Báo cáo Y học: Puri®cation and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli doc

Eur. J. Biochem. 269, 82±92 (2002) Ĩ FEBS 2002

Puri®cation and characterization of the human adenosine A2a
receptor functionally expressed in Escherichia coli
H. Markus Weiû and Reinhard Grisshammer*
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK

The adenosine A2a receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is
abundant in striatum, vasculature and platelets and is
involved in several physiological processes such as blood
pressure regulation and protection of cells during anoxia.
For structural and biophysical studies we have expressed
the human adenosine A2a receptor (hA2aR) at high levels
inserted into the Escherichia coli inner membrane, and
established a puri®cation scheme. Expression was in fusion
with the periplasmic maltose-binding protein to levels of
10±20 nmol of receptor per L of culture, as detected with
the speci®c antagonist ligand [3H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a
protease-resistant but still functional receptor was created
by truncation to Ala316. Addition of the sterol, cholesteryl
hemisuccinate, allowed a stable preparation of functional

Adenosine is a paracrine modulator of cell function that is
important for local regulatory processes in virtually all
mammalian organs. Adenosine is involved in protection of
cells during anoxia and is an ubiquitous neuromodulator in
the central nervous system [1±4]. So far, four human
adenosine receptors have been identi®ed (A1, A2a, A2b, and
A3) belonging to the family of G-protein-coupled receptors
(GPCRs). Like many other GPCRs, adenosine receptors
are potential drug targets. Drugs acting on the human

adenosine A2a receptor (hA2aR) are expected to have a
therapeutic potential in CNS disorders, in¯ammation or
stroke [5]. Mice lacking the adenosine A2a receptor show
increased aggression, hypoalgesia, faster platelet aggregation, high blood pressure and reduced exploratory activity
indicating involvement of the receptor in a variety of
physiological functions [6]. Direct structure determination
of hA2aR by X-ray or electron crystallography, or infor-

Correspondence to H. M. Weiû, Aesku.lab Diagnostika, Mikroforum
Ring 2, 55234 Wendelsheim, Germany. Fax: + 49 6734 9627 27,
Tel.: + 49 6734 9627 11, E-mail:
Abbreviations: CHS, cholesteryl hemisuccinate; DDM, n-dodecyl-b-Dmaltoside; DeM, n-decyl-b-D-maltoside; GPCR, G-protein-coupled
receptor; hA2aR, human adenosine A2a receptor; IMAC, immobilized
metal anity chromatography; IPTG, isopropyl thio-b-D-galactoside;
MBP, maltose-binding protein; NECA, 5¢-N-ethylcarboxamidoadenosine; R-PIA, R(±)N6-(2-phenylisopropyl)-adenosine; UM,
n-undecyl-b-D-maltoside; XAC, xanthine amine congener.
*Present address: Laboratory of Molecular Biology, NIDDK, NIH,
Building 50/4503, 50 South Drive, Bethesda, MD 20892-8030, USA.
(Received 6 August 2001, accepted 22 October 2001)

hA2aR solubilized in dodecylmaltoside to be obtained,
and, increased the stability of the receptor solubilized in
other alkylmaltosides. Puri®cation to homogeneity was
achieved in three steps, including ligand anity chromatography based on the antagonist xanthine amine congener. The puri®ed hA2aR fusion protein bound
[3H]ZM241385 with a Kd of 0.19 nM and an average Bmax
of 13.7 nmolámg)1 that suggests 100% functionality.
Agonist anities for the puri®ed solubilized receptor were
higher than those for the membrane-bound form. Sucient
pure, functional hA2aR can now be prepared regularly for
structural studies.

Keywords: adenosine A2a receptor; [3H]ZM241385;
G-protein-coupled receptor; maltose-binding protein
fusion; functional solubilization.

mation on the bound ligand conformation obtained by
NMR spectroscopy, would assist in the design of subtype
speci®c compounds and improve the understanding of
GPCR function.
GPCRs constitute a large protein family, but from the
thousands of members, only rhodopsin has been puri®ed in
large quantities from natural tissue. Functional heterologous expression and puri®cation of large quantities of
GPCRs has proved to be very dif®cult [7±10]. Crystallization, NMR spectroscopy, and other work that depends on
milligram quantities of puri®ed protein, are therefore
hindered. Structure determination of GPCRs is successful
when suf®cient pure protein is available, as shown for
rhodopsin [11±13]. Hence, functional over-expression and
stable puri®cation are the keys to more rapid progress in
understanding GPCR structure and function.
No well documented puri®cation of functional adenosine
A2a receptor and characterization of the puri®ed protein has
yet been reported. The A2a receptor has been heterologously
expressed but puri®ed only in small amounts for antibody
production [14]. The puri®cation of microgram quantities of
adenosine A1 receptor from different tissues has been
reported previously based on ef®cient ligand af®nity chromatography using the antagonist xanthine amine congener
(XAC) [15±17], but this procedure has never been used for
the adenosine A2a receptor.
We report here the expression of the hA2aR fused at its
N-terminus with the maltose-binding protein (MBP) and its
puri®cation in milligram quantities. The receptor is fully

functional according to ligand binding analysis. This is the
®rst puri®cation of an adenosine receptor in a functional
form and in suf®cient quantity for structural and biophysical
work.


Ó FEBS 2002

Puri®cation and characterization of A2a receptor (Eur. J. Biochem. 269) 83

MATERIALS AND METHODS
Materials
[3H]ZM241385 (629 GBqámmol)1) and ZM241385 were
obtained from Tocris (Bristol, UK). Theophylline, XAC,
R(±)N6-(2-phenylisopropyl)-adenosine (R-PIA), and 5¢-Nethylcarboxamidoadenosine (NECA) were purchased from
Sigma RBI. Chelating Sepharose, HiTrap Q Sepharose,
PD10 and NICK spin columns were from Amersham
Pharmacia (Uppsala, Sweden), Ni-nitrilotriacetic acid agarose was from Qiagen (Hilden, Germany) and Af®-Gel 10
gel was from Bio-Rad (Hercules, CA, USA). Adenosine
deaminase was purchased from Boehringer (Mannheim,
Germany). Cholesteryl hemisuccinate (CHS) was from
Sigma, n-dodecyl-b-D-maltoside (DDM) was from Anatrace (Maume, OH, USA) and n-undecyl-b-D-maltoside
(UM) and n-decyl-b-D-maltoside (DeM) were from Calbiochem (Nottingham, UK). The cDNA coding for hA2aR
[18] was generously provided by J. Coote (GlaxoWellcome,
Stevenage, UK).
Expression of hA2aR fusion proteins
Escherichia coli strain DH5a (Gibco BRL) [19] was used as
the host for recombinant plasmids. Cells were grown in
2 ´ TY medium [19] containing ampicillin (100 lgámL)1)
and glucose (0.2%, w/v). The cDNA coding for the hA2aR

[18] was modi®ed by standard cloning techniques and PCR
as follows. The start codon was replaced by a BamHI
restriction enzyme site, encoding the amino-acid residues
Gly-Ser, in-frame with the codon for Pro2 of the receptor
cDNA. The codon for the last used amino acid of the
receptor (Ser412 in case of the full-length receptor and
Ala316 for the truncated receptor) was followed by the
nucleotide sequence GCGGCCGCA that contains a NotI
restriction site and encodes three Ala residues in the ®nal
construct. The regions coding for the C-terminal tags
(Fig. 1) and two stop codons were ¯anked by NotI and
HindIII restriction sites at the 5¢ and 3¢ ends, respectively.
Cassettes were cloned into a pBluescript KS vector
(Stratagene) and were con®rmed by DNA sequencing.
For obtaining the ®nal expression vector, the cassettes were
cloned as BamHI/HindIII fragments into the E. coli
expression vector pRG/III-hs-MBP [20]. This vector contains the coding region for MBP including the N-terminal
signal sequence; Thr366 is followed by a BamHI site used
for introduction of the receptor cassette.
For expression, cells with the respective expression vector
were grown at 37 °C in 2-L ¯asks containing 500 mL
of 2 ´ TY medium supplemented with ampicillin
(100 lgámL)1), glucose (0.2%, w/v) and theophylline
(100 lM). At a D600  0.7, isopropyl thio-b-D-galactoside
(IPTG) was added to a ®nal concentration of 0.3 mM and the
temperature was reduced to 22 °C. Cells were harvested 22±
28 h later, frozen in liquid nitrogen, and stored at )70 °C.
Synthesis of XAC-agarose gel
The ligand af®nity gel was prepared based on the method
described by Nakata [15]. XAC was dissolved in dimethylsulfoxide at a concentration of 0.5 mgámL)1. Af®-Gel 10

resin was washed extensively with ice-cold isopropanol and

Fig. 1. hA2aR-fusion proteins used in this study. (A) Schematic representation of hA2aR fusion proteins. The boxes shown are not drawn to
scale. The names used in the text are given on the right. MBP, mature
E. coli maltose-binding protein (Lys1 to Thr366) followed by glycine
and serine encoded by a BamHI restriction site; hA2aR, human
adenosine A2a receptor (Pro2 to Ser412); hA2aRTr316, C-terminally
truncated human adenosine A2a receptor (Pro2 to Ala316); TrxA,
E. coli thioredoxin (Ser2 to Ala109); H10, 10 histidine residues; H10F,
10 histidine residues followed by the Flag-peptide. (B) C-terminal tags.
Amino-acid residues are given in the one-letter code. The sequence
AAA is encoded by a NotI restriction site, the sequence GT is encoded
by a KpnI site, the sequence EF is encoded by a EcoRI site, the
sequence DYKDDDDK corresponds to the Flag peptide.

then brie¯y with dimethylsulfoxide before adding it to the
XAC solution (12 mL XAC in dimethylsulfoxide for 1 mL
of packed gel). The suspension was slowly stirred overnight
at room temperature. The amount of covalently bound
XAC was estimated to be about 14 lmol per mL of gel by
monitoring the absorbance at 310 nm in 10 mM HCl of the
XAC solution before and after incubation with the gel. This
is about eight times more ligand per volume of gel than
reported by Nakata [15]. The gel was washed with
dimethylsulfoxide and then extensively with buffer (50 mM
Tris/HCl, pH 7.4), before ®nally being washed and stored in
20% ethanol.
Membrane preparation and solubilization
from membranes
All work was carried out at 0±4 °C. E. coli cells were thawed

and resuspended in lysis buffer (20 mM Hepes/KOH,
pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM phenylleupeptin,
methanesulfonyl
¯uoride,
0.5 lgámL)1
)1
0.7 lgámL pepstatin, 20 lgámL)1 DNaseI) using 2±3 mL
of buffer per gram of cells. The suspension was twice passed
through a French press. Membranes were pelleted by
centrifugation at 100 000 g for 1 h, suspended in buffer
(50 mM Hepes/KOH, pH 7.4, 100 mM NaCl, 30%
glycerol, 1 mM phenylmethanesulfonyl ¯uoride, 0.5
lgámL)1 leupeptin, 0.7 lgámL)1 pepstatin), snap-frozen in
aliquots and stored at )70 °C.
Solubilization of the hA2aR from membranes was
carried out at a protein concentration of about 6 mgámL)1
in membrane solubilization buffer (50 mM Hepes/KOH,
pH 7.4, 200 mM NaCl, 30% glycerol, 40 lM theophylline,
1 mM phenylmethanesulfonyl ¯uoride, 0.5 lgámL)1 leupeptin, 0.7 lgámL)1 pepstatin and 1% of the respective
detergent (DDM, UM or DeM) with or without 0.2%


84 H. M. Weiû and R. Grisshammer (Eur. J. Biochem. 269)

CHS. After incubation with slow rotation for 1 h at 4 °C,
samples were centrifuged for 45 min at 125 000 g. The
supernatant was saved as the solubilized fraction. Aliquots
were used for ligand binding assays.
Receptor solubilization from intact cells and puri®cation
All work was performed at 0±4 °C. One-hundred grams of

E. coli cells expressing M-A2aTr316-H10 (from 9 L of
culture) were thawed and resuspended, using a Kenwood
BL350 blender, in 175 mL of buffer (100 mM Hepes/KOH,
pH 7.4, 60% glycerol) supplemented with 700 lL leupeptin
(stock solution 0.25 mgámL)1), 700 lL pepstatin
(0.35 mgámL)1), 700 lL phenylmethanesulfonyl ¯uoride
(500 mM), 14 mL NaCl (5 M), 200 lL DNaseI
(10 mgámL)1), 700 lL theophylline (25 mM) and 1.75 mL
MgCl2 (1 M). Water was added afterwards to give a ®nal
volume of 315 mL. Then 35 mL of detergent stock solution
(10% DDM, 2% CHS) were added with stirring. The
suspension was sonicated with stirring on ice, using a
sonicator ultrasonic processor XL (Misonix, Farmingdale,
NY, USA), level 5, pulsing 1 s on/1 s off, with 12 s pulsing on
per gram of cells. Then the suspension was stirred for 30 min
on ice and centrifuged at 100 000 g for 1 h. The supernatant
(about 300 mL) was supplemented with protease inhibitors
(concentrations as above) and then added in batch to 50 mL
of chelating Sepharose, loaded with Ni2+ ions and equilibrated in buffer NiA (50 mM Hepes/KOH, pH 7.4, 200 mM
NaCl, 30% glycerol, 50 mM imidazole, 0.1% DDM, 0.02%
CHS), resulting in a ®nal imidazole concentration of less
than 15 mM. The suspension was slowly stirred for 3 h and
then packed into an Econo-column (Bio-Rad) with 5 cm
diameter. The resin was washed at 9 mLámin)1 with 600 mL
of buffer NiA supplemented with protease inhibitors (see
above). Bound receptor was eluted with 150 mL of buffer
NiB (50 mM Hepes/KOH, pH 7.4, 200 mM NaCl, 30%
glycerol, 400 mM imidazole, 0.1% DDM, 0.02% CHS,
0.5 mM phenylmethanesulfonyl ¯uoride, 0.25 lgámL)1 leupeptin, 0.35 lgámL)1 pepstatin) at 5 mLámin)1. NaCl and
imidazole concentrations were reduced by adding 150 mL of

buffer XDil (50 mM Hepes/KOH, pH 7.4, 30% glycerol,
0.1% DDM, 0.02% CHS, 0.5 mM phenylmethanesulfonyl
¯uoride, 0.25 lgámL)1 leupeptin, 0.35 lgámL)1 pepstatin)
and the solution was passed through a 0.22-lm ®lter
(Stericup, Millipore). XAC-agarose gel (10 mL), packed
into an XK 26 column (Amersham Pharmacia), was
equilibrated with buffer XA (50 mM Hepes/KOH, pH 7.4,
100 mM NaCl, 30% glycerol, 0.1% DDM, 0.02% CHS).
The ®ltered sample (about 300 mL) was loaded onto the
column overnight at 0.35 mLámin)1 and the column was
washed at 0.8 mLámin)1 with about 60 mL of buffer XA
supplemented with protease inhibitors. The receptor was
eluted at 25 °C in buffer XA, supplemented with 20 mM
theophylline and protease inhibitors, at a ¯ow rate of
0.6 mLámin)1. Due to the strong absorption of theophylline
at 280 nm, elution could not be monitored spectroscopically.
However, as judged from protein gels, the receptor was
almost completely eluted after 70 mL of elution buffer.
70 mL of the XAC-agarose gel eluate were diluted with
70 mL of buffer QDil (50 mM Hepes/KOH, pH 7.4, 30%
glycerol, 0.1% DDM, 0.02% CHS, 0.5 mM phenylmethanesulfonyl ¯uoride, 0.25 lgámL)1 leupeptin,
0.35 lgámL)1 pepstatin) and passed through a 0.22-lm ®lter.

Ĩ FEBS 2002

A prepacked 5 mL HiTrap Q Sepharose column was
washed with 25 mL of buffer QA (50 mM Hepes/KOH,
pH 7.4, 50 mM NaCl, 30% glycerol, 0.05% DDM, 0.01%
CHS) followed by 25 mL of buffer QB (50 mM Hepes/
KOH, pH 7.4, 1 M NaCl, 30% glycerol, 0.05% DDM,

0.01% CHS) and then equilibrated with 30 mL of buffer
QA. The sample (140 mL) was loaded at 1.5 mLámin)1.
After washing with 60 mL of buffer QA at 2.5 mLámin)1,
the receptor was eluted with 14% of buffer QB (183 mM
NaCl) at 1 mLámin)1 and 1.5 mL fractions were collected.
Peak fractions were pooled and concentrated using an
Ultrafree-15 centrifugal concentrator (50 000 molecular
weight cut-off, Millipore). Concentrated material was either
used immediately for 2D crystallization trials (not shown),
stored at 4 °C, or frozen in liquid nitrogen for long-term
storage.
Radioligand binding
All binding assays were carried out in polystyrol tubes
using LBA buffer (20 mM Hepes/KOH, pH 7.4, 100 mM
NaCl) and the A2a receptor speci®c antagonist
[3H]ZM241385. Samples were incubated on ice unless
stated otherwise. The incubation time was 3 h for competition binding assays, 1.5 h for one-point saturation assays
and for saturation experiments, and 1 h for saturation
experiments performed at room temperature. These times
were suf®cient to reach equilibrium as association and
dissociation of ZM241385 to the A2a receptor is fast [21]
even at low temperature (0±4 °C) (data not shown).
Nonspeci®c binding was determined in the presence
of 2.5±3.2 mM theophylline. Adenosine deaminase
(0.5 UámL)1) was included in all assays to remove
adenosine, except for saturation and competition binding
experiments on puri®ed receptor.
Amounts of tritiated antagonist were determined by
liquid scintillation counting. Binding data were analysed by
nonlinear least-squares ®tting using the program GRAPHPAD

PRISM. Competition curves were ®tted to the four-parameter
logistic function.
Assays on membrane-bound receptors. For one-point
saturation assays, membranes or E. coli cells were incubated
in a total volume of 400 lL containing 6±9 nM
[3H]ZM241385. Competition binding assays using membranes were performed in a ®nal volume of 1.2 mL with
[3H]ZM241385 at a concentration of 0.75 nM. In saturation
experiments on membranes, the total volume was 1.5 mL.
Bound and free ligand were separated by rapid vacuum
®ltration over GF/B ®lters soaked in 0.3% polyethylenimine. Filtration was carried out with a Brandel cell
harvester at 4 °C. Filters were washed three times with
ice-cold buffer (20 mM Hepes/KOH, pH 7.4). For calculation of the parameter Ôreceptors per cellÕ, the number of
cells growing in suspension was estimated by measuring
D600: a D600 of 1 was assumed to correspond to
109 cellsámL)1.
Assays on solubilized receptors. Assays were performed in
LBA buffer containing 0.1% DDM and 0.02% CHS in a
volume of 200 lL (one-point saturation assays) or 300 lL
(saturation and competition experiments). The concentration of [3H]ZM241385 was 0.75 nM in competition assays


Ó FEBS 2002

Puri®cation and characterization of A2a receptor (Eur. J. Biochem. 269) 85

and 12±18 nM in one-point saturation assays. Bound and
free ligand were separated by gel ®ltration using NICK
Spin columns. Columns were equilibrated in LBA buffer
containing 0.1% DDM and 0.02% CHS, precooled to
4 °C and precentrifuged for 3 min at 500 g (4 °C).

Aliquots of the assay mix (100 lL for one-point saturation
assays, and 170 lL in saturation and competition experiments) were loaded and receptor-bound ligand was eluted
by spinning at 630 g (4 °C). Binding analysis on receptors
solubilized in DeM or UM were performed as above
except that DDM was substituted for 0.1% DeM or 0.1%
UM in the assay mix and NICK Spin equilibration buffer.
Theophylline, present in samples eluted from the ligand
af®nity column, was removed by gel ®ltration using
PD10 columns equilibrated in buffer consisting of 20 mM
Hepes/KOH, pH 7.4, 100 mM NaCl, 0.1% DDM and
0.02% CHS.
SDS/PAGE and N-terminal sequencing
Proteins were incubated in sample buffer (125 mM Tris/
HCl, pH 8.1, 3.75% SDS, 12.5% glycerol, 6% 2-mercaptoethanol, 0.002% Bromophenol Blue) at room temperature for at least 15 min and separated by SDS/PAGE on
high-molarity Tris buffered gels [22]. For N-terminal
sequencing, the protein was electro-blotted onto poly(vinylidene di¯uoride) membranes (Immobilon-P, Millipore) as
described previously [23]. Sequence analyses were performed with an Edman automated N-terminal protein
sequencer (Procise 494, Applied Biosystems).
Protein assay and amino-acid analysis
Protein concentrations were determined by the Amido black
assay [24] using BSA as a standard. For the puri®ed
receptor, amino-acid analysis was performed on a Biochrom
20 amino-acid analyser (Amersham Pharmacia) after
hydrolysis in 6 M HCl for 18 h at 110 °C. Comparing
results from amino-acid analysis and Amido black assays
indicated that the latter underestimated the amount of
puri®ed receptor fusion protein by 12%. Protein concentrations of ®nal puri®ed receptor given in the text and tables
have been corrected accordingly.

RESULTS

Expression
For work aiming at structural and biophysical studies on
GPCRs, high expression levels are essential. As the expression level of a given receptor is dif®cult to predict, we
performed an initial screen, expressing the cDNAs of four
different human GPCRs (somatostatin receptors S2 and S4,
and adenosine receptors A1 and A2a). Two expression
systems were investigated. In E. coli, all four receptors were
N-terminally fused to MBP, whereas in the yeast Pichia
pastoris they were N-terminally fused to the a-factor
prepropeptide (S2 and A1 receptors only). We employed
vectors previously used for the successful expression at high
levels of the rat neurotensin receptor in E. coli and of the
mouse 5HT5A 5-hydroxytryptamine and human b2-adrenergic receptors in P. pastoris [20,25]. All receptor fusion
proteins could be detected by immunoblot-analyses.

Radioligand binding-analyses on E. coli and P. pastoris
membrane preparations, containing the S2 or the S4
somatostatin receptor fusion protein did not reveal speci®c
binding of the agonist somatostatin. In contrast, the
adenosine A1 receptor fusion proteins displayed high
af®nity binding of the antagonist [3H]8-cyclopentyl-1,3dipropylxanthine in both expression systems; expression
levels of functional A1 receptor were 3±4 nmol and
1±2 nmol per litre of shake ¯ask culture in E. coli and in
P. pastoris, respectively (data not shown).
Best expression levels, deduced from immunoblot- and
radioligand binding-analyses, were achieved with the
hA2aR using E. coli as the expression host. This receptor
and expression system were therefore pursued. Expression
of the hA2aR (construct M-A2a-H10F; Fig. 1) was suf®ciently high to start puri®cation. However, the receptor
C-terminus was sensitive to proteolysis after solubilization.

A second construct, M-A2a-TrxA-H10F (Fig. 1), allowed
identi®cation of the major C-terminal degradation product.
This information was used to make a number of protease
resistant constructs truncated at the C-terminus. One of
those (M-A2aTr316-H10; Fig. 1) was used for the puri®cation and characterization described here.
Culture conditions were optimized for functional expression of hA2aR using the construct M-A2a-H10F (Fig. 1).
Addition of glucose (0.2% (w/v) or more) to the medium
increased the ®nal cell density from an D600 of about 1.5 to a
D600 of about 6. The number of receptors per cell, monitored
by binding of [3H]ZM241385 to whole E. coli cells, increased
about fourfold by including 0.2% glucose, but decreased
again at higher glucose concentrations (0.4%). Addition of
theophylline (50±100 lM) to the medium improved the
expression level by another 10±30% per volume. Induction
at a D600 of 0.6±1.0 was optimal whereas earlier induction
resulted in poor cell growth. Neither the concentration of
IPTG within a certain range (100 lM)1 mM), nor the use of
different E. coli strains (BL21, CAG627, KS474), had a
signi®cant effect on expression levels. To achieve the highest
levels of functional receptors, cells were grown for 22±28 h
after induction; longer growth (40 h) did not improve
expression further. The conditions described in the Materials
and methods section resulted in receptor concentrations of
10±20 nmol per L of culture, or 1000±2000 receptors per cell.
This corresponds to 17±34 pmol receptor per mg of membrane protein. Constructs coding for full-length receptors
gave on average better expression than the truncated form
used for puri®cation.
Solubilization
Solubilization from membranes is necessary for puri®cation
of a membrane protein. However, for fragile membrane

proteins such as GPCRs, it is dif®cult to ®nd conditions that
allow ef®cient extraction and maintain structural integrity.
We used radioligand binding assays to monitor hA2aR
integrity and stability. Approximately 70±90% of speci®c
[3H]ZM241385 binding sites were solubilized from membrane preparations using DDM. Use of the shorter chain
derivatives UM and DeM resulted in poorer recoveries, and
the solubilized receptor was less stable (Fig. 2). Addition of
the cholesterol derivative CHS to solubilization experiments
increased the recovery to nearly 100% for all three
alkylmaltoside detergents, and increased the stability of


86 H. M. Weiû and R. Grisshammer (Eur. J. Biochem. 269)

Fig. 2. Solubilization and stability of hA2aR (M-A2aTr316-H10) in
di€erent alkylmaltosides, with or without addition of CHS. Solubilization from membranes and one-point saturation assays were carried out
as described in the Materials and methods section. Solubilized fractions were stored at 4 °C in membrane solubilization bu€er for the
time indicated. Solubilization was in 1% DeM (j,h), 1% UM (.,,)
or 1% DDM (d,s) with (®lled symbols) or without (open symbols)
the addition of 0.2% CHS. In many cases, the error bars are smaller
than the symbols. Results shown are from one of two experiments.

receptors solubilized in UM and DeM (Fig. 2). Receptor
half-lives in the solubilized fraction (deduced from two
experiments, one of which is given in Fig. 2) were 7 days
using DeM, 26 days using UM and 40±130 days using
DDM. When CHS was employed in combination with any
of the three alkylmaltosides, half-lives were in the range
40±130 days. Both the full-length hA2aR (M-A2a-H10F)
and the truncated form (M-A2aTr316-H10) behaved

identically in these experiments. For puri®cation of
M-A2aTr316-H10, solubilization was carried out with
DDM and CHS, starting with whole cells instead with
membrane preparations. Recoveries were lower (50±60%);
however, the time consuming membrane preparation and
losses in this step (usually more than 40%) were avoided.
Truncation of the receptor C-terminus
Compared to other members of the rhodopsin family,
hA2aR has a long C-terminus consisting of amino acids
294±412 [26]. Its susceptibility to proteolysis has been
discussed previously [14,27]. Immunoblot analysis indicated
that the C-terminus of the fusion protein M-A2a-H10F was
rapidly cleaved upon solubilization, whereas the membrane-

Ó FEBS 2002

bound receptor was more resistant. Proteolysis could be
reduced by neither the use of protease de®cient E. coli
strains (CAG 627, KS 474), nor with additional protease
inhibitors (DFP, Pefabloc, a2 macroglobulin, complete
protease inhibitor mix (Boehringer), (4-amidinophenyl)methanesulfonyl ¯uoride, results not shown). IMAC starting with solubilized M-A2a-TrxA-H10F fusion protein
allowed the isolation of a C-terminal degradation product
that was subject to N-terminal sequence analysis. The
resulting amino-acid sequence Leu-Val-Ser-Gly-Gly-SerAla-Gln is found in the receptor C-terminus starting from
Leu365. As shortening of the C-terminus by 95 residues has
been shown to affect neither ligand binding properties nor
adenylate cyclase activation nor functional desensitization
[26], we focused on the fusion protein M-A2aTr316-H10
where the C-terminus is truncated by 95 residues. This
truncated fusion protein was puri®ed without any sign of

proteolytic degradation.
Puri®cation
The optimized large-scale puri®cation of the fusion protein
M-A2aTr316-H10 from 100 g of E. coli cells is documented
in Table 1 and Fig. 3. The ®nal recovery from three
preparations carried out on identical scale was 29 (‹ 2)%
of the total solubilized fraction. The experimental value for
speci®c radioligand binding was 13.7 (‹ 0.8) nmolámg)1,
which is in agreement with the theoretical value of
13 nmolámg)1 for the 77-kDa fusion protein, assuming
one ligand binding site per molecule. Puri®cation of protein
starting from 70 g of cells gave similar results. The following
parameters were investigated to optimize the puri®cation
procedure. Batch loading of the IMAC resin resulted in
much better recoveries (> 50%) compared to column
loading (< 20%). Batch binding of the receptor fusion
protein to the IMAC gel was relatively slow, requiring 3 h to
achieve greater than 80% binding. The presence of E. coli
thioredoxin between the truncated receptor C-terminus and
the deca-histidine tag accelerated binding to the IMAC gel
(data not shown), probably by improving the accessibility of
the tag. However, this was not investigated further as good
recoveries were achieved for M-A2aTr316-H10 by batch
loading for 3 h using suf®cient amounts of Ni2+ loaded
chelating Sepharose. The binding capacity of the gel was
found to be low ( 100 lg of fusion protein per mL of gel
as judged from speci®c [3H]ZM241385 binding). The
binding capacity of Ni2+ loaded chelating Sepharose was
slightly higher compared to that of Ni-nitrilotriacetic acid
agarose but so was the background binding.

Table 1. Puri®cation of the fusion protein M-A2aTr316-H10 from 100 g of E. coli cells (data from one representative experiment). The puri®cation
was performed as described in the Materials and methods section. Total receptor was determined by one-point saturation binding of
[3H]ZM241385. Total protein was determined with the Amido black assay [24] and this was corrected by the results from amino-acid analysis
(+ 12%) for the puri®ed receptor (Q Sepharose eluate).
Total receptor
(pmol)
Solubilized material
IMAC eluate
XAC ¯ow through
XAC eluate
Q Sepharose eluate

Relative yield
(%)

Protein concn
(mgámL)1)

Total protein
(mg)

Speci®c binding
(pmolámg)1)

70896
43790
8735
23657
18091

100
62
(12)
33
26

24.6
0.35
0.128
0.045
0.200

7306
53.6
44.8
3.2
1.5

9.7
817
195
7393
12061

Puri®cation
(fold)
1
84
±

762
1243


Ó FEBS 2002

Puri®cation and characterization of A2a receptor (Eur. J. Biochem. 269) 87

Fig. 3. Puri®cation of hA2aR (M-A2aTr316-H10) from E. coli cells.
The puri®cation was performed as described in the Materials and
methods section and is quantitatively documented in Table 1. The
following fractions were analysed by 10% SDS/PAGE and silver
staining. Lane 1, high molecular mass standard (Sigma); lane 2, 1.5 lg
of the solubilized fraction; lane 3, 0.5 lg of IMAC eluate; lane 4, 0.5 lg
of XAC ¯ow through; lane 5, 0.2 lg of XAC eluate; lane 6, 0.2 lg of Q
Sepharose eluate (eluted with 14% bu€er QB, ®nal puri®ed fraction);
lane 7, 0.2 lg of protein eluted with 100% bu€er QB from the Q
Sepharose.

IMAC puri®ed M-A2aTr316-H10 bound almost quantitatively to the ligand af®nity matrix when loaded slowly
(0.23 mLámin)1, Fig. 4), indicating that the majority of the
receptor protein was correctly folded. For scaling up, ¯ow
rates were increased to 0.35 mLámin)1 to allow the preparation to be completed within 2 days, leading to slightly
poorer binding to the XAC-agarose (Fig. 3; Table 1).
Elution from the ligand af®nity column was slow even at
increased temperature (25 °C) resulting in a dilute eluate.
The low af®nity antagonist theophylline was used for
elution as high af®nity hA2aR antagonists are hardly
soluble in aqueous buffers. In some experiments the
antagonist ZM241385 was used for elution. However, it

was found to bind nonspeci®cally to the XAC-agarose.
Theophylline was removed by gel ®ltration before quantifying speci®c [3H]ZM241385 binding in the XAC-agarose
eluate. The value obtained was only 50±80% of the
theoretical value calculated for pure and fully functional
receptor. Possible reasons for this ®nding are: (a) quanti®cation of binding sites is inaccurate (presence of theophylline) or (b) denaturation of some receptor due to the
increased temperature used for elution from the ligand
column. However, the ®nal ion-exchange step increased the
speci®c radioligand binding to its theoretical value. This
might result from the complete removal of theophylline in
this step or the separation of denatured protein from
functional receptor. Indeed, receptor protein with low
speci®c binding (0.2±8 nmolámg)1) is eluted from the ionexchange column at high salt concentrations (Fig. 3, lane 7).
Integrity and stability of the puri®ed protein
M-A2aTr316-H10
The puri®ed receptor runs in SDS/PAGE gels as a single,
slightly diffuse band with an apparent molecular mass of

Fig. 4. E. coli expressed and solubilized hA2aR (M-A2aTr316-H10)
binds speci®cally and quantitatively to a XAC-agarose gel. Shown is a
section of a 10% silver-stained SDS/PAGE gel. Lane 1, 0.4 lg of the
fraction loaded onto the XAC-agarose (IMAC puri®ed); lane 2, 0.4 lg
of the XAC-agarose ¯ow through fraction; lane 3, 0.2 lg of protein
eluted with 20 mM theophylline. The arrow points to the
M-A2aTr316-H10 fusion protein. Note that the main contamination,
seen in lane 1, runs only marginally above the hA2aR fusion protein
and is the main band in lane 2. In this puri®cation, 7% of speci®c
ligand binding sites loaded onto the XAC-agarose were detected in the
¯ow through fraction. The puri®cation was carried out as outlined in
the Materials and methods section but starting from 70 g of cells.
Ni-nitrilotriacetic acid agarose was used for the IMAC step and

washing and elution were carried out with 35 and 200 mM imidazole,
respectively. The XAC-agarose was loaded at a ¯ow rate of
0.23 mLámin)1.

65 kDa (Fig. 3, lane 6), which deviates by 12 kDa from the
calculated value of 77 kDa. To show that this resulted from
atypical running behaviour, frequently observed with
membrane proteins, rather than from proteolysis, we
veri®ed the identity of the hA2aR fusion protein by speci®c
radioligand binding (see below), N-terminal sequencing and
amino-acid analysis. The sequence obtained, namely LysIle-Glu-Glu-Gly-Lys-Leu-Val-Ile-Trp corresponds to the
N-terminus of the mature maltose-binding protein. Binding
of the fusion protein to the IMAC gel in buffer containing
50 mM imidazole indicates the presence of the C-terminal
histidine tag. We conclude that the 65-kDa band observed
in SDS acrylamide gels corresponds to the 77-kDa fusion
protein.
When stored at 4 °C in buffer consisting of 50 mM
Hepes/KOH, pH 7.4, 200 mM NaCl, 30% glycerol, 0.1%
DDM and 0.02% CHS, speci®c [3H]ZM241385 binding of
the puri®ed receptor decreased to 81% over 40 days
corresponding to a half-life of 3.5±6.0 months (one experiment, ®ve time points). In another experiment, the in¯uence
of speci®c ligands on the stability of puri®ed receptor was
tested at 4 °C in buffer consisting of 50 mM Hepes/KOH,
pH 7.4, 100 mM NaCl, 6% glycerol, 0.05% DDM and
0.01% CHS. Ligands were added at concentrations of 10±20
times their Ki value. Without ligand, a half-life of 33 days
was obtained. This increased to 77 days in the presence of
NECA (agonist) and 53 days in the presence of theophylline
(antagonist) (one experiment, eight time points over a

period of 116 days, a one-phase exponential decay curve
was ®tted to the data). Addition of the antagonist
CGS15943 had no effect.


88 H. M. Weiû and R. Grisshammer (Eur. J. Biochem. 269)

Ĩ FEBS 2002

Signi®cance of CHS for receptor stability
during puri®cation
DDM-solubilized M-A2aTr316-H10 (no CHS added)
had a half-life of more than 40 days when stored at 4 °C
(Fig. 2, s). However, subsequent puri®cation performed in
DDM without CHS resulted in low recovery of functional
receptor (0.4 nmol of speci®c [3H]ZM241385 binding sites,
starting from 54 nmol). Despite near homogeneity, as seen
on SDS/PAGE gels, the DDM-puri®ed receptor displayed
low values for speci®c binding (465 pmolámg)1), indicating
loss of receptor functionality.
A detailed investigation showed that CHS was essential
to maintain receptor functionality during the course of
puri®cation. The half-live of DDM-solubilized receptor was
reduced to less than 1 day by enrichment using IMAC
(Fig. 5, j). Addition of CHS during solubilization and into
the puri®cation buffers increased the stability of IMAC
puri®ed receptor by at least 20-fold (Fig. 5B, s). Addition
of CHS during solubilization, but not during the subsequent
IMAC step, was not suf®cient to maintain the receptor
stable (Fig. 5B, m).

The presence of CHS not only improved solubilization
recoveries using different alkylmaltoside detergents (Fig. 2),
but also allowed a pure and stable receptor preparation to
be achieved.
Ligand binding studies

Fig. 5. The stability of solubilized hA2aR (M-A2aTr316-H10) during
puri®cation is dependent on the presence of CHS. (A) Solubilized fraction. (B) IMAC puri®ed fraction. Solubilization and puri®cation by
IMAC was performed as described in the Materials and methods
section starting with 10 g of cells for each condition described. Suspension of cells for solubilization was achieved using a potter instead of
a blender. Ni-nitrilotriacetic acid agarose was used for the IMAC step.
Washing and elution were carried out with 35 and 200 mM imidazole,
respectively. One-point saturation assays with [3H]ZM241385 were
performed as outlined in the Materials and methods section, with
0.02% CHS in all binding assays. The results shown are from one of
two independent experiments. In many cases, the error bars are smaller
than the symbols. j, no CHS added for solubilization and puri®cation; m, 0.2% CHS in solubilization bu€er and no CHS in bu€er NiA
and NiB; s, 0.2% CHS in solubilization bu€er and 0.02% in bu€ers
NiA and NiB.

The quanti®cation of interactions with speci®c ligands
allows the structural integrity and homogeneity of a puri®ed
receptor protein to be judged. The M-A2aTr316-H10 fusion
protein displayed high af®nity binding to all ligands tested
both in membrane-bound and puri®ed form. Af®nities to
agonists were about one order of magnitude higher for the
puri®ed receptor compared to those for the membranebound form, whereas antagonist af®nities were similar in
both cases (Table 2).
Ligand binding on puri®ed receptor protein was carried
out at 0±4 °C to avoid receptor denaturation at higher

temperatures. Binding to membrane-bound receptors was
performed on ice and at room temperature. Puri®ed
receptors displayed a single af®nity for the antagonist
[3H]ZM241385 (Fig. 6C) with a Kd value of
0.19 ‹ 0.02 nM and a Bmax value of 12.4 ‹ 0.5 nmolámg)1
(n ˆ 3). In contrast, data from saturation experiments on

Table 2. Pharmacological pro®le of M-A2aTr316-H10 in membrane-bound and puri®ed form. Values are from at least two independent experiments
analysed using the four-parameter logistic function and given as mean ‹ standard error. Membrane preparation, puri®cation and competition
experiments were carried out as outlined in the Materials and methods section. Incubation of assays was on ice for 3 h with [3H]ZM241385 at a
concentration of 0.75 nM. Ki values for binding to the puri®ed receptor were calculated according to Cheng & Pruso€ [53] with a Kd for
[3H]ZM241385 of 0.19 nM.
Membrane-bound receptor
Ligand
Agonists
NECA
R-PIA
Antagonists
Theophylline
XAC

Puri®ed receptor

IC50 (M)

n

IC50 (M)

Ki (M)

n

3.6 ‹ 0.2 ´ 10)6
6.5 ‹ 0.4 ´ 10)5

0.73 ‹ 0.04
0.87 ‹ 0.18

3.9 ‹ 0.2 ´ 10)7
6.5 ‹ 0.1 ´ 10)6

7.9 ´ 10)8
1.3 ´ 10)6

0.96 ‹ 0.06
1.01 ‹ 0.02

2.3 ‹ 0.3 ´ 10)5
1.7 ‹ 0.4 ´ 10)7

0.94 ‹ 0.11
0.96 ‹ 0.06

1.3 ‹ 0.3 ´ 10)5
1.6 ‹ 0.2 ´ 10)7

2.7 ´ 10)6
3.2 ´ 10)8

1.19 ‹ 0.15
1.12 ‹ 0.11


Ó FEBS 2002

Puri®cation and characterization of A2a receptor (Eur. J. Biochem. 269) 89

Fig. 6. Saturation binding of [3H]ZM241385 to membrane-bound and puri®ed hA2aR (M-A2aTr316-H10). Membrane preparation, receptor puri®cation and ligand binding analyses were performed as outlined in the Materials and methods section. The results shown are from one of six (A),
two (B) or three (C) experiments performed in duplicate. In many cases, the error bars are smaller than the symbols. (A) Saturation binding to
membrane-bound M-A2aTr316-H10 at 0±4 °C. (B) Saturation binding to membrane-bound M-A2aTr316-H10 at room termperature ( 25 °C).
(C) Saturation binding to puri®ed M-A2aTr316-H10 at 0±4 °C. Insets: Scatchard transformation of the binding data. B, bound; F, free. Kd values
determined by nonlinear least-squares ®tting are given in the results section.

ice, using membrane-inserted receptors were signi®cantly
(P < 0.01) better ®tted assuming two binding sites (®ve out
of six experiments) with a Kd1 value of 0.10 ‹ 0.03 nM
(44 ‹ 5% of binding sites) and a Kd2 value of
1.42 ‹ 0.47 nM (56 ‹ 5% of binding sites) (Fig. 6A).
However, at room temperature, membrane-bound receptors
displayed only one af®nity for the antagonist (Kd value of
0.65 ‹ 0.02 nM, n ˆ 2) (Fig. 6B). Bmax values for membrane-bound receptors were 10±20% higher when measurements were carried out on ice rather than at room
temperature.
Unlabeled adenosine receptor ligands, tested at 0 °C,
inhibited speci®c [3H]ZM241385 binding to both membrane-bound and puri®ed hA2aR by more than 90% at the
highest concentrations used (6.7 mM theophylline, 200 lM
XAC, 2 mM NECA, 2 mM R-PIA; Fig. 7). The agonist
NECA bound with higher af®nity to membrane-bound and
puri®ed hA2aR than the agonist R-PIA, which is a typical
feature of the adenosine A2a receptor. The rank order of

potency for the puri®ed receptor (XAC > NECA > R-PIA
> theophylline; Fig. 7B; Table 2) is identical to that
reported for membrane-bound hA2aR expressed in CHO
cells [28,29]. In contrast, the rank order was found to be
different for the membrane-bound M-A2aTr316-H10
receptor (XAC > NECA > theophylline > R-PIA;
Fig. 7A). Pseudo-Hill coef®cients, derived from agonist
competition curves on membrane-bound receptors, were
smaller than unity, despite the absence of G proteins in
E. coli. For antagonist competition curves, pseudo-Hill
coef®cients were  1. A similar behaviour, with agonist, but
not antagonist, pseudo-Hill coef®cients < 1 and no guanine
nucleotide effect, was reported for the dopamine D2S
receptor expressed in insect cells [30]. All competition curves
obtained for puri®ed receptors displayed pseudo-Hill coef®cients  1.

DISCUSSION
Puri®cation of a hA2aR fusion protein (M-A2aTr316-H10),
expressed in E. coli, resulted in 1.5 mg of homogenous, fully
functional protein from 100 g of wet cells. The amount of
puri®ed receptor protein is at least 100-fold greater than that
for adenosine A1 receptor obtained from different tissues or

Fig. 7. Displacement of [3H]ZM241385 binding to membrane-bound
and puri®ed hA2aR (M-A2aTr316-H10). Membrane preparation,
receptor puri®cation and ligand binding analyses were performed as
outlined in the Materials and methods section. Incubation was on ice
for 3 h in LBA bu€er which was supplemented with 0.1% DDM/
0.02% CHS for the analysis of puri®ed receptors. [3H]ZM241385 was
present at a concentration of 0.75 nM. The results shown are from one

of at least two independent experiments. Ligands used for displacement were: m, NECA; j, R-PIA; ,, theophylline; s, XAC. (A)
Displacement curves for membrane-bound M-A2aTr316-H10. (B)
Displacement curves for puri®ed M-A2aTr316-H10. IC50 values for
the displacing drugs are given in Table 2.


90 H. M. Weiû and R. Grisshammer (Eur. J. Biochem. 269)

for tagged A1 receptor puri®ed from stably transfected
CHO cells [17,31]. To date, no quanti®ed puri®cation of the
adenosine A2a receptor has been reported. The availability
of milligram quantities of functional M-A2aTr316-H10
fusion protein will allow extensive crystallization trials.
The adenosine A2a receptor is expressed to high levels in a
functional form in insect cells using the baculovirus
expression system [32]. In contrast, expression levels in
mammalian cells are low [28,29]. We have achieved
expression of correctly folded hA2aR in E. coli by using a
vector system optimized for expression of functional
GPCRs [20,33]. The level of functional expression is
amongst the highest reported for a GPCR in the E. coli
system [7,8,10]. Remarkably, the fraction of correctly folded
receptors in the solubilized material was close to 100% as
indicated by the high degree of speci®c binding to a ligand
af®nity column (Fig. 4). In contrast, a high proportion of
misfolded protein in membranes and sometimes also in the
solubilized fraction is reported for some membrane proteins
expressed in insect cells [34,35]. Furthermore, the lack of
glycosylation in E. coli excludes one source of heterogeneity
and can therefore be considered as an advantage for

crystallization.
Shortening of the hA2aR C-terminus was necessary in
this work to achieve protease resistance and to allow a
homogeneous receptor preparation. As shown previously in
mammalian cells, a deletion after Ala316, corresponding to
the truncation used in this study, did not in¯uence receptor
properties such as ligand binding, adenylate cyclase activation and functional desensitization [26]. In agreement with
this, previous work also showed that shortening of the
C-terminus in combination with prevention of receptor
glycosylation did not alter the ligand binding properties of
the canine adenosine A2a receptor [27]. This indicates that
the truncated receptor puri®ed by us is suf®cient to ful®l the
main receptor functions and is appropriate for structural
and biophysical investigations. Comparison of the aminoacid sequences of adenosine receptors with those of other
GPCRs indicates that the C-terminus of the adenosine A2a
receptor consists of about 120 amino acids, which compares
to only 30±45 amino acids for the other three known
adenosine receptors. The functional signi®cance of this long
C-terminus is so far unclear, but it is likely to be involved in
A2a receptor speci®c protein±protein interactions.
Ef®cient functional extraction from membranes and
stability of solubilized GPCRs are crucial to obtain
suf®cient protein for structural studies. Solubilization of
functional GPCRs has in many cases been achieved with
digitonin [15,36,37]. However, impurities and batch variations make it unsuitable for reproducible puri®cation and
subsequent crystallization experiments [38]. In contrast, the
alkylmaltosides employed in this study are chemically well
de®ned and commercially available at high purity. The
combination of the cholesterol derivative CHS and different
alkylmaltosides allowed highly ef®cient solubilization of A2a

receptor in functional form. The reported recoveries of
speci®c ligand binding sites (100% from membranes and
50±60% from whole cells) compare favourably to the
 25% solubilization ef®ciency of adenosine A2a receptor
from striatal membranes obtained with Chaps/cholic acid,
with Chaps and with digitonin [39±41]. Using our detergent
combinations, hA2aR was much more stable in solubilized
as well as puri®ed form than A2a receptor solubilized with

Ó FEBS 2002

Chaps [40] that lost 45% of binding sites within 15 days.
CHS has been bene®cial for the stability of solubilized
somatostatin receptor from pancreatic acinar cells [42], for
puri®cation of the rat neurotensin receptor [20] and for
functional solubilization of the mouse 5HT5a hydroxytryptamine receptor expressed in yeast [43]. The bene®cial effects
of CHS on receptor integrity could be due to its structural
similarity to cholesterol for which a direct or indirect
in¯uence on receptor function has been shown for a number
of GPCRs [44,45]. CHS in combination with different
alkylmaltosides is thus an alternative to digitonin in
functional puri®cation of GPCRs. However, preliminary
results from analytical gel ®ltration experiments show that
addition of CHS results in broad peaks indicating a
substantial heterogeneity in micelle size.
Even in cases of good expression, a 250- to 2500-fold
enrichment is typically necessary to purify a GPCR from a
recombinant source. This level of puri®cation is usually not
possible in a single step [20,32,34,36]. We used IMAC as an
ef®cient ®rst step to capture the fusion protein

M-A2aTr316-H10. The deca-histidine tag allowed IMAC
to be performed under more stringent conditions compared
to shorter histidine tags, resulting in better enrichment [46].
However, in contrast to results obtained with a decahistidine tagged neurotensin receptor [46], the binding of
M-A2aTr316-H10 to Ni-nitrilotriacetic acid resin packed
into a column was extremely poor, with recoveries not
exceeding 20% testing a variety of conditions. In contrast,
good binding was achieved by batch-loading allowing
suf®cient time. The subsequent ligand af®nity step resulted
in an almost homogenous preparation (Fig. 3, lane 5). A
XAC-based af®nity gel has been used for puri®cation of
adenosine A1 receptors [15±17] but not for enrichment of
functional A2a receptors. The M-A2aTr316-H10 fusion
protein bound very ef®ciently to the XAC gel (Fig. 4). Fast
elution to give a high receptor concentration was dif®cult,
and best results were obtained by increasing temperature
and using high concentrations of the weak antagonist
theophylline combined with low ¯ow rates. The ®nal ionexchange step allowed removal of theophylline, and a
reduction of volume and detergent concentration. The
higher degree of receptor homogeneity obtained by the ionexchange step will help crystallization. This step also allows
fast detergent exchange in preparation for crystallization
experiments.
The M-A2aTr316-H10 fusion protein was characterized
by ligand binding analyses to judge its identity and integrity.
Binding of the antagonist [3H]ZM241385 was speci®c and
saturable for both membrane-bound and puri®ed receptors.
The Kd value obtained for the membrane-bound hA2aR
fusion protein at room temperature (0.65 nM) is in good
agreement with a Ki value of 0.8 nM measured at 25 °C for
the hA2aR expressed in HEK-293 cells [47]. In contrast to

experiments performed at room temperature, two af®nity
states were resolved when binding assays were performed at
0±4 °C (Fig. 6A,B). The rank order of potency was also
found to be altered in competition experiments with
membrane-bound receptor conducted at 0 °C (Fig. 7,
Table 2) compared to that reported for hA2aR assayed at
higher temperature [28,29]. These observations are in
agreement with data from binding experiments performed
with membrane-bound adenosine A2a receptor at 21 and
0 °C using a eukaryotic expression system [48]. The


Ó FEBS 2002

Puri®cation and characterization of A2a receptor (Eur. J. Biochem. 269) 91

observed changes could result from an altered membrane
¯uidity and lateral pressure on the membrane-bound
receptor at low temperature, similar to effects due to a
changed lipid composition [49,50].
The puri®ed receptor displayed a single apparent af®nity
for each ligand tested, including [3H]ZM241385 (Table 2)
and an identical rank order of potency as the hA2aR in
CHO cell membranes (XAC > NECA > R-PIA >
theophylline), determined by using agonist or antagonist
radioligands [28,29]. Ki values for the puri®ed receptor
compare to Ki values determined for membrane-bound
hA2aR on platelets or expressed in CHO cells [28,29] as
follows. R-PIA: 1.3 lM (E. coli), 0.86 and 0.68 lM (CHO),
1.6 lM (platelets); NECA: 79 nM (E. coli), 66 nM (CHO),

30 nM (platelets); XAC: 32 nM (E. coli), 1 and 4 nM
(CHO), 5 nM (platelets); theophylline: 2.7 lM (E. coli), 1.7
and 5 lM (CHO). The presence of 100 mM NaCl in our
assays, but not in the assays performed with platelet and
CHO cell membranes may explain the reduced af®nity
towards XAC. NaCl (100 mM) was shown to reduce
binding of [3H]XAC (1 nM) to striatal A2a receptor by about
50% in membrane-bound and solubilized form and to
reduce agonist binding [40]. Agonist af®nities increased by
one order of magnitude upon solubilization and puri®cation, whereas antagonist af®nities remained similar
(Table 2). Increased agonist af®nities of solubilized GPCRs
have been observed before [37,51,52]. In case of the striatal
adenosine A2a receptor high agonist af®nity of solubilized
receptor was believed to result from G protein coupling
[39,41]. This was supported by a reversible reduction of the
number of agonist binding sites by addition of GTP [39]. We
conclude that solubilized A2a receptor fusion protein
displays high agonist af®nity in absence of G proteins.

CONCLUSION
In this report we describe methods for the expression,
solubilization and puri®cation of a hA2aR fusion protein in
quantity and quality suf®cient for biophysical characterization and crystallization. The following points made the
puri®cation of large amounts of functional hA2aR possible:
(a) ®nding conditions for high level functional expression of
hA2aR; (b) creating a protease resistant receptor form; (c)
®nding conditions that allow solubilization and puri®cation
without denaturation; and (d) employing methods that
allow good enrichment at large scale with minimal losses of
functional receptor protein.

The hA2aR was characterized pharmacologically for the
®rst time in puri®ed form. The ligand binding properties of
the puri®ed receptor were found to be similar to those in its
natural environment or expressed in an eukaryotic system.
All puri®ed protein molecules bound the tested ligands with
one apparent af®nity, indicating a high degree of conformational homogeneity. The methodology we describe opens
the way to a wide range of biophysical and structural
studies. We have started to use the puri®ed material for 2D
crystallization trials.

ACKNOWLEDGEMENTS
We thank Sew Peak-Chew for N-terminal sequencing and David Owen
for the amino acid analysis. Jim Coote (GlaxoWellcome) has provided
the receptor cDNAs and given valuable advice. We are grateful to Joyce

Baldwin, Wasyl Feniuk (Glaxo Institute for Applied Pharmacology)
and Mike Sheehan (GlaxoWellcome) for helpful discussions, and to
Awinder Sohal, Richard Henderson and Chris Tate for comments on
the manuscript and helpful discussions. This project was supported by
GlaxoWellcome, AstraZeneca and the Medical Research Council (UK)
with Link Grant G9712367.

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