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: weiss@aeskulab.de 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 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 Ĩ 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 ´ 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 ´ 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 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 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, mM MgCl2, 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 h, suspended in buffer (50 mM Hepes/KOH, pH 7.4, 100 mM NaCl, 30% glycerol, 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 mgámL)1 in membrane solubilization buffer (50 mM Hepes/KOH, pH 7.4, 200 mM NaCl, 30% glycerol, 40 lM theophylline, 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 h at °C, samples were centrifuged for 45 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 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 s on/1 s off, with 12 s pulsing on per gram of cells Then the suspension was stirred for 30 on ice and centrifuged at 100 000 g for 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 h and then packed into an Econo-column (Bio-Rad) with cm diameter The resin was washed at 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 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 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, 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 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 °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 h for competition binding assays, 1.5 h for one-point saturation assays and for saturation experiments, and 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 °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 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 °C and precentrifuged for 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 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 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 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 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 °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 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 and Fig 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 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 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 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) 84 ± 762 1243 Ĩ FEBS 2002 Puri®cation and characterization of A2a receptor (Eur J Biochem 269) 87 Fig 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 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 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 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 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 °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 °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 °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 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 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 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 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 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 °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, mM NECA, 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  A similar behaviour, with agonist, but not antagonist, pseudo-Hill coef®cients < 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  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 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 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 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 °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 °C using a eukaryotic expression system [48] The Ĩ 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), and nM (CHO), nM (platelets); theophylline: 2.7 lM (E coli), 1.7 and 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) 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characterization and crystallization The following points made the puri®cation of large amounts of