Puri®cation and characterization of the human adenosine A
2a
receptor functionally expressed in
Escherichia coli
H. Markus Weiû and Reinhard Grisshammer*
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK
The adenosine A
2a
receptor belongs to the seven trans-
membrane 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 p rotection of cells during anoxia.
For structural and bioph ysical studies we have expressed
the human adenosine A
2a
receptor (hA2aR) at high levels
inserted in to the Escherichia c oli inner membrane, and
established a puri®cation scheme. Expression was in fusion
with the p eriplasmic maltose-binding protein to levels of
10±20 nmol of receptor per L of culture, as detected with
the speci®c antagonist ligand [
3
H]ZM241385. As the re-
ceptor C -terminus was proteolyzed upon solubilization, a
protease-resistant but still functional r eceptor was c reated
by truncation t o Ala316. Addition of the s terol, cholesteryl
hemisuccinate, allowed a stable preparation of f unctional
hA2aR solubilized in dodecylmaltoside to be obtained,
and, increased t he stability o f the rec eptor solubilized in
other alkylmaltosides. Puri®cation to homogeneity was

adenosine receptors have been identi®ed (A
1
,A
2a
,A
2b
,and
A
3
) belonging to the family of G-protein-coupled receptors
(GPCRs). Like many other GPCRs, adenosine receptors
are potential drug targets. Dr ugs acting on the human
adenosine A
2a
receptor ( hA2aR) are expected to have a
therapeutic potential in CNS disorders, in¯ammation or
stroke [5]. Mice lacking t he adenosine A
2a
receptor show
increased aggression, hypoalgesia, f aster platelet aggrega-
tion, high blood pressure and reduced exploratory activity
indicating involvement of the receptor in a variety of
physiological functions [6]. D irect structure determination
of hA2aR by X-ray or electron crystallography, or infor-
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 c onstitute a large protein family, but from the
thousands of members, only rhodopsin has been puri®ed in

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.
Correspondence to H. M. Weiû, Aesku.lab D iagnostika, M ikroforum
Ring 2, 55234 Wendelsheim, Germany. Fax: + 49 6734 9627 27,
Tel.: + 49 6734 9627 11, E-mail: w
Abbreviations: CHS, cholesteryl hemisuccinate; DDM, n-dodecyl-b-
D
-
maltoside; DeM, n-decyl-b-
D
-maltoside; GPCR, G-protein-coupled
receptor; hA2aR, human adeno s ine A
2a
receptor; IMAC, immobilized
metal anity chromatography; IPTG, isopropyl thio-b-
D
-galactoside;
MBP, maltose-binding protein; NECA, 5¢-N-ethylcarboxamidoade-
nosine; R-PIA, R(±)N
6
-(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)

[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 i n
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 c odon was r eplaced by a BamHI
restriction enzyme s ite, 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 th e
receptor (Ser412 in case of the full-length receptor and
Ala316 for the truncated r eceptor) was followed b y the
nucleotide sequence GCGGCCGCA that contains a NotI
restriction s ite and encodes three Ala residues i n the ®nal
construct. The regions coding for the C-terminal tags
(Fig. 1) and two s top codons wer e ¯anked b y NotIand
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 i nto the E. coli
expression vector pRG/III-hs-MBP [20]. This vector con-
tains 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 e xpression vector

M
HCl of t he
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 m
M
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 m
M
Hepes/KOH,
pH 7.4, 100 m
M
NaCl, 5 m
M
MgCl
2
,1m
M
phenyl-
methanesulfonyl ¯uoride, 0.5 lgámL
)1
leupeptin,
0.7 lgámL
)1
pepstatin, 20 lgámL

1m
M
phenylmethanesulfonyl ¯uoride, 0.5 lgámL
)1
leu-
peptin, 0.7 lgámL
)1
pepstatin and 1% of the respective
detergent (DDM, UM o r DeM) with or without 0.2%
Fig. 1. hA2aR-fusion proteins used in this study. (A) Schematic repre-
sentation of h A2aR fusion proteins. The boxes shown are n ot drawn to
scale. The n ames used in the text are given on t he righ t. MBP, mature
E. coli maltose-binding pro tein (Lys1 to Thr366) f ollowed by glyci ne
and serine encoded by a BamHI restriction site; hA2aR, human
adenosine A
2a
receptor (Pro2 to Ser412); hA2aRTr316, C-terminally
truncated hum an adenosine A
2a
receptor (Pro2 to Ala316); T rxA,
E. coli thioredoxin (Ser2 to Ala109); H10, 10 histidine r esidues; 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, th e sequence GT is encoded
by a Kpn I s ite, the sequence EF is e ncoded by a Ec oRI s it e, the
sequence DYKDDDDK corresponds to the Flag peptide.
Ó FEBS 2002 Puri®cation and characterization of A
2a
receptor (Eur. J. Biochem. 269)83
CHS. After incubation with slow rotation for 1 h at 4 °C,

M
). Water was added afterwards t o give a ®nal
volume of 315 mL. Then 35 mL of d etergent stock solution
(10% DDM, 2 % C HS) w ere a dded w ith s tirring. T he
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 p rotease inhibitors
(concentrations as above) and then a dded in batch to 50 mL
of chelating Sepharose, loaded with Ni
2+
ions and equili-
brated in buffer NiA (50 m
M
Hepes/KOH, pH 7.4, 200 m
M
NaCl,30%glycerol,50m
M
imidazole, 0.1% DDM, 0.02%
CHS), resulting in a ®nal imidazole concentration of less
than 15 m
M
. The suspension was slowly stirred for 3 h and
then packed into an Econo-column (Bio-Rad) with 5 cm
diameter. The re sin was washed at 9 mLámin
)1
with 600 mL
of buffer NiA supplemented with protease i nhibitors (see

pepstatin)
and the solution was passed through a 0.22-lm®lter
(Stericup, Millipore). XAC-agarose gel (10 mL), p acked
into an XK 26 column (Amersham Pharmacia), was
equilibrated with buffer XA (50 m
M
Hepes/KOH, pH 7.4,
100 m
M
NaCl, 3 0% 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 o f buffer X A
supplemented with protease inhibitors. The receptor was
eluted at 25 °C in buffer XA, supplemented with 20 m
M
theophylline and protease inhibitors, at a ¯ow rate of
0.6 m Lámin
)1
. Due to the strong absorption of theophylline
at 280 nm, elution could not be monitored spectroscopically.
However, as judged from protein g els, the receptor was
almost completely eluted after 70 mL o f elution buffer.
70mLoftheXAC-agarosegeleluateweredilutedwith
70 mL of buffer QDil (50 m
M

the r eceptor was eluted with 14% of buffer QB (183 m
M
NaCl)at1mLámin
)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 e ither
used immediately for 2D crystallization trials (not shown),
stored at 4 °C, or frozen in liquid nitrogen for long-term
storag e.
Radioligand binding
All b inding assays were carried out in polystyrol t ubes
using LBA buffer (20 m
M
Hepes/KOH, pH 7.4, 100 m
M
NaCl) and the A
2a
receptor speci®c antagonist
[
3
H]ZM241385. Samples were incubated on ice unless
stated otherwise. The incub ation time was 3 h for compe-
tition binding assays, 1.5 h for one-point saturation assays
and for saturation experiments, and 1 h fo r saturation
experiments performed at room temperature. These times
were suf®cient to reach equilibrium as association and
dissociation of ZM241385 to the A
2a

. In saturation
experiments on membranes, the total volume was 1.5 mL.
Bound and free ligand were separated by rapid v acuum
®ltration over GF/B ® lters soaked in 0 .3% polyethyleni-
mine. F iltration was carried out with a Brandel cell
harvester at 4 °C. Filters w ere washed three times with
ice-cold buffer (20 m
M
Hepes/KOH, pH 7.4). For calcula-
tion of the parameter Ôreceptors per cellÕ, the number of
cells growing in suspension was estimated by measuring
D
600
:a D
600
of 1 was assumed to correspond to
10
9
cellsámL
)1
.
Assays on solubilized receptors. Assays were performed i n
LBA buffer containing 0.1% DDM and 0.02% CHS in a
volume of 200 lL (one-point saturation assays) or 300 lL
(saturation a nd competition experiments). The concentra-
tion of [
3
H]ZM241385 was 0.75 n
M
in competition assays

high-molarity Tris buffered gels [22]. For N-terminal
sequencing, the protein was electro-blotted onto poly(viny-
lidene di¯uoride) membranes (Immobilon-P, Millipore) as
described previously [23]. Sequence analyses were per-
formed 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) af ter
hydrolysis in 6
M
HCl for 18 h at 110 °C. Comparing
results f rom amino-acid ana lysis and A mido black a ssays
indicated that the latter un derestimated the amount of
puri®ed receptor f usion protein b y 12% . Protein c oncen-
trations of ®nal puri®ed receptor given in the text and tables
have been corrected accordingly.
RESULTS
Expression
For work a iming at s tructural and bio physical studies on
GPCRs, high expression levels are e ssential. As the expres-
sion level of a given receptor is dif®cult t o predict, we
performed an initial screen, expressing the cDNAs of four
different human GPCRs (somatostatin receptors S
2
and S
4
,

1
receptor fusion p roteins displayed high
af®nity binding of the antagonist [
3
H]8-cyclopentyl-1,3-
dipropylxanthine i n both expression systems; expression
levels of functional A
1
receptor were 3±4 nmol and
1±2 nmol per litre of shake ¯ask c ulture in E. coli and i n
P. pastoris, respectively (data not shown).
Best expression levels, deduced from immunoblot- a nd
radioligand b inding-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 (const ruct M -A2a-H10F; Fig. 1) was suf®-
ciently high to start puri®cation. However, the r eceptor
C-terminus was sensitive to proteolysis after solubilization.
A second const ruct, M-A2a-TrxA-H10F ( Fig. 1), a llowed
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. O ne of
those (M-A2aTr316-H10; Fig. 1) was used for the puri®ca-
tion and characterization described here.
Culture conditions were optimized for functional e xpres-
sion of hA2aR using the construct M-A2a-H10F (Fig. 1).
Addition of glucose (0.2% (w/v) o r more) to the medium
increased the ®nal cell density from an D
600
of about 1.5 to a

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. H owever, f or fragile membrane
proteins such as GPCRs, it is dif®cult to ®nd conditions that
allow e f®cient extraction and maintain structural integrity.
We used radioligand binding assays to monitor hA2aR
integrity and stability. Approximately 70±90% of speci®c
[
3
H]ZM241385 binding sites were solubilized from mem-
brane 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 t he stability of
Ó FEBS 2002 Puri®cation and characterization of A
2a
receptor (Eur. J. Biochem. 269)85
receptors solubiliz ed in UM and DeM (Fig. 2). Receptor
half-lives in the s olubilized fraction (deduced from two
experiments, one of which is given in F ig. 2) were 7 days
using D eM, 2 6 days using UM and 40± 130 days using
DDM. When CHS was employed in combination with any
of the three alkylm altosides, h alf-lives were in the range
40±130 days. Both the full-length hA2aR (M-A2a-H10F)
and t he truncated f orm (M-A2aTr316-H10) behaved
identically in these experiments. For puri®cation of

truncated fusion protein w as 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 e xperimental value for
speci®c radioligand b inding was 13.7 ( 0.8) nmolámg
)1
,
which is in agreement with the theoretical value of
13 n molámg
)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 r esulted in
much better recoveries (> 50%) compared to column
loading (< 20%). Batch binding of the r eceptor 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 b y improving the accessibility of
the tag. However, this was not investigated further as g ood
recoveries were achieved for M-A2aTr316-H10 by batch
loading for 3 h using suf®cient amounts o f Ni
2+

Protein concn
(mgámL
)1
)
Total protein
(mg)
Speci®c binding
(pmolámg
)1
)
Puri®cation
(fold)
Solubilized material 70896 100 24.6 7306 9.7 1
IMAC eluate 43790 62 0.35 53.6 817 84
XAC ¯ow through 8735 (12) 0.128 44.8 195 ±
XAC eluate 23657 33 0.045 3.2 7393 762
Q Sepharose eluate 18091 26 0.200 1.5 12061 1243
86 H. M. Weiû and R. Grisshammer (Eur. J. Biochem. 269) Ó FEBS 2002
IMAC puri®ed M-A2aTr316-H10 bound almost quantita-
tively 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 prepa-
ration to be completed within 2 days, leading to slightly
poorer binding to the XAC-agarose (Fig. 3; Table 1).
Elution from t he ligand af®nity column was slow even a t

calculated value of 77 kDa. To show that this resulted from
atypical running behaviour, f requently 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 Lys-
Ile-Glu-Glu-Gly-Lys-Leu-Val-Ile-Trp corresponds to the
N-terminus of the m ature maltose-binding protein. Binding
of the fusion protein to the IMAC gel in buffer containing
50 m
M
imidazole indicates the presence of the C-terminal
histidine tag. W e 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 m
M
Hepes/KOH, pH 7.4, 200 m
M
NaCl, 30% glycerol, 0.1%
DDM and 0.02% CHS, speci®c [
3
H]ZM241385 binding of
the puri®ed re ceptor decreased to 81% over 40 days
corresponding to a half-life of 3.5±6.0 months (one exper-
iment, ®ve time points). In another experiment, the i n¯uence
of speci®c ligands on the stability of puri®ed receptor was
tested at 4 °C in buffer consisting of 50 m
M
Hepes/KOH,

theophylline. The arrow points to the
M-A2aTr316-H10 fusion protein. Note that the main contamination,
seen in lane 1, run s only m arginally above the hA2aR fusion pro tein
and is the main band in lane 2. In this pu ri®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 a s outlined in
the Materials and m ethods section but starting from 70 g of cells.
Ni-nitrilotriacetic a cid agarose was used for the IMAC step and
washing and elution were carried out with 3 5 and 200 m
M
imidazole,
respectively. The XAC-agarose was loaded at a ¯ow rate of
0.23 mLámin
)1
.
Ó FEBS 2002 Puri®cation and characterization of A
2a
receptor (Eur. J. Biochem. 269)87
Signi®cance of CHS for receptor stability
during puri®cation
DDM-solubilized M-A2aTr316-H10 (no CHS added)
had a half-life of more t han 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 [
3
H]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

performed on ice and at room temperature. Puri®ed
receptors displayed a single af®nity for the antagonist
[
3
H]ZM241385 (Fig. 6C) with a K
d
value of
0.19  0.02 n
M
and a B
max
value of 12.4  0.5 nmolámg
)1
(n  3). In contrast, data from saturation experiments on
Fig. 5. The stability of solubilized hA2aR (M-A2aTr316-H10) during
puri®cation is depende nt on the presence of CHS. (A) Solubilized frac-
tion. (B) IMAC puri®ed fraction. Solubilization and puri®cat ion by
IMAC was performed as descr ibed in the Materials and methods
section starting with 10 g of cells for each condition described. Sus-
pension of cells for solub ilizatio n w as ach iev ed using a potter i nstead of
a blender. Ni-nitrilotriacetic acid agarose was used for the IMAC step.
Washing a nd elution were c arried out with 3 5 and 200 m
M
imidazole,
respectively. One-point saturation assays with [
3
H]ZM241385 were
performed as o utlined in the Materials and methods section, w ith
0.02% CHS in all binding assays. The results shown are from one of
two independent e xperime nts. In many cases, the error bars are smaller

M
) K
i
(
M
) n
Agonists
NECA 3.6  0.2 ´ 10
)6
0.73  0.04 3.9  0.2 ´ 10
)7
7.9 ´ 10
)8
0.96  0.06
R-PIA 6.5  0.4 ´ 10
)5
0.87  0.18 6.5  0.1 ´ 10
)6
1.3 ´ 10
)6
1.01  0.02
Antagonists
Theophylline 2.3  0.3 ´ 10
)5
0.94  0.11 1.3  0.3 ´ 10
)5
2.7 ´ 10
)6
1.19  0.15
XAC 1.7  0.4 ´ 10

temperature.
Unlabeled a denosine receptor ligands, tested a t 0 °C,
inhibited speci®c [
3
H]ZM241385 binding to bot h mem-
brane-bound and puri®ed hA2aR by more than 90% at the
highest concentrations used (6.7 m
M
theophylline, 200 l
M
XAC, 2 m
M
NECA, 2 m
M
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 o f t he adenosine A
2a
receptor. The rank order of
potency for t he 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 t han unity, d espite the a bsence of G proteins in
E. coli. For antagonist competition curves, pseudo-Hill

outlined in the Materials and method s section. Incubation was o n ice
for3hinLBAbuerwhichwassupplementedwith0.1%DDM/
0.02% CHS for t he an alysis of puri®ed r eceptors. [
3
H]ZM241385 was
present at a co ncentration of 0.75 n
M
. The results shown are from one
of at least two independent experiments. Ligands used for displace-
ment were: m,NECA;j,R-PIA;,, t heophylline; s, XAC. (A)
Displacemen t curv es for membrane-bou nd M-A2aTr316-H10. (B)
Displacement curves for puri®ed M-A2aTr316-H10. IC
50
values for
the displacing drugs are given in Table 2.
Ó FEBS 2002 Puri®cation and characterization of A
2a
receptor (Eur. J. Biochem. 269)89
for tagged A
1
receptor puri®ed from stably transfected
CHO cells [17,31]. To date, no quanti®ed puri®cation of the
adenosine A
2a
receptor has be en reported. The availability
of milligram quantities of functional M-A2aTr316-H10
fusion protein will allow extensive crystallization trials.
The ad enosine A
2a
receptorisexpressedtohighlevelsina

the truncated receptor puri®ed by us is suf®cient to ful®l the
main receptor functions and i s appro priate for structu ral
and biophysical investigations. Comparison of the amino-
acid sequences of adenosine r eceptors with those o f other
GPCRs indicates that t he C-terminus of the adenosine A
2a
receptor consists of about 120 a mino acids, which compares
to only 30±45 a mino acids f or the o ther three known
adenosine receptors. The functional sign i®cance of this long
C-terminus is so far unclear, but it is likely to be involved in
A
2a
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 G PCRs has in m any cases been achieved with
digitonin [15,36,37]. However, impurities and b atch varia-
tions make it unsuitable for reproducible puri®cation and
subsequent crystalliz ation experiments [38]. In contrast, the
alkylmaltosides employed in this study are chemically well
de®ned a nd commercially available at high purity. The
combination of the cholesterol derivative CHS and different
alkylmaltosides allowed highly ef®cient solubilization of A
2a
receptor i n f unctional 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 A
2a

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 d eca-
histidine tagged neurotensin receptor [46], the binding of
M-A2aTr316-H10 to Ni-n itrilotriacetic acid resin packed
into a c olumn was extremely poor, with r ecoveries 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 A
1
receptors [15±17] but not for enrichment of
functional A
2a
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 r esults were ob tained by increasing temperature
and using high concentrations of the weak antagonist
theophylline combined with low ¯ow rates. The ®nal ion-
exchange step allowed removal of theophylline, and a
reduction of volume and detergent concentration. The
higher degree of re ceptor homogeneity obtained by the ion-
exchange step will help crystallization. This step also allows
fast detergent exchange in preparation for c rystallization
experiments.
The M-A2aTr316-H10 fusion protein was characterized
by ligan d b inding analyses to judge its identity and integrity.

¯uidity and l ateral pressure on the membrane-bound
receptor at low temperature, similar to effects due to a
changed lipid composition [49,50].
The puri®ed receptor d isplayed a single a pparent af®nity
for e ach ligand tested, including [
3
H]ZM241385 ( Table 2)
and an identical rank order of potency as the h A2aR in
CHOcellmembranes(XAC>NECA>R-PIA>
theophylline), d etermined by using agonist or antagonist
radioligands [28,29]. K
i
values for the puri®ed receptor
compare to K
i
values determined for m embrane-b ound
hA2aR on platelets or expressed i n CHO cells [28,29] as
follows. R-PIA: 1.3 l
M
(E. coli), 0.86 and 0.68 l
M
(CHO),
1.6 l
M
(platelets); NECA: 79 n
M
(E. coli), 66 n
M
(CHO),
30 n

tion, 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 A
2a
receptor high agonist af®nity of solubiliz ed
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 A
2a
receptor fusion protein
displays high agonist af®nity in absence of G proteins.
CONCLUSION
In this repo rt we describe meth ods fo r the expression,
solubilization and puri®cation of a hA2aR fusion protein in
quantity and quality suf®cient for biophysical characteriza-
tion and crystallization. The f ollowing points m ade 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 enrich ment at large scale with minimal l osses 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

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