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Properties of solubilized and reconstituted A1 adenosine receptors from bovine brain
Pharmacological Research, Vol . 24, No . 1, 1991
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PROPERTIES OF SOLUBILIZED AND RECONSTITUTED A, ADENOSINE RECEPTORS FROM BOVINE BRAIN EUGENIO RAGAZZI*, JOHN SHRYOCK* and KRZYSZTOF PALCZEWSKIt$ Departments of *Medicine and tOphthalmology, College of Medicine, University of Florida, Gainesville, FL 32610, USA Received in final form 25 October 1990
SUMMARY A simple method for solubilization and reconstitution of the A, adenosine receptor from bovine brain is presented . Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A, adenosine receptor antagonist [ 3 H]-DPCPX . The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins . Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0 .4 M) . The fractions corresponding to the peak of [ 3 H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis . The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA>S-PIA>NECA) as the native receptor, although its thermal stability was remarkably reduced . The binding of [ 3 H]DPCPX to A, adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins . KEY WORDS :
adenosine receptor, solubilization, chromatography, reconstitution .
[3H]-DPCPX, ['H]-8-cyclopentyl-l,3-dipropylxanthine ; R-PIA, N'-(R)phenylisopropyladenosine ; S-PIA, N 6 -(S)-phenylisopropyladenosine ; NECA, 5'-N-ethylcarboxamido-adenosine ; BTP, (1,3-bis[tris(hydroxymethyl)methylaminolpropane) : PC, i a-phosphatidylcholine ; CHAPS, (3-[(3-cholamidopropyl)dimethylammonioll-propanesulphonate) ; ADA, adenosine deaminase : HA, hydroxyapatite ; DEAE-cellulose, diethylaminoethylcellulose . ABBREVIATIONS :
INTRODUCTION The A, adenosine receptor is found in a wide variety of tissues and has recently been purified to homogeneity from rat brain using a multiple-step
*Present address : Robert S . Dow Neurological Sciences Institute . 1120 N .W . 20th Ave ., Portland, OR 97209, USA . Correspondence to : E . Ragazzi, Department of Pharmacology . University of Padova, Largo E . Meneghetti 2, 1-35131 Padova, Italy . 1043-6618/91/050015-09/$03 .00/0
C- 1991
The Italian Pharmacological Society
16
Pharmacological Research, Vol . 24, No . 1, 1991
chromatographic method [1,2] . The receptor is tightly linked to an inhibitory Gprotein [3-6] that mediates inhibition of adenylyl cyclase [7,8] . To study receptor regulation by pharmacological agents and receptor-G-protein-adenylyl cyclase coupling, a functional and stable reconstituted receptor preparation is required . Here we present a simple method for solubilization and reconstitution of the A, adenosine receptor in phosphatidylcholine . The binding of agonists and antagonists, and the regulation of binding by G-proteins were similar for reconstituted A, adenosine receptors and receptors in native membranes .
MATERIALS AND METHODS Chemicals
[ 3 H]-DPCPX (99 .4 Ci/mmol) was obtained from Amersham (Arlington Hts, IL) . Adenosine analogues, GTP, CHAPS, BTP, PC (type III-S) and ADA (type VI) were from Sigma (St Louis, MO) . HA (Bio-Gel HT) was from Bio-Rad Laboratories (Richmond, CA) . DEAE-cellulose (DE 52) was from Whatman (Maidstone, UK) . Membrane preparation
Pieces of frozen bovine brain cortex (Pel-Freez Biological, Rogers, AR) were immersed in 10 vol of ice cold 10 mm HEPES buffer, pH 7 .4, containing 10 mm EDTA, 1 mm dithiothreitol, 0 .1 mm benzamidine, 10 pg/ml phenylmethylsulphonyl fluoride and 10% (w/v) sucrose, and homogenized (Polytron, Brinkmann Instruments) for 10 s . The homogenate was filtered through 210,um nylon mesh and pelleted at 31 000 g for 15 min at 4°C . The supernatant was discarded and the pellet was resuspended in 10 vol of the same buffer and washed three times . The washed crude membrane pellet was resuspended in 3 vol of 25 mm BTP buffer, pH 7 .4, containing 0 .1 mm EDTA, 1 mm MgCl, and 50 mm potassium sulphate, and stored at -80°C until use . Receptor solubilization
Receptors in washed crude membranes were solubilized in buffer (25 mm BTP, pH 7 .4, containing 0 .1 mm EDTA, 1 mm MgCl,, 50 mm potassium sulphate and 10 ,ug/ml each of leupeptin, pepstatin, benzamidine and aprotinin) with CHAPS (final concentration 0 .5-1 %) plus L-a-phosphatidylcholine (PC, 0 .8 mg/ml) to give a final protein concentration of 5-15 mg/ml . The suspension was gently mixed and incubated on ice for 1 h ; insoluble material was pelleted at 48 000 g for 2 h at 4 ° C . The supernatant was either used immediately, stored on ice for 2 days, or frozen at -80°C . Binding assay
Crude membranes, solubilized receptors, or reconstituted receptor preparations were incubated with ADA (2 U/ml) for 30 min at 21 °C to degrade endogenous adenosine . Aliquots (100,1) containing 0 .05-0 .15 mg protein were incubated in
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glass tubes with 100, u1 of [ 3 H]-DPCPX solution (0 .002-2 nM in 25 mm BTP+l mm MgCl,, pH 7 .4) for 90 min at 21'C . In competition binding studies, 25 ,ul solution of adenosine analogue were added to each assay tube in the presence of 1 nM [ 3 H]DPCPX, and incubated for 120 min at 21'C . Incubation was terminated by rapid filtration (Brandel Cell Harvester M-24R ; Brandet Scientific, Gaithersburg, MD) through Whatman GF/B glass fibre filters presoaked with 0 .3% polyethyleneimine . Filters were washed three times with 4-ml aliquots of ice-cold buffer (10 mm tris(hydroxymethyl)aminomethane chloride, 4 mm MgCl 2 , 100 mm NaCI, pH 7 .4) and placed in 10 ml of scintillation cocktail for determination of radioactivity . Non-specific binding was defined as radioligand bound in the presence of 10 uM R-PIA . Chromatography Hydrox_yapatite
(HA) column chromatography . Solubilized membrane preparation (10-25 ml, 1 .5-2 mg protein/ml) was applied at a flow rate of 10 ml/h on a HA column (1 x 10 cm) equilibrated with 25 mm BTP buffer (pH 7 .4) containing 1 mm MgCl,, 0 .75% (w/v) CHAPS and 0 .8 mg/ml PC . After washing, a linear gradient of KH 2PO 4 (0-0 .5 M) or KCI (0-0 .6 M) in buffer at a flow rate of 7 ml/h was used to elute protein from the column . Eluate fractions of 1-1 .5 ml were collected for determination of protein content and [ 3 H]-DPCPX binding . All procedures were done at 4°C . DEAF-cellulose column chromatography . Detergent- solubilized membranes (10-28 ml, 1 .5-2 mg protein/ml) were applied at a flow rate of 10 ml/h on a DEAE-cellulose column (1x20 cm) equilibrated with buffer at 4° C . Protein was eluted with a KCI gradient (0-0 .4 M) in 25 mm BTP buffer (pH 7 .4) containing 1 mm MgCl, and 0 .75% CHAPS with or without 0 .8 mg/ml PC at a flow rate of 7 ml/h . No difference was observed in the elution peak appearance if PC was present throughout the chromatography or only during the elution phase . Eluate fractions of 1-1 .5 ml were collected . Protein content and [ 3 H]-DPCPX binding were determined .
Reconstitution of the receptor
Fractions corresponding to the peak of [3 H]-DPCPX binding activity (range 20-100% of maximal activity) were pooled and dialysed in a dialysis bag (10 000 molecular size cut-off) against 25 mm BTP buffer containing 1 mm MgCl, for 3-4 days at 4 ° C under continuous mixing to remove KCI and detergent and obtain the reconstituted receptor preparation . Protein assay
Protein concentrations were determined according to the method of Bradford [9] using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin as standard . Data analysis
Binding data were fit to one- or two-site binding models using the EBDA-
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Pharmacological Research, Vol. 24, No . 1, 1991
LIGAND program (Biosoft, Cambridge, UK) . The program utilizes a non-linear least squares curve fitting technique with the Marquardt-Levenberg modification of the Gause-Newton method . Estimated parameters are given as means±SEM . The statistical evaluation of data was performed using a two-tailed Student t-test for unpaired or paired data .
RESULTS AND DISCUSSION Solubilization of A, adenosine receptor from bovine brain
CHAPS was used to solubilize the A, adenosine receptor because it can be easily removed from the preparation by dialysis . PC was added during all the procedures to help stabilize the receptor molecule . CHAPS (0 .5-1%) with PC (0 .8 mg/ml) solubilized about 50% of initial specific [ 3 H]-DPCPX binding sites and 50% of membrane protein . Recovery of receptors determined by specific [ 3H]-DPCPX binding was similar using 0 .5 and 1 % CHAPS . Binding of the A, adenosine receptor antagonist [3 H]-DPCPX to solubilized receptors (Ko =0 .41±0 .10 nM and B max =1213±147 fmol/mg protein, n=11), and native membranes (Kp=0 .22±0 .04 nM and B max =1084±129 fmol/mg protein, n=8) was similar and not statistically different . The CHAPS/PC solubilized receptor preparation was stable for several weeks when frozen, and freezing/thawing did not alter [ 3 H]-DPCPX binding to the receptor . This preparation was used in all further experiments . GTP (0 .1 mm) increased the binding of the specific A, antagonist [ 3 H]-DPCPX to native and solubilized A, adenosine receptors . In native membranes Bmax was 1269±40 fmol/mg protein in the absence, and 1755±42 fmol/mg protein in the presence of GTP (n=3, P<0 .01, Student t-test for paired data) . Receptor number in solubilized preparations was 1631±164 and 2106±248 fmol/mg protein in the absence and presence of GTP, respectively (n=4, P<0 .05) . The KD of binding to adenosine receptors in either preparation was not altered by GTP . This effect was specific for GTP ; 0 .1 mm ATP, GDP and inosine did not alter antagonist binding (2 nM [ 3 H]-DPCPX) to the A, adenosine receptor . Conversely, GTP decreased binding of the agonist [ 3 H]-CHA (data not shown) . GTP was previously reported to decrease the binding of agonists [3, 5, 10] and increase the binding of antagonists [4, 6, 11, 12] to A, adenosine receptors . Our findings indicate that adenosine receptors solubilized in CHAPS with PC retain their association with G-proteins . Moreover, the results support the A, receptor radiolabelling data [13], indicating that agonists and antagonists are associated with distinct conformations of the A, receptor . Chromatography of CHAPS/PC-solubilized membranes on hydroxyapatite (HA) and DEAE-cellulose
Two chromatographic procedures were developed for removal of native membrane lipids . CHAPS/PC-solubilized adenosine receptors bound almost completely to HA and were not eluted with 0 .6 M KCI ; however, 200 mm potassium phosphate by step-wise or continuous gradient (0-0 .5 M) eluted the
Pharmacological Research, Vol. 24, No . 1, 1991
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receptor rapidly with an overall yield of greater than 40% . The ratio of receptors to total protein (fmol/mg) was unchanged compared to the loaded material . Chromatography on DEAE-cellulose is presented in Fig . 1 . Adenosine A, receptor binding activity was retained on the column and eluted with a continuous gradient of KCI (0-0 .4 M) as a single peak of activity at 200 mm KCI . The majority of proteins eluted before the peak of adenosine receptor binding activity . The overall yield of adenosine receptors was 61±9% (range 37-84, n=4), with a two- to three-fold peak increase of the ratio of receptors to protein . A lower overall yield, but a slightly greater receptor/protein ratio was observed if PC was omitted during the chromatography . These results from HA and DEAE-cellulose chromatography show that the solubilized adenosine receptor acts like an acidic protein [14] . Reconstitution of the receptor and its characterization A, adenosine receptors partially purified by DEAE-cellulose chromatography were reconstituted into PC vesicles by dialysis to remove detergent, as reported in the Methods section . Saturation binding curves for [ 3 H]-DPCPX using native and reconstituted receptors are shown in Fig . 2 . K 0 for DPCPX binding was determined to be 0.52 nM and 0.21 nM for the native and reconstituted receptors, respectively . Bmax was 820 fmol/mg protein for native and 460 fmol/mg protein for reconstituted receptors . The binding per mg protein of the reconstituted adenosine receptor preparation was lower than that of native membranes, indicating either a partial denaturation of the receptor or a different incorporation and orientation of some of the receptors in the pure PC matrix such that they are unavailable to the radioligand . However, there was no loss in binding affinity but apparently an increase . This may be the result of a different sterical modification of the reconstituted receptor or a different coupling to regulatory proteins . In a competition study (Fig . 3), the order of binding potency (R-PIA>S-PIA :> 1400 1 .40-
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Fig . 1 . DEAE-cellulose chromatography of CHAPS-solubilized A, adenosine receptors from bovine cerebral cortex membranes . Protein was eluted from the column with a KCI gradient (0-0 .4 M) . Protein content (O), and 2 nM ['H]-DPCPX binding ( •) by eluted fractions . W, wash with KCI-free buffer ; E, elution .
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Pharmacological Research, Vol . 24, No. 1, 1991
700 600 500 400 300 200 100-/
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° I f 0 m.°~n-I 0.0 0.5 1 .0 1 .5 2 .0 Concentration of free ligand (nM)
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300200- •' 100-9 I ° 0 0.0 0 .5 1 .0 1 .5 2 .0 Concentration of free ligand (nM) Saturation of [ ; H]-DPCPX binding to bovine cerebral cortex native membranes (top) and reconstituted receptors (bottom) . Specific ( •) , total (O) and non-specific (A) binding are presented . Insets : Scatchard plots of specific binding . Fig. 2 .
NECA) was the same for both native and reconstituted receptors, suggesting that the receptor retained the initial binding selectivity when reconstituted in PC . Similar findings and similar rank order of potency in the displacement of DPCPX were reported by Olah et al . in a bovine brain preparation [15] . This demonstrates that native membrane components other than PC are not required for ligand binding to the receptor . The addition of GTP (0 .1 mm) decreased the affinity of R-PIA competition
Pharmacological Research, Vol. 2 4 , No . 1, 1991
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Fig . 3 . Displacement of [`H]-DPCPX (1 nM) binding to native bovine brain membranes (right) and reconstituted receptors (left) . Ordinate values are presented as the percentage of radioligand bound in the absence of competing ligand . The reported data are the mean value from a competition experiment performed in duplicate . ( •) R-PIA ; (O) R-PIA+GTP 0 .1 mu : (A) S-
PIA ; (A) NECA .
binding to both native and reconstituted receptors (Fig . 3) . An increase in the specific binding of [3 H]-DPCPX to the reconstituted receptor was found after GTP addition (data not shown) . These results further support the hypothesis [16] that agonists can discriminate different affinity states also of DPCPX binding sites . confirming that the reconstituted receptor is associated with G-proteins . Thermal stability
The native receptor was somewhat more thermostable than the reconstituted receptor (Fig . 4) . Only a moderate decrease in the amount of [ 3 H]-DPCPX binding was found to native receptor preparations after their exposure to 30-50°C for I h . On the other hand, binding to the reconstituted receptor was decreased by previous exposure of the receptor preparation to 30°C and nearly abolished after incubation of receptor preparations at 40 and 50 ° C . No specific binding to either receptor preparation occurred after exposure to 60°C . A reduction of binding may be explained by receptor denaturation or loss of receptor-G-protein coupling . A marked reduction in thermal stability at 25°C was previously found by Nakata [1] in purified A, adenosine receptors from rat brain membranes . This result and our findings that A, adenosine receptor in native membranes is more thermostable than reconstituted receptor, indicate that the native lipids are important to give an optimal stability of the receptor . In conclusion bovine brain A, adenosine receptors retain affinity and selectivity for agonists and antagonists, and association with G-proteins after reconstitution into phosphatidylcholine . The excellent yield and simplicity of the methods
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Pharmacological Research, Vol. 24, No . 1, 1991
Native
. L
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temperature (°C) Fig. 4 . Thermal stability of A, adenosine receptors . Native bovine brain membranes (A) and receptors reconstituted in phosphatidylcholine (B) were exposed for 1 h to the indicated temperatures, then incubated at 21°C with 2 nm [ 3 H]-DPCPX for 90 min . Ordinate values are expressed as per cent of binding to control preparations exposed to 20°C : hatched bars, total binding ; solid bars, non-specific binding .
presented here represent a convenient procedure for reconstitution of A, adenosine receptors, and should facilitate further pharmacological and biophysical characterization of receptor-lipid-G-protein interactions .
ACKNOWLEDGEMENTS This research was supported by National Institute of Health grant HL-35272 and the Florida Affiliate of the American Heart Association . Dr Palczewski was supported by NIH grant EY-08061 . We would like to thank Drs Paul A . Hargrave and Luiz Belardinelli for support, encouragement and use of their laboratories .
Pharmacological Research, Vol. 24, No . 1, 1991
REFERENCES I . Nakata H . Affinity chromatography of A, adenosine receptors of rat brain membranes . Molec Pharmacol 1989 ; 35 : 780-6 . 2 . Nakata H . Purification of A, adenosine receptor from rat brain membranes . .1 Biol Chem 1989 ;264 :16545-51 . 3 . Gavish M, Goodman RR, Snyder SH . Solubilized adenosine receptors in the brain : regulation by guanine nucleosides . Science 1982 ; 215 : 1633-5 . 4 . Stiles GL . A, adenosine receptor-G protein coupling in bovine brain membranes : effects of guanine nucleosides, salt, and solubilization . J Neurochem 1988 ; 51 : 1592-8 . 5 . Klotz K-N, Lohse MJ, Schwabe U . Characterization of the solubilized A, adenosine receptor from rat brain membranes . J Neurochem 1986 ; 46 : 1528-34 . 6 . Munshi R, Linden J. Co-purification of A, adenosine receptors and guanine nucleotidebinding proteins from bovine brain . J Biol Chem 1989 ; 264 : 14853-9 . 7 . Gilman AG . G proteins : transducers of receptor-generated signals . A Rev Biochem 1987 : 56 :615-49 . 8 . Weiss ER, Kelleher DJ, Woon CW, et al . Receptor activation of G proteins . FASEB J 1988 ; 2 : 2841-8 . 9 . Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem 1976 ; 72 : 248-54 . 10 . Yeung S-MH, Perez-Reyes E, Cooper DMF . Hydrodynamic properties of adenosine A, receptors solubilized from rat cerebral-cortical membranes . Biochem .1 1987 : 248 : 635-42 . 11 . Yeung S-MH, Green RD . Agonist and antagonist affinities for inhibitory adenosine receptors are reciprocally affected by 5'-guanylylimidodiphosphate or Nethylmaleimide . J Biol Chem 1983 ; 258 : 2334-9 . 12 . Ramkumar V, Stiles GL . Reciprocal modulation of agonists and antagonists binding to A, adenosine receptors by guanine nucleotides is mediated via a pertussis toxin-sensitive G protein . .1 Pharmacol Exp Ther 1988 ; 246 : 1194-200 . 13 . Barrington WW, Jacobson KA, Stiles GL . Demonstration of distinct agonist and antagonist conformation of the A, adenosine receptor . J Biol Chem 1983 ; 264 : 13157-64 . 14 . Gorbunoff MJ . The interaction of proteins with hydroxyapatite . I . Role of protein charge and structure . Anal Biochem 1984 ; 136 : 425-32 . 15 . Olah ME . Jacobson KA, Stiles GL . Affinity chromatography of the bovine cerebral cortex A, adenosine receptor . FEES Lett 1989 ; 257 : 292-6 . 16 . Martens D, Lohse MJ, Schwabe U . ['H]-8-Cyclopentyl-l,3-dipropylxanthine binding to A, adenosine receptors of intact rat ventricular myocytes . Circulation Res 1988 : 63 : 613-20 .
15
PROPERTIES OF SOLUBILIZED AND RECONSTITUTED A, ADENOSINE RECEPTORS FROM BOVINE BRAIN EUGENIO RAGAZZI*, JOHN SHRYOCK* and KRZYSZTOF PALCZEWSKIt$ Departments of *Medicine and tOphthalmology, College of Medicine, University of Florida, Gainesville, FL 32610, USA Received in final form 25 October 1990
SUMMARY A simple method for solubilization and reconstitution of the A, adenosine receptor from bovine brain is presented . Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A, adenosine receptor antagonist [ 3 H]-DPCPX . The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins . Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0 .4 M) . The fractions corresponding to the peak of [ 3 H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis . The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA>S-PIA>NECA) as the native receptor, although its thermal stability was remarkably reduced . The binding of [ 3 H]DPCPX to A, adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins . KEY WORDS :
adenosine receptor, solubilization, chromatography, reconstitution .
[3H]-DPCPX, ['H]-8-cyclopentyl-l,3-dipropylxanthine ; R-PIA, N'-(R)phenylisopropyladenosine ; S-PIA, N 6 -(S)-phenylisopropyladenosine ; NECA, 5'-N-ethylcarboxamido-adenosine ; BTP, (1,3-bis[tris(hydroxymethyl)methylaminolpropane) : PC, i a-phosphatidylcholine ; CHAPS, (3-[(3-cholamidopropyl)dimethylammonioll-propanesulphonate) ; ADA, adenosine deaminase : HA, hydroxyapatite ; DEAE-cellulose, diethylaminoethylcellulose . ABBREVIATIONS :
INTRODUCTION The A, adenosine receptor is found in a wide variety of tissues and has recently been purified to homogeneity from rat brain using a multiple-step
*Present address : Robert S . Dow Neurological Sciences Institute . 1120 N .W . 20th Ave ., Portland, OR 97209, USA . Correspondence to : E . Ragazzi, Department of Pharmacology . University of Padova, Largo E . Meneghetti 2, 1-35131 Padova, Italy . 1043-6618/91/050015-09/$03 .00/0
C- 1991
The Italian Pharmacological Society
16
Pharmacological Research, Vol . 24, No . 1, 1991
chromatographic method [1,2] . The receptor is tightly linked to an inhibitory Gprotein [3-6] that mediates inhibition of adenylyl cyclase [7,8] . To study receptor regulation by pharmacological agents and receptor-G-protein-adenylyl cyclase coupling, a functional and stable reconstituted receptor preparation is required . Here we present a simple method for solubilization and reconstitution of the A, adenosine receptor in phosphatidylcholine . The binding of agonists and antagonists, and the regulation of binding by G-proteins were similar for reconstituted A, adenosine receptors and receptors in native membranes .
MATERIALS AND METHODS Chemicals
[ 3 H]-DPCPX (99 .4 Ci/mmol) was obtained from Amersham (Arlington Hts, IL) . Adenosine analogues, GTP, CHAPS, BTP, PC (type III-S) and ADA (type VI) were from Sigma (St Louis, MO) . HA (Bio-Gel HT) was from Bio-Rad Laboratories (Richmond, CA) . DEAE-cellulose (DE 52) was from Whatman (Maidstone, UK) . Membrane preparation
Pieces of frozen bovine brain cortex (Pel-Freez Biological, Rogers, AR) were immersed in 10 vol of ice cold 10 mm HEPES buffer, pH 7 .4, containing 10 mm EDTA, 1 mm dithiothreitol, 0 .1 mm benzamidine, 10 pg/ml phenylmethylsulphonyl fluoride and 10% (w/v) sucrose, and homogenized (Polytron, Brinkmann Instruments) for 10 s . The homogenate was filtered through 210,um nylon mesh and pelleted at 31 000 g for 15 min at 4°C . The supernatant was discarded and the pellet was resuspended in 10 vol of the same buffer and washed three times . The washed crude membrane pellet was resuspended in 3 vol of 25 mm BTP buffer, pH 7 .4, containing 0 .1 mm EDTA, 1 mm MgCl, and 50 mm potassium sulphate, and stored at -80°C until use . Receptor solubilization
Receptors in washed crude membranes were solubilized in buffer (25 mm BTP, pH 7 .4, containing 0 .1 mm EDTA, 1 mm MgCl,, 50 mm potassium sulphate and 10 ,ug/ml each of leupeptin, pepstatin, benzamidine and aprotinin) with CHAPS (final concentration 0 .5-1 %) plus L-a-phosphatidylcholine (PC, 0 .8 mg/ml) to give a final protein concentration of 5-15 mg/ml . The suspension was gently mixed and incubated on ice for 1 h ; insoluble material was pelleted at 48 000 g for 2 h at 4 ° C . The supernatant was either used immediately, stored on ice for 2 days, or frozen at -80°C . Binding assay
Crude membranes, solubilized receptors, or reconstituted receptor preparations were incubated with ADA (2 U/ml) for 30 min at 21 °C to degrade endogenous adenosine . Aliquots (100,1) containing 0 .05-0 .15 mg protein were incubated in
Pharmacological Research, Vol. 24, No . /, 199/
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glass tubes with 100, u1 of [ 3 H]-DPCPX solution (0 .002-2 nM in 25 mm BTP+l mm MgCl,, pH 7 .4) for 90 min at 21'C . In competition binding studies, 25 ,ul solution of adenosine analogue were added to each assay tube in the presence of 1 nM [ 3 H]DPCPX, and incubated for 120 min at 21'C . Incubation was terminated by rapid filtration (Brandel Cell Harvester M-24R ; Brandet Scientific, Gaithersburg, MD) through Whatman GF/B glass fibre filters presoaked with 0 .3% polyethyleneimine . Filters were washed three times with 4-ml aliquots of ice-cold buffer (10 mm tris(hydroxymethyl)aminomethane chloride, 4 mm MgCl 2 , 100 mm NaCI, pH 7 .4) and placed in 10 ml of scintillation cocktail for determination of radioactivity . Non-specific binding was defined as radioligand bound in the presence of 10 uM R-PIA . Chromatography Hydrox_yapatite
(HA) column chromatography . Solubilized membrane preparation (10-25 ml, 1 .5-2 mg protein/ml) was applied at a flow rate of 10 ml/h on a HA column (1 x 10 cm) equilibrated with 25 mm BTP buffer (pH 7 .4) containing 1 mm MgCl,, 0 .75% (w/v) CHAPS and 0 .8 mg/ml PC . After washing, a linear gradient of KH 2PO 4 (0-0 .5 M) or KCI (0-0 .6 M) in buffer at a flow rate of 7 ml/h was used to elute protein from the column . Eluate fractions of 1-1 .5 ml were collected for determination of protein content and [ 3 H]-DPCPX binding . All procedures were done at 4°C . DEAF-cellulose column chromatography . Detergent- solubilized membranes (10-28 ml, 1 .5-2 mg protein/ml) were applied at a flow rate of 10 ml/h on a DEAE-cellulose column (1x20 cm) equilibrated with buffer at 4° C . Protein was eluted with a KCI gradient (0-0 .4 M) in 25 mm BTP buffer (pH 7 .4) containing 1 mm MgCl, and 0 .75% CHAPS with or without 0 .8 mg/ml PC at a flow rate of 7 ml/h . No difference was observed in the elution peak appearance if PC was present throughout the chromatography or only during the elution phase . Eluate fractions of 1-1 .5 ml were collected . Protein content and [ 3 H]-DPCPX binding were determined .
Reconstitution of the receptor
Fractions corresponding to the peak of [3 H]-DPCPX binding activity (range 20-100% of maximal activity) were pooled and dialysed in a dialysis bag (10 000 molecular size cut-off) against 25 mm BTP buffer containing 1 mm MgCl, for 3-4 days at 4 ° C under continuous mixing to remove KCI and detergent and obtain the reconstituted receptor preparation . Protein assay
Protein concentrations were determined according to the method of Bradford [9] using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin as standard . Data analysis
Binding data were fit to one- or two-site binding models using the EBDA-
18
Pharmacological Research, Vol. 24, No . 1, 1991
LIGAND program (Biosoft, Cambridge, UK) . The program utilizes a non-linear least squares curve fitting technique with the Marquardt-Levenberg modification of the Gause-Newton method . Estimated parameters are given as means±SEM . The statistical evaluation of data was performed using a two-tailed Student t-test for unpaired or paired data .
RESULTS AND DISCUSSION Solubilization of A, adenosine receptor from bovine brain
CHAPS was used to solubilize the A, adenosine receptor because it can be easily removed from the preparation by dialysis . PC was added during all the procedures to help stabilize the receptor molecule . CHAPS (0 .5-1%) with PC (0 .8 mg/ml) solubilized about 50% of initial specific [ 3 H]-DPCPX binding sites and 50% of membrane protein . Recovery of receptors determined by specific [ 3H]-DPCPX binding was similar using 0 .5 and 1 % CHAPS . Binding of the A, adenosine receptor antagonist [3 H]-DPCPX to solubilized receptors (Ko =0 .41±0 .10 nM and B max =1213±147 fmol/mg protein, n=11), and native membranes (Kp=0 .22±0 .04 nM and B max =1084±129 fmol/mg protein, n=8) was similar and not statistically different . The CHAPS/PC solubilized receptor preparation was stable for several weeks when frozen, and freezing/thawing did not alter [ 3 H]-DPCPX binding to the receptor . This preparation was used in all further experiments . GTP (0 .1 mm) increased the binding of the specific A, antagonist [ 3 H]-DPCPX to native and solubilized A, adenosine receptors . In native membranes Bmax was 1269±40 fmol/mg protein in the absence, and 1755±42 fmol/mg protein in the presence of GTP (n=3, P<0 .01, Student t-test for paired data) . Receptor number in solubilized preparations was 1631±164 and 2106±248 fmol/mg protein in the absence and presence of GTP, respectively (n=4, P<0 .05) . The KD of binding to adenosine receptors in either preparation was not altered by GTP . This effect was specific for GTP ; 0 .1 mm ATP, GDP and inosine did not alter antagonist binding (2 nM [ 3 H]-DPCPX) to the A, adenosine receptor . Conversely, GTP decreased binding of the agonist [ 3 H]-CHA (data not shown) . GTP was previously reported to decrease the binding of agonists [3, 5, 10] and increase the binding of antagonists [4, 6, 11, 12] to A, adenosine receptors . Our findings indicate that adenosine receptors solubilized in CHAPS with PC retain their association with G-proteins . Moreover, the results support the A, receptor radiolabelling data [13], indicating that agonists and antagonists are associated with distinct conformations of the A, receptor . Chromatography of CHAPS/PC-solubilized membranes on hydroxyapatite (HA) and DEAE-cellulose
Two chromatographic procedures were developed for removal of native membrane lipids . CHAPS/PC-solubilized adenosine receptors bound almost completely to HA and were not eluted with 0 .6 M KCI ; however, 200 mm potassium phosphate by step-wise or continuous gradient (0-0 .5 M) eluted the
Pharmacological Research, Vol. 24, No . 1, 1991
19
receptor rapidly with an overall yield of greater than 40% . The ratio of receptors to total protein (fmol/mg) was unchanged compared to the loaded material . Chromatography on DEAE-cellulose is presented in Fig . 1 . Adenosine A, receptor binding activity was retained on the column and eluted with a continuous gradient of KCI (0-0 .4 M) as a single peak of activity at 200 mm KCI . The majority of proteins eluted before the peak of adenosine receptor binding activity . The overall yield of adenosine receptors was 61±9% (range 37-84, n=4), with a two- to three-fold peak increase of the ratio of receptors to protein . A lower overall yield, but a slightly greater receptor/protein ratio was observed if PC was omitted during the chromatography . These results from HA and DEAE-cellulose chromatography show that the solubilized adenosine receptor acts like an acidic protein [14] . Reconstitution of the receptor and its characterization A, adenosine receptors partially purified by DEAE-cellulose chromatography were reconstituted into PC vesicles by dialysis to remove detergent, as reported in the Methods section . Saturation binding curves for [ 3 H]-DPCPX using native and reconstituted receptors are shown in Fig . 2 . K 0 for DPCPX binding was determined to be 0.52 nM and 0.21 nM for the native and reconstituted receptors, respectively . Bmax was 820 fmol/mg protein for native and 460 fmol/mg protein for reconstituted receptors . The binding per mg protein of the reconstituted adenosine receptor preparation was lower than that of native membranes, indicating either a partial denaturation of the receptor or a different incorporation and orientation of some of the receptors in the pure PC matrix such that they are unavailable to the radioligand . However, there was no loss in binding affinity but apparently an increase . This may be the result of a different sterical modification of the reconstituted receptor or a different coupling to regulatory proteins . In a competition study (Fig . 3), the order of binding potency (R-PIA>S-PIA :> 1400 1 .40-
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Fig . 1 . DEAE-cellulose chromatography of CHAPS-solubilized A, adenosine receptors from bovine cerebral cortex membranes . Protein was eluted from the column with a KCI gradient (0-0 .4 M) . Protein content (O), and 2 nM ['H]-DPCPX binding ( •) by eluted fractions . W, wash with KCI-free buffer ; E, elution .
20
Pharmacological Research, Vol . 24, No. 1, 1991
700 600 500 400 300 200 100-/
200 400 600 Bound (fmoleefmg protein)
° I f 0 m.°~n-I 0.0 0.5 1 .0 1 .5 2 .0 Concentration of free ligand (nM)
500 400-
o
300200- •' 100-9 I ° 0 0.0 0 .5 1 .0 1 .5 2 .0 Concentration of free ligand (nM) Saturation of [ ; H]-DPCPX binding to bovine cerebral cortex native membranes (top) and reconstituted receptors (bottom) . Specific ( •) , total (O) and non-specific (A) binding are presented . Insets : Scatchard plots of specific binding . Fig. 2 .
NECA) was the same for both native and reconstituted receptors, suggesting that the receptor retained the initial binding selectivity when reconstituted in PC . Similar findings and similar rank order of potency in the displacement of DPCPX were reported by Olah et al . in a bovine brain preparation [15] . This demonstrates that native membrane components other than PC are not required for ligand binding to the receptor . The addition of GTP (0 .1 mm) decreased the affinity of R-PIA competition
Pharmacological Research, Vol. 2 4 , No . 1, 1991
100-
•
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Fig . 3 . Displacement of [`H]-DPCPX (1 nM) binding to native bovine brain membranes (right) and reconstituted receptors (left) . Ordinate values are presented as the percentage of radioligand bound in the absence of competing ligand . The reported data are the mean value from a competition experiment performed in duplicate . ( •) R-PIA ; (O) R-PIA+GTP 0 .1 mu : (A) S-
PIA ; (A) NECA .
binding to both native and reconstituted receptors (Fig . 3) . An increase in the specific binding of [3 H]-DPCPX to the reconstituted receptor was found after GTP addition (data not shown) . These results further support the hypothesis [16] that agonists can discriminate different affinity states also of DPCPX binding sites . confirming that the reconstituted receptor is associated with G-proteins . Thermal stability
The native receptor was somewhat more thermostable than the reconstituted receptor (Fig . 4) . Only a moderate decrease in the amount of [ 3 H]-DPCPX binding was found to native receptor preparations after their exposure to 30-50°C for I h . On the other hand, binding to the reconstituted receptor was decreased by previous exposure of the receptor preparation to 30°C and nearly abolished after incubation of receptor preparations at 40 and 50 ° C . No specific binding to either receptor preparation occurred after exposure to 60°C . A reduction of binding may be explained by receptor denaturation or loss of receptor-G-protein coupling . A marked reduction in thermal stability at 25°C was previously found by Nakata [1] in purified A, adenosine receptors from rat brain membranes . This result and our findings that A, adenosine receptor in native membranes is more thermostable than reconstituted receptor, indicate that the native lipids are important to give an optimal stability of the receptor . In conclusion bovine brain A, adenosine receptors retain affinity and selectivity for agonists and antagonists, and association with G-proteins after reconstitution into phosphatidylcholine . The excellent yield and simplicity of the methods
22
Pharmacological Research, Vol. 24, No . 1, 1991
Native
. L
co
100-
Q
80-
B
60`J (il c C
.a
40 200
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r 40r
20 30
50
60
temperature (°C)
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60 40 20 -
o rRM 20
30
40
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50
60
temperature (°C) Fig. 4 . Thermal stability of A, adenosine receptors . Native bovine brain membranes (A) and receptors reconstituted in phosphatidylcholine (B) were exposed for 1 h to the indicated temperatures, then incubated at 21°C with 2 nm [ 3 H]-DPCPX for 90 min . Ordinate values are expressed as per cent of binding to control preparations exposed to 20°C : hatched bars, total binding ; solid bars, non-specific binding .
presented here represent a convenient procedure for reconstitution of A, adenosine receptors, and should facilitate further pharmacological and biophysical characterization of receptor-lipid-G-protein interactions .
ACKNOWLEDGEMENTS This research was supported by National Institute of Health grant HL-35272 and the Florida Affiliate of the American Heart Association . Dr Palczewski was supported by NIH grant EY-08061 . We would like to thank Drs Paul A . Hargrave and Luiz Belardinelli for support, encouragement and use of their laboratories .
Pharmacological Research, Vol. 24, No . 1, 1991
REFERENCES I . Nakata H . Affinity chromatography of A, adenosine receptors of rat brain membranes . Molec Pharmacol 1989 ; 35 : 780-6 . 2 . Nakata H . Purification of A, adenosine receptor from rat brain membranes . .1 Biol Chem 1989 ;264 :16545-51 . 3 . Gavish M, Goodman RR, Snyder SH . Solubilized adenosine receptors in the brain : regulation by guanine nucleosides . Science 1982 ; 215 : 1633-5 . 4 . Stiles GL . A, adenosine receptor-G protein coupling in bovine brain membranes : effects of guanine nucleosides, salt, and solubilization . J Neurochem 1988 ; 51 : 1592-8 . 5 . Klotz K-N, Lohse MJ, Schwabe U . Characterization of the solubilized A, adenosine receptor from rat brain membranes . J Neurochem 1986 ; 46 : 1528-34 . 6 . Munshi R, Linden J. Co-purification of A, adenosine receptors and guanine nucleotidebinding proteins from bovine brain . J Biol Chem 1989 ; 264 : 14853-9 . 7 . Gilman AG . G proteins : transducers of receptor-generated signals . A Rev Biochem 1987 : 56 :615-49 . 8 . Weiss ER, Kelleher DJ, Woon CW, et al . Receptor activation of G proteins . FASEB J 1988 ; 2 : 2841-8 . 9 . Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem 1976 ; 72 : 248-54 . 10 . Yeung S-MH, Perez-Reyes E, Cooper DMF . Hydrodynamic properties of adenosine A, receptors solubilized from rat cerebral-cortical membranes . Biochem .1 1987 : 248 : 635-42 . 11 . Yeung S-MH, Green RD . Agonist and antagonist affinities for inhibitory adenosine receptors are reciprocally affected by 5'-guanylylimidodiphosphate or Nethylmaleimide . J Biol Chem 1983 ; 258 : 2334-9 . 12 . Ramkumar V, Stiles GL . Reciprocal modulation of agonists and antagonists binding to A, adenosine receptors by guanine nucleotides is mediated via a pertussis toxin-sensitive G protein . .1 Pharmacol Exp Ther 1988 ; 246 : 1194-200 . 13 . Barrington WW, Jacobson KA, Stiles GL . Demonstration of distinct agonist and antagonist conformation of the A, adenosine receptor . J Biol Chem 1983 ; 264 : 13157-64 . 14 . Gorbunoff MJ . The interaction of proteins with hydroxyapatite . I . Role of protein charge and structure . Anal Biochem 1984 ; 136 : 425-32 . 15 . Olah ME . Jacobson KA, Stiles GL . Affinity chromatography of the bovine cerebral cortex A, adenosine receptor . FEES Lett 1989 ; 257 : 292-6 . 16 . Martens D, Lohse MJ, Schwabe U . ['H]-8-Cyclopentyl-l,3-dipropylxanthine binding to A, adenosine receptors of intact rat ventricular myocytes . Circulation Res 1988 : 63 : 613-20 .