Neurochemistry International 55 (2009) 467–475

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Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint

Comparative pharmacology of adrenergic a2C receptors coupled to

Ca2+ signaling through different Ga proteins
Dalma Kurko *, Zsófia Bekes, Anikó Gere, Andrea Baki, András Boros, Sándor Kolok,
Gyula Bugovics, József Nagy, Zsolt Szombathelyi, Györgyi Ignácz-Szendrei
Pharmacological and Drug Safety Research, Gedeon Richter Plc., Budapest, Hungary

A R T I C L E I N F O A B S T R A C T

Article history: Adrenergic a1, a2 and b receptors are members of the G-protein-coupled receptor families (GPCRs)
Received 9 March 2009 mediating physiological responses to adrenaline (epinephrine) and noradrenaline (norepinephrine).
Received in revised form 24 April 2009 Since GPCRs are major targets for potential therapeutic agents, development of robust, reliable and cost
Accepted 28 April 2009
effective functional screening methods for these receptors is in the focus of pharmacological research.
Available online 6 May 2009
For this reason, the aim of the present study was to develop an intracellular calcium assay for
investigating the pharmacology of the a2C type of adrenergic receptors (a2C-AR). Although activation of
Keywords:
a2C-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Gaqi5
a2C-AR
Chimeric Gaqi5 protein
protein, containing the five carboxyl-terminal amino acids from Gi, or promiscuosus Ga16 protein can
Promiscuosus Ga16 protein divert receptor signaling to the Gq pathway generating Ca2+ release from intracellular stores.
Fluorometric Ca2+ assay In order to assess the functional potency of a2-AR agonists and antagonists, we established a
High-throughput screening fluorometric Ca2+ assay using cell lines stably and constitutively co-expressing a2C-AR and Gaqi5 or Ga16
proteins (Gaqi5/a2C and Ga16/a2C). As part of the pharmacological characterization, we measured the
changes in cytoplasmic Ca2+ levels due to activation of the chimeric Gaqi5 or Ga16 coupled recombinant
a2C receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine,
oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of
a2-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in a2C-
AR expressing cells were also measured. These results confirmed that the Gaqi5/a2C and Ga16/a2C
recombinant systems can be useful for modelling the native Gi-coupled system. Our results indicate that
a plate-reader based fluorometric Ca2+ assay may be suitable in high-throughput screening for a2C-AR
ligands as well.
ß 2009 Elsevier Ltd. All rights reserved.

1. Introduction depression, obesity, diabetes (Serle et al., 1991; Ruffolo et al.,

The a2-adrenergic receptors (a2-ARs) constitute a distinct
family of catecholamine receptors which mediate physiological
responses to noradrenaline and adrenaline. Pharmacological and
molecular biological studies indicate that a2-ARs consist of three
homologous subtypes: namely a2A, a2B, and a2C (Bylund, 1992,
2005; Hieble et al., 1995; Docherty, 1998; Calzada and Artinano,
2001). There are some reports of existence of a fourth subtype, a2D,
but it is generally acknowledged to be the rodent homologue of the
human a2A-AR subtype (Link et al., 1992; MacDonald et al., 1997).
The a2-adrenergic receptor subtypes play a pivotal role in many
diverse cell functions and physiological processes, therefore these
receptors have been considered as important drug targets for
treatment of pain, hypertension, ischemia, glaucoma, spasticity,
* Corresponding author. Fax: +36 1 8898400.
E-mail address: d.kurko@richter.hu (D. Kurko).
1993,1995; Ruffolo and Hieble, 1994; Lakhlani et al., 1997;
Arnsten, 1998; Kable et al., 2000; Crassous et al., 2007; Sanders
and Maze, 2007). Several line of evidence suggests that a2-ARs may
also play a role in gastric mucosal defense pointing to the
gastroprotective effect of the a2-AR agonists (Gyires et al., 2007).
Moreover, as presynaptic release-modulating receptors represent
promising targets for pharmacological intervention it is possible
that a new generation of drugs with actions on transmitter release
through autoreceptors and heteroreceptors may become a
generation of novel drugs with useful therapeutic properties,
including some unmet medical needs of today (Langer, 2008).
However, the therapeutic use of the currently available a2-
drugs is limited because of severe side effects, including sedation
and cardiovascular effects (Kamibayashi and Maze, 2000; Maze
and Fujinaga, 2000). Recent studies on transgenic animals lacking
or overexpressing different a2-AR subtypes have greatly enhanced
our understanding of subtype specific physiological functions and
have highlighted the potential for the development of subtype

0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2009.04.015
468 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475
selective therapeutic agents with improved efficacy and/or
reduced side effects when compared to existing drugs (Lakhlani
et al., 1997; Kable et al., 2000; Scholz and Tonner, 2000; Philipp
et al., 2002; Knaus et al., 2007). Therefore, screening of compound
libraries, molecular modeling and synthesis of novel adrenergic
agonists and antagonists without therapy-limiting side effects,
with better subtype selectivity and specificity are still under
thorough investigation (Wong et al., 1994, 2000; Balogh et al.,
2007). As such, cell lines that stably express a2C-ARs are extremely
useful in identifying selective ligands of this receptor class.
a2-ARs belong to the GPCR superfamily, which comprises one
of the largest gene families in the human genome (Ji et al., 1998;
Perez, 2005). Physiological signaling pathways linked to the a2-
AR subtypes are mostly mediated by pertussis toxin-sensitive G-
proteins (Gi1, Gi2, Gi3 and Go) resulting in inhibition of adenylyl-
cyclase (AC) activity and consequent decreases in cellular cAMP,
activation of receptor operated K+ channels and inhibition of
voltage gated Ca2+ channels (Limbird, 1988; Abdulla and Smith,
1997; Saunders and Limbird, 1999). Stimulatory effects of a2-AR
agonists have also been described, including coupling to
stimulatory cholera toxin-sensitive Gs proteins with increased
cAMP generation (Eason et al., 1992, 1994; Pohjanoksa et al.,
1997; Jasper et al., 1998) and Ca2+ mobilization (Kukkonen et al.,
1998; Reynen et al., 2000). Although the preferential transduc-
tion pathway of the a2C-subtype is through the inhibitory Gi/o
proteins (Pohjanoksa et al., 1997), in recombinant systems they
can also activate Gq when aq subunit is overexpressed in HEK-
293 cells (Conklin et al., 1992). Ca2+ mobilization by a2C-ARs has
also been demonstrated in CHO-K1 cells stably expressing the
receptor; however, the pertussis toxin sensitivity of this
response points that it is likely to be mediated by the bg
subunits of Gi/o proteins (Kukkonen et al., 1998). Furthermore,
GPCR signaling exhibits even greater diversity and complexity if
several receptors are simultaneously activated since the signal
transduction pathways can cross-talk or interact (Fresco et al.,
2007).
Since GPCRs represent one of the most important drug
discovery targets, a variety of functional assays are available for
screening GPCR ligands (Thomsen et al., 2004, 2005). Historically,
screening for GPCR agonists or antagonists has been performed by
filter binding assays utilizing high-affinity radioligands. However,
an ideal screening system should be simple, non-radioactive,
robust (i.e. characterized by high signal-to-noise ratio), homo-
genous, suitable in a microtiter plate format (96-, 384- or 1536-
well) and also utilizing minimal reagent addition steps to facilitate
robotic automation (Milligan and Rees, 1999). Further, these
screening methods should allow a simple functional discrimina-
tion between agonists and antagonists and be applicable for a great
variety of receptors. One of the widely used methods for studying
signaling through Gi proteins is the measurement of incorporation
of radiolabelled GTPgS into activated G-proteins. Since this
method uses radioisotopes and requires purification of membrane
components, furthermore, it lacks the advantages of intrinsic
signal amplification that occurs in a whole cell (Cerione, 1991), it is
not the most appropriate method as a drug screening assay.
Moreover, this assay is generally limited to Gi/o-coupled receptors,
because Gi/o proteins have a faster GDP–GTP exchange rate than
other G-proteins (Milligan, 2003). As activation of the Gi proteins
results in a decreased AC activity, measuring the inhibition of
forskolin-stimulated cAMP accumulation is another popular
method for studying Gi signaling (Wong, 1994; Gabriel et al.,
2003). Since this assay generally has a low signal-to-noise ratio
(Williams, 2004) and a limited dynamic range, furthermore it is
expensive, time-consuming, and because the inhibition of the
stimulation is rarely exceeds 60%, it is not ideal for higher
throughput screening.
More recently, functional assays that measure various signaling
events downstream to receptor activation have become widely
utilized (Chambers et al., 2003; Monteith and Bird, 2005). Ca2+
assays are now recognized as efficient tools for the characteriza-
tion of GPCRs signaling via Gq proteins. Ca2+ measurement can be
an option for evaluation of non-Gq-coupled receptors as well using
chimeric and/or promiscuous G-proteins (Kassack et al., 2002;
Eglen, 2005; New and Wong, 2005). It has been reported that the
carboxyl-terminal end of the a subunit of G-proteins plays an
important role in the specific interaction between the G-protein
and the receptor. Accordingly, the exchange of three to nine amino
acids of the C-terminal residues of the PLC-b-activating Ga
subunit, i.e. the Gaq, by those of either Gai or Gao is sufficient to
allow these chimeric subunits to alter the signaling pathways of
many GPCRs (Conklin et al., 1993; Conklin et al., 1996; Liu et al.,
1995; Kostenis et al., 1997). It has also been reported that the
promiscuous Ga15 and Ga16 proteins, that activate PLC-b, can
activate different GPCRs, including those recognized not to
stimulate PLC-b per se (Offermanns and Simon, 1995; Milligan
et al., 1996; Kostenis, 2001). To date, however, it is impossible to
predict a priori whether a particular GPCR can interact effectively
with a specific chimeric or promiscuous G-protein.
In order to identify an efficient receptor–G-protein combination
and to provide increased sensitivity as well as a high signal-to-
noise ratio for ligand screens at a2C-ARs, we developed stable
transfected cell lines co-expressing a2C-ARs with either chimeric
Gaqi5 or promiscuous Ga16 proteins. We report the successful
application of these G-proteins for functional characterization of
ligands acting at a2C-AR by establishing a fluorometric Ca2+ assay
compatible even with high-throughput evaluation.
2. Experimental procedures
2.1. Generation of stably transfected cell lines expressing recombinant human a2C-ARs
and chimeric Gaqi5 or promiscuous Ga16 proteins
Chinese hamster ovary (CHO-K1) cells expressing a2C-ARs (a2C-C1 cell line) were
purchased from Euroscreen (cat. nr. ES-032-C, Bruessels, Belgium). Sequencing
revealed that the DNA region encoding human a2C-AR was identical to the gene
published by GenBank with accession number of AY605898.1, corresponding to the
haplotype 1 of the human a2C-AR (i.e. an isoform where the sequence 322–325 is
not deleted); it codes for a protein identical to that available in GenBank under the
accession number of AAT02221.1. Expression vectors pCEP-Gqi5-HA and pCEP-
Ga16 encoding the chimeric Gaqi5 and promiscuous Ga16 proteins, respectively,
were obtained from Molecular Devices (Sunnyvale, CA, USA). For transfection a2C-
C1 cells were grown on 96-well plates to 50% confluency in Ham’s F12 medium
(nutrient mixture Ham’s F12 + L-Glutamine (GIBCO BRL, Gaithersburg, MD, USA),
containing 10% fetal bovine serum (GIBCO BRL), 2.5 mg/ml amphotericin B, 100 U/
ml penicillin G, 100 mg/ml streptomycin and 400 mg/ml G418 (Invitrogen, Carlsbad,
CA, USA). Cells were incubated with Gaqi5 or Ga16 cDNAs containing vectors (5 mg/
ml) for 4 h in serum-free F12 in the presence of lipofectamine (Pfx-7, Invitrogen) in
a humidified atmosphere with 5% CO2 at 37 8C. Following the 4 h transfection
period cells were placed in serum containing F12 medium and next day they were
exposed to selection medium, containing hygromycin (200 mg/ml, Invitrogen) and
G418 (400 mg/ml, Invitrogen). The medium was replaced every 2 or 3 days for 2
weeks. Stable cell colonies were isolated and assayed for calcium responses using
the FlexStation II (described below).
2.2. Determination of cytosolic free concentration of calcium ions
Changes in the cytoplasmic calcium ([Ca2+]i) were measured using the Ca2+-
sensitive dye, fluo-4AM (Molecular Probes, Eugene, OR). Cells were seeded into 96-
well plates at a density of 40,000 cells/well and maintained overnight in a tissue
culture incubator at 37 8C under an atmosphere of 5% CO2. The growth medium was
aspirated from the cells and replaced with 2 mM fluo-4AM in extracellular solution
(140 mM NaCl, 5 mM KCl, 5 mM HEPES-Na, 5 mM HEPES, 2 mM CaCl2, 2 mM MgCl2,
10 mM glucose, pH 7.4). Cell loading was enhanced by premixing the hydrophobic
dye AM-ester with low levels (final concentration 0.02%) of Pluronic-127 (Sigma–
Aldrich, St. Louis, MO, USA). To inhibit the efflux of the dye from the cells during and
following loading 2 mM probenecid (anion transporter inhibitor) was added to the
cells. After the incubation period (60 min, 37 8C, 5% CO2/95% air), unloaded dye was
removed from the cells by washing and 100 ml fresh extracellular medium with or
without an a2-AR antagonist was added to the wells and the plate was incubated at
37 8C for an additional 10 min. The plate was transferred to FlexStation II (Molecular
 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475
Devices, Sunnyvale, CA), a plate-reader fluorometer with integrated 8-channel fluid
addition capability. Fluorescence measurements were carried out at 37 8C. The dye
was excited at 485 nm, emission was sampled at 538 nm at 1.4-s intervals. The basal
(unstimulated) level of cytosolic Ca2+ ion concentration was recorded for 20 s
followed by agonist stimulation. The 25 ml 5 concentrated agonist solution was
added to the cells using the pipettor of FlexStation and fluorescence was monitored for
an additional 40 s. Results were expressed as DF/F values using SOFTMax Pro software
(Molecular Devices), where F was the resting fluorescence preceding agonist
application and DF was the increase in fluorescence at a given time (DF = maximum
fluorescence intensity values after stimulation minus average fluorescence intensity
values for unstimulated level of cytosolic Ca2+ ion concentration). In all experiments,
all treatments on the plates were measured in multiple wells (4–12) in parallel, and
the mean DF/F values or inhibition percentages of those were used for analysis.
Sigmoid curves were fitted to the concentration–response data to obtain EC50 or IC50
values. Curve fitting was done by Microcal Origin 6.0 software. For agonists, relative
efficacy (Emax) values were calculated as fitted maximum of each concentration–effect
curve expressed as a percentage of the response observed with a maximally effective
concentration of brimonidine (UK14,304; 1 mM) from the same plate. All experiments
were performed at least three times, on different days. Data were expressed as
mean  S.E.M., unless otherwise stated.
2.3. Measurement of intracellular cAMP levels
Chinese hamster ovary CHO-K1 cells expressing recombinant a2C-ARs were
grown as described above. For the measurements cells were seeded in 384-well plates
and cultured overnight at 37 8C and 5% CO2. Following medium replacement with
HBSS (Hank’s buffered saline solution) supplemented with 50 mM Ro 20-1724
(phosphodiesterase inhibitor, Sigma) cells were incubated for 20 min at 37 8C/5% CO2.
Cells were then treated with an a2-AR agonist, UK14,304, in a concentration range of
3 nM to 3 mM for 15 min and were stimulated with forskolin (1 mM) for an additional
15 min at 37 8C. In antagonists experiments, cells were treated first with MK912
(25 mM), and after 15 min incubation, with UK14,304 (25 mM). After agonist
treatment cells were also incubated for 15 min, followed by forskolin (0.03–100 mM)
treatment and an additional 15 min incubation (37 8C; 5% CO2). Stimulated cells were
lysed and cAMP levels were measured immediately via a competitive, fluorescence
intensity based immunoassay. Concentration of cAMP was determined with the
CatchPointTM kit (Molecular Devices) according to the manufacturer’s instructions.
cAMP, derived from treated and lysed cells competes with horseradish peroxidase-
labelled cAMP conjugate for binding sites on the anti-cAMP antibodies. After a single
wash step, any unbound HRP–cAMP is removed. In the absence of cAMP, most of the
HRP–cAMP is bound to the antibody. Increasing concentrations of cAMP competi-
tively decrease the amount of bound conjugate, thus decreasing the measured HRP
activity. In the presence of HRP and hydrogen peroxide, a fluorescent substrate is
oxidized. The recorded fluorescent intensity (excitation: 530 nm; emission: 590 nm)
is proportional to the amount of HRP and inversely proportional to the cAMP
concentration present in the reaction. Fluorescence was measured with the Polarstar
Optima (BMG Labtech) multimode microplate reader. The fluorescence intensity
values of the forskolin-stimulated samples were extracted from those of the
unstimulated control samples (the highest value). The DRFU calculated is
proportional to the cAMP concentration present in the sample.
2.4. Radioligand binding assay
Membranes were prepared from CHO-K1 cells stably expressing human a2C-ARs.
Cells were harvested and centrifuged at 500  g. The cell pellet was broken with a
Polytron PTA 10 S homogenizer (setting 5, 30 s burst) in ice-cold homogenization buffer
containing 5 mM Tris–HCl, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl
fluoride (PMSF); pH 7.4, followed by two consecutive centrifugation steps at 30,000  g
for 15 min separated by a wash step in the same buffer. Membranes were resuspended
in TRIS buffer containing 50 mM Tris–HCl, 1 mM EDTA, 5 mM MgCl2, 0.1 mM PMSF; pH
7.4. Protein content was determined and was adjusted to 1 mg protein/ml. The final
suspension was aliquoted and was frozen at 70 8C. For saturation analysis the
radiolabelled agonist ligand [3H]UK14,304 was used in a concentration range of 0.1–
15 nM with an incubation time of 30 min at room temperature.
To determine the binding affinities of some reference drugs the radioligand
[3H]UK14,304 was used in a concentration of 2 nM. Non-specific binding was
determined in the presence of phentolamine (10 mM). The binding procedure was
stopped by filtration on Unifilter GF/B glass-fiber filter presoaked in 0.5%
polyethyleneimine. The filters were washed five times with 1 ml TRIS buffer and
the radioactivity of the filters was counted by liquid scintillation spectrometry in a
Pachard TopCount NXT microplate scintillation 8 luminescence counter. All
experiments were performed at room temperature. IC50 values were calculated
from concentration displacement curves by sigmoidal fitting using Microcal Origin
6.0 software, Ki values were calculated using the Cheng–Prusoff equation (Cheng
and Prusoff, 1973).
2.5. Determination of mRNA expression by RT-PCR
Total RNA from transfected and non-transfected a2C-C1 cells was extracted using
the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription (RT) into
469
cDNA was performed using the Ready-To-Go You-Prime First-Strand Beads Kit
(Amersham Biosciences, New Jersey, USA) following the manufacturer’s instruc-
tions. The 20 ml total volume of the PCR reaction solution contained 1 ml cDNA
template from each sample, 1–1 ml forward and reverse primer, 10 ml 2 Master
Mix (Promega Corporation, Madison, WI, USA) and 7 ml dH2O.
Primers were designed using the Primer 2.0 software and were synthesized by
IDT (Leuven, Belgium). Sequences of Gaqi5 and Ga16 specific primers generating an
293 and 186 base-pair (bp) amplicons, respectively, were as follows: forward
primer: 50 -CTCTGGAGTCCATCATGGCA-30 , reverse primer: 50 -GATCTTGAGCGTGTC-
CATCG-30 ; and forward primer: 50 -GCTACCTGGACGAGATCAAC-30 , reverse primer:
50 -TGAGAGGCAGAGAAGAGAGC-30 . PCR analysis was performed with a Biometra T
Gradient instrument. The PCR program consisted of an initial denaturation phase of
5 min at 95 8C, 20 cycles of denaturation at 94 8C (60 s), annealing at 55 8C (30 s) and
extension at 72 8C (60 s) and a Wnal synthesis step of 10 min at 72 8C. The PCR assay
was performed simultaneously with negative control samples (cDNA obtained from
non-transfected a2C-C1 cells and sterile water). The resulting PCR products were
separated using a Bioanalyzer 2100 Instrument (Agilent Technologies, Santa Clara,
CA, USA) and a DNA 7500 LabChip Kit. The specific PCR products were analyzed with
Agilent Technologies 2100 Expert Software (Version Nr: B.02.02.SI238)
2.6. Immunocytochemical analysis of Gaqi5
Internal hemagglutinin (HA) epitope in Gaqi5 cDNA allows analysis of protein
expression using flow cytometry based immunocytochemistry. CHO-K1 cells stably
expressing a2C-ARs and Gaqi5 proteins were cultured on 24-well plates. Cells were
suspended in Dulbecco’s phosphate buffered saline (D-PBS), and then fixed in 4%
buffered paraformaldehyde for 30 min at room temperature. After three washes,
samples were incubated overnight at 4 8C with mouse monoclonal anti-
hemagglutinin (HA)–FITC antibody (1:20, Roche, Basel, Switzerland) in D-PBS
containing 0.2% Triton X-100 and 2% BSA. The same staining procedure was applied
to Gaqi5 non-transfected a2C-C1 cells as well to determine the non-specific staining
properties of the antibody. After washing, the FITC fluorescence was analyzed using
a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), equipped with
15 mW air-cooled 488 nm argon ion laser. Fluorescence signals were measured in
the Fl-1 window (530  30 nm band pass filter). Data of 3000 events per sample were
recorded in the list mode at a flow rate of approximately 300 cell/s following
logarithmic amplification. Data were analyzed using CellQuest software (version: 3.1.f;
Becton Dickinson, San Jose, CA, USA). Cells were gated in the forward scatter (FSC) vs.
side scatter (SSC) dot plot. The arithmetic means of the FITC-fluorescence intensities
were calculated from fluorescence histograms for the gated population.
2.7. Drugs
UK14,304 (5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine),
oxymetazoline (2-(3-hydroxy-2,6-dimethyl-4-t-butylbenzyl)-2-imidazoline), clo-
nidine (2-[2,6-dichloroaniline]-2-imidazoline HCl), moxonidine (4-chloro-N-(4,5-
dihydro-1H-imidazol-2-yl)-6-methoxy-2-methylpyrimidin-5-amine), yohimbine,
MK912 ((2S,12bS)10 ,30 -dimethylspiro(1,3,4,50 ,6,60 ,7,12b-octahydro-2H-benzo[b]-
furo[2,3-a]quinolizine)-2,40 -pyrimidin-20 -one), U-73122 (1-(6-((17b-3-methox-
yestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) were obtained
from Sigma–Aldrich.
3. Results
3.1. Cell lines co-expressing recombinant human a2C-ARs with
chimeric Gaqi5 or promiscuous Ga16 proteins
After transfection of a2C-C1 cells with pCEP-Gqi5-HA or pCEP-
Ga16 expression vectors, we obtained cell lines stably co-
expressing human a2C-ARs with chimeric Gaqi5 or promiscuous
Ga16 proteins, respectively. More than two dozens of cell colonies
resistant for the selecting agents (i.e. hygromycin and neomycin)
were picked and pre-screened for functional activity by assessing
UK14,304-evoked cytoplasmic Ca2+ release. Stable expression of
the a2C-AR in CHO-K1 cells alone did not result in Ca2+ elevation in
response to 1 mM UK14,304 stimulation, but co-expression of the
receptor with chimeric Gaqi5 or promiscuous Ga16 proteins
resulted in robust Ca2+ responses with a high signal-to-background
ratio. The maximal changes in relative fluorescence units were up
to 45,000 and 20,000, respectively (Fig. 1). UK14,304 generated
typical responses consisting of a rapid initial peak in fluorescence
(maximal 8–12 s after agonist addition) followed by a rapid decline
towards the basline (over a period of 40 s).
Two clones, designated as Gaqi5/H7 and Ga16/B6, which
exhibited the highest agonist-induced [Ca2+]i responses, were
470 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475

Fig. 1. Ca2+ response in CHO-K1 cells expressing the a2C-AR alone (circle) or co-
transfected with chimeric Gaqi5 (square) or promiscuous Ga16 proteins (triangle)
following stimulation with 1 mM UK14,304. Cells were loaded with the Ca2+
indicator dye Fluo-4, transferred to FlexStationII and after recording baseline (0–
20 s) stimulated with UK14,304. Raw fluorescence data from a representative
experiment is shown with each data point representing the mean value  S.E.M.
from 6 wells.

chosen and characterized further in subsequent experiments. RT-

PCR analysis of the expression of Gaqi5 or Ga16 mRNA clearly
verified the expression of these mRNAs in both cells lines (data not
shown). The expression of the Gaqi5 protein tagged with
hemagglutinin (HA) could be detected by a flow cytometry based
immunocytochemical method as well. The level of expression was
assessed by comparing the fluorescence histograms obtained from
measurements on the non-expressing (a2C-C1) and the Gaqi5
expressing (Gaqi5/H7) cells stained with anti-HA–FITC antibody.
The intensity of specific immunolabelling was about six times
higher in Gaqi5/H7 compared to a2C-C1 cells (data not shown).
The mechanism of agonist-evoked increase of cytoplasmic
calcium was also examined in the two different G-protein
expressing cell lines and was found to be consistent with Gq
signal transduction. 5 min preincubation with the PLC inhibitor U-
73122 reduced Ca2+ elevation in response to UK14,304 (IC50 values
were 1.51  0.18 and 1.36  0.12 mM in Gaqi5/H7 and Ga16/B6 cell
lines, respectively).
Functional stability of the generated cell lines was also
monitored by comparing the maximum responses to UK14,304
observed in cells following several passages. We could detect
proper calcium responses up to 30 passages and the DF/F values
(see Section 2) were stable during this period.
3.2. Pharmacological properties of recombinant human a2C-ARs
Further detailed characterization of the a2C-AR-coupled Ca2+
signal facilitated by chimeric Gaqi5 or promiscuous Ga16 proteins
was undertaken using established receptor agonists and antago-
nists. All of the agonists tested caused a concentration-dependent
increase in [Ca2+]i; the generated concentration–response curves
are illustrated in Fig. 2. The rank order of potency for examined
agonists was UK14,304 (EC50, 1.7  0.1 nM) = noradrenaline (2.1 
0.3 nM) > oxymetazoline (2.9  0.5 nM) > clonidine (7.0  0.6 nM)
> moxonidine (41.0  4.7 nM) for Gaqi5/H7 cell line and UK14,304
(EC50, 5.1  0.3 nM) > oxymetazoline (7.4  1.2 nM) > noradrenaline
(15.3  2.7 nM) > clonidine (29  3.8 nM) > moxonidine (321 
34 nM) for Ga16/B6 cell line, respectively. Although UK14,304 and
noradrenaline were equally efficacious in all the clones, marked
differences in response to the other agonists were seen resulting in a
rank order of efficacy as follows in both cell lines: UK14,304 = nor-
Fig. 2. (a) Concentration-dependence of agonist-induced Ca2+ responses in Gaqi5/
H7 cells co-expressing the a2C-AR and chimeric Gaqi5 proteins. Agonist responses
were expressed as a percentage of the response observed with a maximally effective
concentration of UK14,304 (1 mM). Data points represent mean  S.E.M. from three
to seven independent experiments. (b) Concentration-dependence of agonist-induced
Ca2+ responses in Ga16/B6 cells co-expressing the a2C-AR and promiscuous Ga16
proteins. Agonist responses were expressed as a percentage of the response observed
with a maximally effective concentration of UK14,304 (1 mM). Data points represent
mean  S.E.M. from three to seven independent experiments.
adrenaline > moxonidine > oxymetazoline > clonidine. In compari-
son to UK14,304, whose efficacy was considered as 100%,
noradrenaline displayed similar maximal efficacies in Gaqi5/H7 and
Ga16/B6 cells (97.4  0.4% and 95.8  2.5%) while moxonidine (90.3 
3.9% and 80.1  2.2%, respectively), oxymetazoline (82.3  2.7% and
61.5  2.7%, respectively) and clonidine (77.3  0.6% and 46.3  7.1%,
respectively), resulted in lower maximal responses suggesting partial
agonist responses for a2C-ARs.
To examine the effect of antagonists, cells were pre-treated
with yohimbine and MK912. The compounds tested in presence of
40 nM UK14,304 (corresponding approximately to the concentra-
tion of the agonist generating 90% activity [EC90]) resulted in a
concentration-dependent inhibition of agonist-induced Ca2+
mobilization, as shown in Fig. 3. Calculated IC50 values for
MK912 were 0.73  0.05 nM and 0.20  0.04 nM in Gaqi5/H7 and
Ga16/B6 cell lines, respectively. Estimated IC50 values for the
inhibitory effect of yohimbine were 17.8  2.9 nM and
3.1  0.8 nM, respectively. None of the antagonists tested increased
[Ca2+]i in any of the cell lines (data not shown). In the case of Ga16/B6
cell line, as it can be seen in Fig. 3b, the maximum inhibition % of
antagonists varied between 94% and 106%. However, the differences
 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475 471

Fig. 4. Determination of forskolin-stimulated cAMP levels by Catch PointTM kit.

Stimulation of membrane bound AC with forskolin resulted in cAMP production and
accumulation in a concentration-dependent manner. The inhibitory effect of the
agonist UK14,304 depends on forskolin concentration. 1 mM forskolin seemed to be
optimal, at higher concentrations the inhibitory efficacy of UK14,304 gradually
decreased. Average raw fluorescence intensity values of unstimulated control
samples were 4013  53 (buffer) and 4182  81 (25 mM UK14,304), respectively.
Each column reperesents the mean value  S.E.M from a representative experiment
performed in triplicate.

between the maximal inhibitions of the tested antagonists were

Fig. 3. (a) Concentration-dependent inhibition of yohimbine and MK912 on the
cytoplasmic Ca2+ concentration rises evoked by 40 nM UK14,304 in Gaqi5/H7
cells. Antagonists were equilibrated with cells for 10 min before agonist
stimulation. Preliminary experiments confirmed that both yohimbine and
MK912 were devoid of agonist activity. Data points represent mean  S.E.M of 3
and 4 independent experiments, respectively. (b) Concentration-dependent
inhibition of yohimbine and MK912 on the cytoplasmic Ca2+ concentration rises
evoked by 40 nM UK14,304 in Ga16/B6 cells. Antagonists were equilibrated
with cells for 10 min before agonist stimulation and preliminary experiments
confirmed that both yohimbine and MK912 were devoid of agonist activity.
Data points represent mean  S.E.M of 3 and 4 independent experiments,
respectively.
not significant. The rank order of potencies and relative efficacies
of various a2-AR ligands for both cell lines are summarized in
Table 1.
UK14,304 was also evaluated for its ability to inhibit forskolin-
induced cAMP elevation in CHO-K1 cells expressing recombinant
a2C-ARs alone. Stimulation of membrane bound adenylyl-cyclase
with forskolin resulted in cAMP production and accumulation in a
dose-dependent manner with an EC50 value of 343 nM. The
inhibitory effect of UK14,304 was dependent on forskolin
concentration. 1 mM forskolin seemed to be optimal for a near
complete inhibition of forskolin-induced cAMP elevation by full
agonists. At higher concentrations (>3 mM) the percentage of
inhibition gradually decreased (Fig. 4). UK14,304 concentration-
dependently decreased the cAMP accumulation induced by
forskolin (1 mM) with an IC50 value of 11.2  3.3 nM (Fig. 5). Pre-
treatment of agonist-stimulated a2C-C1 cells with 25 mM MK912
reversed the agonist effect, as shown in Fig. 6.
For comparative purposes we have developed a competitive
binding assay in membranes derived from a2C-AR expressing
CHO-K1 cells using [3H]UK14,304 as a radioligand. All compounds

Table 1
Pharmacological profile of a2C-ARs as determined in fluorometric [Ca2+]i and radioligand binding experiments.

Compounds [Ca2+]i [3H]UK14,304 binding

EC50/IC50 (nM) Emax (%) Ki (nM)

Gaqi5/H7 Ga16/B6 Gaqi5/H7 Ga16/B6 a2C-C1

UK14,304 1.7  0.1 5.1  0.3 100 100 1.7  0.4
Noradrenaline 2.1  0.3 15.3  2.7 97.4  0.4 95.8  2.5 2.0  0.2
Oxymetazoline 2.9  0.5 7.4  1.2 82.3  2.7 61.5  2.7 6.9  2.5
Clonidine 7.0  0.6 29  3.8 77.3  0.6 46.3  7.1 7.3  2.2
Moxonidine 41  4.7 321  34 90.3  3.9 80.1  2.2 63  5.4
MK912 0.73  0.05 0.20  0.04 0.21  0.04
Yohimbine 17.8  2.9 3.1  0.8 4.33  0.9

Agonist activity (EC50, nM) was deduced from concentration response curves of various compounds and percent efficacy (representing the relative maximum response, Emax
%) was normalized to 1 mM UK14,304. IC50s (nM) were determined against 40 nM UK14,304 (corresponding to an EC90 concentration). a2C-AR ligand affinities (Ki, nM) were
determined by inhibition studies using the radioligand [3H]-UK14,304. FlexStationII data were derived from cells co-expressing the a2C-AR and the chimeric Gaqi5 or
promiscuous Ga16 proteins, while binding data were derived from cells expressing only the a2C-ARs. Data in the table represent the mean  S.E.M. from three to seven
independent experiments.
472 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475
Fig. 5. Inhibition of the forskolin-stimulated cAMP production by UK14,304. Agonist
binding to a2C-ARs prior to forskolin-induced AC activation inhibits cAMP
production in a dose-dependent manner with an IC50 value of 11.2 nM. Each
point represents the mean  S.E.M. of 3 separate experiments.
Fig. 6. Forskolin-induced cAMP elevations (square) and reversal of UK14,304
(25 mM, circle) reduced cAMP levels by the a2-AR antagonist MK912 (25 mM,
triangle) in the original a2C-AR expressing CHO cells. Each point represents the
mean  S.E.M. of 3 separate experiments.
tested in Ca2+ assay bound to the receptor and concentration-
dependently inhibited [3H]UK14,304 binding with the following
rank order of affinity (Ki) MK912 (0.21  0.04 nM) > UK14,304
(1.7  0.4 nM) > noradrenaline (2  0.2 nM) > yohimbine (4.3 
0.9 nM) > oxymetazoline (6.9  2.5 nM)  clonidine (7.3  2.2
nM) > moxonidine (63  5.4 nM). The Hill slopes for adrenergic
receptors ligands did not differ significantly from unity, indicating their
competition with [3H]UK14,304 radioligand for the a2C-ARs (data not
shown). Data deduced from concentration–response curves for
inhibition of [3H]UK14,304 binding are summarized in Table 1.
4. Discussion
Recombinant receptors expressed in heterologous cellular
systems are extensively used as experimental models for studying
receptor function and mechanism of action. Functional cell-based
assays are also widely employed to assess the pharmacological
properties of the expressed receptor and their ligands (Sautel and
Milligan, 2000).
Thus, our primary objective was the generation of an in vitro
model system in which pharmacology of a2C-ARs could be
investigated. Therefore, in addition to constructing appropriate
cell lines, we also developed an assay system by which functional
interaction of compounds with this receptor subtype could be
conveniently evaluated. Since a2C-ARs naturally use a Gai
signaling pathway, in this study we examined the feasibility of
using the chimeric as well as the promiscuous G-protein approach
to couple this adrenergic receptor subtype to a Ca2+signaling
readout facilitating ligand screening and applying fluorometry
beneficial for high-throughput evaluations.
Fluorometric Ca2+ assays have been used efficiently for
characterization of GPCRs signaling via Gaq proteins mobilizing
cytoplasmic [Ca2+]i. There are growing amount of evidence for their
potential use for investigating non-Gaq-coupled receptors as well
since it has been reported that replacement of five amino acids of
the C-terminal of the Gaq sequence with the corresponding Gai
sequence can convert the signaling pathway of Gi/o-coupled
receptors towards the PLC-b pathway. A significant number of
Gi-linked GPCRs have been successfully coupled to such chimeric
G-proteins, e.g. A1 adenosine, D2 dopamine, a2-adrenergic
receptors (Conklin et al., 1993), neuropeptide Y receptors (hY1,
hY2, hY4; Dauzenberg et al., 2005), mGluR2, mGluR4 (Gomeza
et al., 1996; Kowal et al., 2003; Downey et al., 2005), H3 histamine
(Uvegas et al., 2002), mel-1a and mel-1b melatonin (Yokoyama
et al., 2003), D3, D4 dopamine receptors (Moreland et al., 2004).
However, there are some receptors (like somatostatin sst1 and
cannabinoid CB1 receptors) where such an exchange failed to
produce functional systems (Conklin et al., 1996). Alternatively,
receptors can be also co-expressed with certain promiscuous G-
proteins (i.e. Ga15, Ga16) that are capable of interacting with a
variety of Gs-, Gi,o-, and Gq-coupled receptors. Calcium release has
been detected upon activation of b2-adrenergic, muscarinic M2,
dopamine D1, vasopressin V2, adenosine A2A, 5-HT1A, fMLP, m-
opioid, thromboxane A2 receptors when co-expressed with
promiscuous G-proteins (Offermanns and Simon, 1995). However,
the promiscuous G-proteins can not be considered as truly
universal adaptors, as they either poorly couple to or fail to
activate the Ca2+ pathway in the case of some Gi-selective GPCRs
(e.g. dopamine D3, somatostatin sst1, melatonin MT1c, chemokines
CCR1 and CCR2) (Milligan et al., 1996; Marchese et al., 1999; Mody
et al., 2000; Kostenis, 2001; Liu et al., 2003). Several Gi/o-coupled
receptors, such as the mGluR2 and mGluR4 (Gomeza, 1996), 5-
HT1A receptors (Offermanns and Simon, 1995; Kowal et al., 2002)
d-, k- and m-opioid (Coward et al., 1999) receptors, efficiently
activate chimeric and promiscuous G-proteins as well, but
depending on the receptors these recombinant systems do not
always activate the different G-proteins equally.
In order to identify an effective receptor–G-protein combina-
tion useful for screening potential a2C-AR ligands, we compared
the pharmacological profile of two cell lines stably expressing the
receptor together with either a chimeric (Gaqi5) or a promiscuous
(Ga16) G-protein in the same cellular background. Although the
transient co-transfection of a2A and a2B subtypes of adrenergic
receptors with Ga15 promiscuous proteins (Pauwels and Colpaert,
2000a; Pauwels et al., 2000) and the transient co-transfection of
a2C-ARs with chimeric Gaq/i1 proteins (Pauwels and Colpaert,
2000b) have been reported previously, our study is the first to
evaluate the use of the promiscuous Ga16 protein co-expression
strategy in the generation of stable cell lines co-expressing the a2C-
ARs.
For characterization of the expression of chimeric Gaqi5 or
promiscuous Ga16 proteins in the cell lines we have obtained, RT-
PCR and immunocytochemical analyses were applied. To test the
functionality and to examine the pharmacological profile of
recombinant a2C-ARs, radioligand binding and fluorometric
 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475
measuring techniques to determine cAMP-inhibition and calcium
levels were used. Although a2C-ARs were able to couple to both
recombinant G-proteins, it seems that receptors most efficiently
couple to the chimeric Gaqi5 proteins. Transfection of Gaqi5 or
Ga16 proteins into CHO-K1 cells expressing a2C-ARs resulted in a
robust agonist-induced transient Ca2+ response displaying similar
kinetics in both cell lines, although the signal-to-background ratio
was approximately 2.5-fold higher in Gaqi5/H7 cells. Ca2+
elevations were sensitive to the phospholipase C inhibitor U-
73122 suggesting that the Ca2+ response is due to PLC-b activation.
Similar differences in preferential coupling have been seen in HEK-
293 cells co-expressing mGluR2 or mGluR4 and either chimeric
(Gaqi5, Gaqi9) or promiscuous G-proteins (Ga15, Ga16) (Gomeza
et al., 1996). In the case of human d-, k- and m-opioid receptors
expressed in CHO-K1 cells, k- and m-opioid receptors signaled
most efficiently when they were co-transfected with Gaqi5. On the
contrary, in the case of the d-opioid receptor, its signaling was
more efficient with Ga16 co-transfection, although it worked well
with Gaqi5 too (Coward et al., 1999).
Stable expression of the a2C-AR in CHO-K1 cells alone did not
result in cytoplasmic Ca2+ elevation in response up to 1 mM
UK14,304 stimulation. On the contrary, Kukkonen et al. (1998)
found that human a2-AR subtypes were coupled to elevation of
[Ca2+]i when heterologously expressed in CHO cells. However, their
results suggest that this Ca2+ response is due to PLC-b activation by
bg subunits from Gi proteins. According to literature data agonists
can differ in their potency to induce activation of the different G-
protein subunits. Results of Kukkonen et al. suggest that there is
also a roughly150-fold decrease in the potency of UK14,304 when
Ca2+ elevation occurs through stimulation of PLC-b by bg subunits
from Gi. As our aim was to examine whether the use of the chimeric
or that of the promiscuous G-protein approach is more suitable to
model the primary signal transduction pathway of native Gi-
coupled system, experiments with UK14,304 at concentrations
higher than 1 mM have not been carried out. In addition, the
marked differences between our and Kukkonen’s data could be due
to the different expression level of the a2C-ARs in the generated cell
lines, as Kukkonen et al. reported a 2040 fmol/mg protein
expression, while our saturation analysis revealed a 589.2 fmol/
mg protein expression.
To examine whether the pharmacology of the receptor is
unaltered in the generated recombinant systems, a2C-coupled
calcium signals were measured in response to a number of
reference agonists and antagonists. UK14,304 and noradrenaline
were full agonists, while oxymetazoline, clonidine and moxonidine
behaved as partial agonist, in agreement with published data
(Munk et al., 1994; MacDonald et al., 1997; Parsley et al., 1999;
Umland et al., 2001; Millan, 2002; Szabo, 2002; Stone et al., 2003).
The efficacies of all partial agonists tended to be higher in the case
of Gaqi5/H7 cells compared with Ga16/B6 cells. The potencies of
the agonists investigated were approximately in the same range in
both cell lines, with all compounds displaying a moderate decrease
in potency when tested in Ga16/B6 cells. The concentration–
response curves were rightward shifted with 3- to 8-fold
differences in EC50 values. Except that oxymetazoline was some-
what more potent than noradrenaline in this model, there were no
differences between the rank orders of potencies. This may be a
result of either a better coupling of these receptors to this
particular chimeric G-protein or of a higher level of expression of
this a subunit, and subsequent a more efficient coupling to PLC-b.
Similar a2C-AR subtype-dependent pharmacological profile
was reported for these agonists in a study using similar
fluorometric techniques (Pauwels and Colpaert, 2000b). Transient
co-transfection of the wild type (wt) a2C-AR with chimeric Gaq/i1
protein in CHO-K1 cells revealed the following rank order of
efficacies and potencies: UK14,304 (102, EC50 = 1.48 nM) = adre-
473
naline (100, EC50 = 3.98 nM) > oxymetazoline (81, EC50 = 4.68
nM) > clonidine (75, EC50 = 13.18 nM). Other earlier investiga-
tions showed a similar rank order of agonists obtained by
measuring extracellular acidification, [35S]-GTPgS binding,
Ca2+ and GTPase responses in CHO and HEK-293 cells stably
transfected with a wt a2C-AR (Pihlavisto and Scheinin, 1999;
Jasper et al., 1998; Umland et al., 2001; Kukkonen et al., 1998;
Jansson et al., 1999). Some modest quantitative differences in
agonist activities were, however, noted presumably due to
differences in host cell lines used, coupling efficiency of native
vs. chimeric G-proteins, and the measured endpoints.
MK912 and yohimbine, known to be potent and selective a2-AR
antagonists with high affinity for the a2C-AR subtype (Schaak et al.,
1997; Uhlén et al., 1994, 1997; Lalchandani et al., 2002; Mustafa
et al., 2005), inhibited UK14,304-mediated Ca2+ responses in a
concentration-dependent manner. However, the antagonists were
4- to 6-fold less potent in the Gaqi5/H7 than the Ga16/B6 cell line,
with higher IC50s in the concentration–inhibition curves. It is
possible that such discrepancy in antagonist potencies reflect the
different coupling efficiencies of the a2C-ARs with the chimeric
Gaqi5 or the promiscuous Ga16 proteins. The IC50 value of
yohimbine obtained from our Ca2+ assay was consistent with that
obtained in a cAMP measurement assay using a cell-based
luciferase reporter gene assay on human a2C-ARs expressing
CHO cells (IC50 = 3.2 nM; Lalchandani et al., 2002). In contrast to
yohimbine, in our Ca2+ assay the logistic slope of the concentra-
tion–inhibition curve of MK912 was significantly greater than 1.
However, in contrast to the Hill slope, the logistic slope of
functional inhibition curves cannot be interpreted directly
(Lazareno and Birdsall, 1993). Also, despite their prevalence, the
physical events underlying steep concentration–effect curves in
functional assays are poorly understood (Shoichet, 2006). It is not
clear what is behind the apparently different inhibitory patterns
produced by yohimbine and MK912 in our Ca2+ assay. Although a
recent study (Görnemann et al., 2007) found yohimbine and
MK912 to be a competitive and noncompetitive antagonist,
respectively, it has not been shown to our knowledge that the
inhibition curve of a noncompetitive antagonist should have
higher logistic slope than a competitive one.
Although agonist pharmacology did not seem altered in the
fluorometric Ca2+ assay, it is important to verify subsequent
evaluation of compounds in assays reflecting endogenous G-
protein coupling of the recombinantly expressed receptors
(Kostenis et al., 2005). Our data indicate that the potency values
of UK14,304 determined in the Ca2+ assay (1.9 nM and 5.1 nM in
Gaqi5/H7 and Ga16/B6 cells, respectively) were relatively close to
that obtained when forskolin-stimulated cAMP accumulation was
measured (11.2 nM). MK912 pre-treatment reversed the agonist
effect. These data are in agreement with those acquired previously
in cAMP assays on native receptors or recombinant a2C-ARs
coupled to endogenous G-proteins. UK14,304 concentration-
dependently inhibited forskolin-stimulated cAMP accumulation
in human neuroblastoma SH-SY5Y cells (EC50 = 7.83 nM, Parsley
et al., 1999) and also in LM(tk-) and CHO-K1 cells stably transfected
with the cloned human a2C-AR (EC50 = 8.02 nM, Jeon et al., 1995;
EC50 = 2.1 nM, Umland et al., 2001).
In addition to observing good agreement between the func-
tional activities, our Ca2+ measurement data also correspond well
to those generated in radioligand binding studies using mem-
branes prepared from the a2C-C1 cell line. Compounds used in the
Ca2+ assay were profiled using [3H]UK14,304 radioligand binding.
With the exception of oxymetazoline in Ga16/B6 cells, concentra-
tion–response curves for inhibition of [3H]UK14,304 binding
yielded essentially identical rank order of compound affinities
when compared with the Ca2+ assay. In the case of the Gaqi5/H7
cell line, EC50/IC50 values from the fluorometric Ca2+ measure-
474 D. Kurko et al. / Neurochemistry International 55 (2009) 467–475
ments and Ki values from binding tests were almost the same, only
yohimbine had a 4-fold higher value in the functional assay. In the
case of Ga16/B6 cells up to 8-fold differences were observed
between radioligand binding and fluorometric Ca2+ assays. More-
over, the radioligand binding data we obtained were very similar to
those reported by other investigators (Piletz et al., 1996; Umland
et al., 2001; Uhlén et al., 1997; Lalchandani et al., 2002; Mustafa
et al., 2005). Binding affinities of noradrenaline (Ki = 1.7 nM),
UK14,304 (Ki = 2.2 nM), oxymetazoline (Ki = 7.4 nM) for displacing
[3H]UK14,304 on human a2C-ARs expressed in CHO cells have been
reported previously (Umland et al., 2001). Radioligand binding
analyses of antagonists revealed a Ki value of 0.063 nM for MK912
(Lalchandani et al., 2002) and Ki values of 0.68 nM (Lalchandani
et al., 2002) and 0.88 nM (Mustafa et al., 2005) for yohimbine by
replacing [3H]rauwolscine.
In summary, in this study we demonstrated that the human
a2C-AR can be coupled to calcium signaling using chimeric Gaqi5 as
well as promiscuous Ga16 proteins. According to the radioligand
binding affinities of a2-AR agonist and antagonists and their
modulatory potential on forskolin-evoked cAMP accumulation,
pharmacological properties of a2-AR ligands observed in our co-
expression systems proved to be similar to those reported in native
Gai-coupled systems. However, our findings indicate that these
recombinant systems activate the chimeric or promiscuous G-
proteins differently, as co-expression of Gaqi5 protein with a2C-AR
provides a more sensitive cellular system for functional assays. The
established Ca2+ assay on dual recombinant protein expressing cell
lines may substitute or complement the existing cAMP assay
formats and can enhance the capacity for characterization of a2C-
AR ligands providing a robust and reliable functional assay more
appropriate for high-throughput screening examinations.
Acknowledgments
We thank for the excellent technical assistance of Mrs. E. Széll,
Mrs. E. Borók, Mrs. P. Unghy and Mrs. A. Garai. We are grateful to
Dr. I. Tarnawa for invaluable help in discussions and for critically
reading the manuscript.
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