow cytometry (16). In order to visu-alize fluorescent proteins fusion constructs distri-bution and trafficking in living cells fluorescentmicroscopy has to be applied. Confocalmicroscopy offers better resolution, comparingwith white-field epifluorescence microscopy and apossibility to measure energy transfer by acceptorphotobleaching. Fluorescence lifetime imaging
microscopy can provide quantitative information,since fluorescence lifetime measurements are inde-pendent of any change of fluorophore concentra-tion or excitation intensity.
EXPRIMENTAL
PlasmidsThe pcDNA 3.1 plasmid, encoding human
adenosine A2A receptor (UMR cDNA ResourceCenter, Rolla, MO) was PCR-amplified using anuniversal primer for pcDNA 3.1 and a reverseprimer without stop codon and carrying EcoRI
Figure 2. Flow cytometric profile of HEK293 cells transfected with EYFP-N1 plasmid.
68 TOMASZ W”JCIK et al.
restriction site. The entire coding sequence wasinserted into pEYFP-N1 plasmid (Clontech, PaloAlto, CA).
Cell cultures and transfection Human embronic kidney 293 (HEK293) cells
were cultured at 37OC and 5% CO2 in DMEM medi-um (Sigma), supplemented with 10% heat-inactivat-ed FBS (Sigma) and 1% L-glutamine (Sigma). Cellswere transiently transfected using FuGene HDreagent (Roche) for radioligand binding assays orusing Lipofectamine (Invitrogen) for epifluores-cence microscopy, accoring to the manufacturers
instructions. 48 h after transfection cells were har-vested for following experiments.
Epifluorescence microscopyCells were grown on poly(D-lysine)-treated
coverslips (number 0) and transiently transfectedwith 2 µg of plasmid. Coverslips were placed into amicroscope chamber containing physiologicalHEPES-buffered saline solution (130 mM NaCl, 5mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mMHEPES, and 10 mMD-glucose, pH 7.4). Cells werethen imaged using an inverted Nikon TE2000-Emicroscope (Nikon Instruments, Melville, NY)
Figure 3. Flow cytometric profile of HEK293 cells transfected with adenosine A2A receptor EYFP fusion construct.
Flow cytometry application for studies on adenosine A2A receptors expression 69
equipped with a 40 (numerical aperture 1.3) oilim-mersion Fluor lens and a cooled digital photometricsCoolSnap-HQ charge-coupled device camera(Roper Scientific, Trenton, NJ). Epifluorescenceexcitation light was generated by an ultra-highpointintensity 75 W xenon arc Optosource lamp (CairnResearch, Faversham, Kent, UK) coupled to a com-puter-controlled Optoscan monochromator (CairnResearch). Monochromator was set at 495/9 nm.
Radioligand binding assay Radioligand binding assay was performed as
described in (17). Briefly, the transfected HEK 293cells were washed with phosphonate-buffered saline(PBS), scraped from dish in PBS and centrifuged at1000 rpm for 5 min. Pellets were resuspended in 50mM Tris-HCl, pH 7.4, containing 10 mM MgCl2,using an Ultra Turrax homogenizer. This homo-genate was recentrifuged at 48000 ◊ g for 10 min at4OC. The resulting pellet was resuspended in buffercontaining 2 IU/mL of adenosine deaminase (TypeIII, Boehringer Mannheim, Indianapolis, IN) to 20mg/mL of original tissue weight and incubated at37OC, in order to remove endogenous adenosine.This membrane homogenate was recentrifuged andthe final pellet was used for the assay. The saturationstudies were carried out in triplicate, in tubes con-taining 100-200 µg of protein per mL in incubationbuffer. Tubes were incubated with 10 different con-centrations of [3H]CGS 21680 ranging from 0.5 to50 nM. Nonspecific binding was defined in the pres-ence of 20 µM 2-CADO. Binding reactions wereterminated by filtration through Whatman GF/B fil-ters. Filters were washed twice with ice-cold bufferand placed in scintillation vials and bound radioac-tivity was determined by liquid scintillation count-ing (Beckman LS 650). Estimation of the radioli-gand binding parameters was carried out using theGraphPad Prism 2.0 program (GraphPad Software,San Diego CA).
Flow cytometryCells were transiently transfected using a calci-
um phosphonate precipitation procedure (18). 48 hafter transfection cells were harvested by tryp-synization and resuspended in phosphonate buffersaline with 1% bovine serum albumin. Data werecollected with FACSCalibur flow cytometer (BDBiosciences, San Jose, CA), equipped with theargon ion laser (wavelength of 488 nm) and a 635nm Spectrum laser. Signals were detected with530/30 nm bandpass filter (into FL1), 585/42 nmbandpass filter (into FL2) and 670LP nm bandpassfilter (into FL3).
RESULTS
The aim of this study was to develop adenosineA2A receptor fusion protein, which can be used invarious fluorescent-based assays. pEYFP-N1 plas-mid, bearing enhanced yellow fluorescent proteinvariant was employed and adenosine A2A receptorwas cloned into it. Obtained construct was sub-cloned in Escherichia coli DH5a bacteria. In order toget receptors expression in their functional form,human HEK 293 cells were transiently transfectedwith obtained construct by different methods, likecalcium phosphonate precipitation, FuGene HD orLipofectamine reagent. HEK 293 are cells of choicein these type of experiments, because they offer highfidelity of exogenous receptors expression, rapidcell division and low patch-clamping (19). Flowcytometric profile of non-transfected HEK 293 cells(Figure 1) shows homogeneous population of non-fluorescent cells. For cells transfected with pEYFPplasmid (Figure 2), fluorescence is observed in alldetection channels, but the highest one is observedin FL1 channel, what agrees with fluorescence emis-sion maximum for yellow fluorescent protein. Forcells transfected with pEYFP plasmid, with adeno-
Figure 4. Fluorescence microscopy images of HEK293 cells trans-fected with adenosine A2A receptor-EYFP fusion construct (left)and EYFP plasmid alone (right). Figure 5. Saturation binding [3H] CGS 21680 to human adenosine
A2A receptors fused with EYFP fluorescent proteins, expressed inHEK293 cells.
70 TOMASZ W”JCIK et al.
sine A2A receptor encoding gene (Figure 3), thehighest fluorescence is observed, comparing withcells transfected with pEYFP plasmid without theinsert. Epifluorescence imaging reveals membranelocalization of adenosine A2A receptors-EYFPfusion proteins (Figure 4, left), comparing with dif-fuse expression of enhanced yellow fluorescent pro-teins alone (Figure 4, right), although, significantfluorescence of fusion construct is observed in thecells organelles.
In order to investigate the effect of the pres-ence of fluorescent tag on receptors function, satura-tion binding of specific adenosine A2A receptorsagonist [3H]CGS 21680 was performed (Figure 5).The C-terminal tag did not influence the bindingparameters. The obtained kd values (14.9 nM) areclose to the values shown for these receptors in therat brain (17).
DISCUSSION
The C-terminally tagged adenosine A2A rece-ptors were expressed in HEK 293 cells, as judgedboth by flow cytometry and by epifluorescencemicroscopy imaging techniques. Most of theexpressed constructs were localized in the plasmamembrane, although significant fraction of expres-sed constructs were localized in endoplasmic reticu-lum and Golgi apparatus. This may indicate mis-folding of some of the receptors constructs or thatportion of the receptor proteins is still on the way tothe final destination in the cell membrane. Satu-ration binding experiments show that the fraction ofreceptors construct present in the plasma membraneis fully functional and able to bind specific ligands.Continuing efforts to investigate obtained constructsexpression, advanced microscopic techniques likeconfocal microscopy and fluorescence lifetimeimaging are planned to be used.
The EYFP, like most of fluorescent proteinsavailable at the market have natural tendency to selfassociate (20). This can be blocked with the A206Kpoint mutation in the amino acids sequence. Themonomeric fluorescent proteins, like for examplemCitrine (21) can replace the EYFP as fusion part-ners and it may improve folding of fusion proteinsconstructs.
Acknowledgments
Epifluorescence imaging was performed inMolecular Pharmacology Group, University ofGlasgow, UK. We thank Dr John D. Pediani for
microscopy assistance and Prof. Graeme Milliganfor inspiring scientific discussion. T.W. is a recipi-ent of a fellowship from the British Council inBritish-Polish Young Scientists Programme.
