A1 Adenosine Receptor of Rat Testis Membranes

Vol. 265, No. 2, Issue of January 15, pp. W-677,1990 Printed in U.S.A.

PURIFICATION AND PARTIAL CHARACTERIZATION*

(Received for publication, August 7, 1989)

Hiroyasu NakataS From the Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chro- matography system (Nakata, H. (1989) Mol. Pharma- col. 35, 780-786). The Al adenosine receptor was sol- ubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine con- gener-immobilized agarose, hydroxylapatite chroma- tography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of M, 41,000 by autoradiography after radioiodination. This M, 4 1,000 peptide was also specifically labeled with an AI adenosine receptor affinity labeling reagent. A high affinity Al adenosine receptor antagonist, &cyclopen- tyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KO of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the af- finities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified AI adenosine receptor exists as a low agonist-high antag- onist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent M, 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some struc- tural homologies.

Adenosine affects many physiological processes throughout the body via its receptors which are generally subclassified into A, and Az. A, adenosine receptor mediates an inhibition and A2 adenosine receptor an activation of adenylate cyclase (Londos and Wolff, 1977; van Calker et al., 1979). Binding studies with a wide range of organs have shown that a sub- stantial adenosine agonist binding was present only in mem- brane fractions of brains and testes (Williams and Risley,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed: Section on Histopharmacology, Laboratory of Clinical Science, National Insti- tute of Mental Health, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892. Tel.: 301-496-1956.

1980; Murphy and Snyder, 1981). Although many biochemi- cal, physiological, and pharmacological studies on the adeno- sine receptors of brains have been performed, little is known about the functions of testicular adenosine receptors. Several ligand binding studies showed that the pharmacological pro- file of the adenosine binding sites in testes is A, type (Murphy and Snyder, 1981; Monaco and Conti, 1986; Stiles et al., 1986; Cushing et al. 1988). Stiles et al. (1986) showed that the adenosine receptors present in testicular membranes are cou- pled to adenylate cyclase in an inhibitory manner in a similar fashion to the A1 adenosine receptors in brains. In addition, it was reported recently that A1 adenosine receptor agonists inhibit the follicle-stimulating hormone-dependent secretion of inhibin in cultured Sertoli cells (Conti et al., 1988) and the inhibitory effects of the adenosine agonists are reversed by pertussis toxin (Monaco et al., 1988). In order to elucidate the molecular mechanism by which adenosine functions in testis tissues, it is essential to isolate and fully characterize the adenosine receptor and other proteins coupled with the recep- tor.

In this report, a purification procedure of the A, adenosine receptor to an apparent homogeneity from rat testis mem- branes is described using a novel affinity chromatography system which has recently been developed for the purification of brain A1 adenosine receptors (Nakata, 1988; Nakata, 1989a).

EXPERIMENTAL PROCEDURES

Materials-8-Cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DP- CPX)1(106 Ci/mmol) was obtained from Amersham Corp. [3H]XAC (170 Ci/mmol) was from Du Pont-New England Nuclear. Other adenosine agonists and antagonists were purchased from Research Biochemicals Inc. Adenosine deaminase, bovine trypsin, bovine brain calmodulin, and guanine nucleotides were from Sigma. Endoglycosi- dase F and Staphylococcus aureus strain V8 protease were obtained from Boehringer. Affi-Gel 10 and hydroxylapatite gel were products of Bio-Rad. Digitonin was from Gallard-Schlesinger. p-DITC was a product of Fluka. All other chemicals were from regular commercial sources. XAC-agarose was prepared by coupling XAC to Affi-Gel 10 in nonaqueous conditions as described previously (Nakata, 1989a). [3H]p-DITC-XAC was synthesized by incubation of [3H]XAC with

p-DITC as described (Stiles and Jacobson, 1988). Purified rat brain A1 adenosine receptors were obtained by a method similar to that for the purification of testis A, adenosine receptors described in this

1 The abbreviations used are: DPCPX, %cyclopentyl-1,3-dipro- pylxanthine; CPA, NG-cyclopentyladenosine; CPT, 8-cyclopentyl- theophylline; IBMX, isobutylmethylxanthine; NECA, 5’-N-ethylcar- boxamidoadenosine; PIA, N’-phenyl-2-propyladenosine; XAC!: xan- thine amine congener, 8-[4-[[[[(2- aminoethyl)amino]carbonyl]meth- yl]oxy]phenyl]-1,3-dipropylxanthine; p-DITC, 1,4-phenylenediiso- thiocyanate; DITC-XAC, 1,3-dipropyl-%(isothiocyanatophenyl- (aminothiocarbonyl(2-aminoethylaminocarbonyl(4-methylloxy- (phenyl)))))xanthine; EDTA, ethylenediaminetetraacetate; Gpp- (NH)p, guanylylimidodiphosphate; HEPES, 4-(2-hydroxyethyl)-l-pi- perazineethanesufonic acid: NEM. N-ethvlmaleimide: PAGE. ~olv- acrylamide gel electrophoresis; SDS, sodium dodecyl sulfate. - -

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672 Testicular A1 Adenosine Receptor

paper. Details will be reported elsewhere (Nakata, 1989b). Membrane Preparation and Solubilization-Rats were killed by

decapitation, and the testes were quickly removed. The testicular tissue was dissected free of epididymis and placed in ice-cold 20 mM Tris acetate buffer, pH 7.2, containing 1 mM EDTA and 320 mM sucrose. The tissue was homogenized with a Polytron, and the ho- mogenate was centrifuged at 1000 X g for 5 min at 4 “C. The supernatant was centrifuged again at 38,000 x g for 20 min at 4 “C. The resulting pellet was washed by resuspension in 10 volumes of 50 ItIM Tris acetate buffer, pH 7.2, containing 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride and by centrifugation at 38,000 X g for 20 min. This washing was repeated three times. The final pellet was resuspended in the same buffer containing 2 units/ml of adeno- sine deaminase and incubated at 30 “C for 10 min. The membrane suspension was then centrifuged at 38,000 X g for 20 min at 4 “C. The pellet was suspended in 16 volumes of 50 GM Tris acetate buffer, pH 7.2, containing 1% digitonin, 0.1% sodium cholate, 100 mM NaCl, 5 mM MgClz, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmeth- anesulfonyl fluoride, and 1 pg/ml each of pepstatin A, leupeptin, chymostatin, and antipain. The mixture was gently homogenized with a Polytron, stirred for 1 h on ice, and centrifuged at 100,000 X g for 1 h at 4 “C. The clear supernatant was saved as the solubilized preparation.

Purification of A, Adenosine Receptor-All the operations were performed at 4-7 “C. The solubilized preparation (75 ml) was loaded onto a 15-ml column of XAC-agarose at 15 ml/h. This was then washed with 8 volumes of 50 mM Tris-acetate buffer, pH 7.2, con- taining 100 mM NaCl, 1 mM EDTA, and 0.1% digitonin (buffer A). The bound receptor was eluted with 3 volumes of buffer A containing 100 FM CPT. The pooled active fractions were loaded on a 0.5-ml hydroxylapatite column at 15 ml/h. The column was washed succes- sively with 5 ml each of 10 and 100 mM potassium phosphate buffer, pH 7.2, containing 100 mM NaCl and 0.1% digitonin, and the receptor was eluted with 6 volumes of 500 mM potassium phosphate buffer, pH 7.0, containing 100 mM NaCl and 0.1% digitonin. The eluate from the hydroxylapatite column was loaded on a l&ml column of XAC- agarose at 15 ml/h. After washing with 5 volumes of buffer A, the column was eluted with 3 volumes of buffer A containing 100 pM CPT at 15 ml/h. The eluate was concentrated by Centricon 10 (Amicon) to 20-fold and injected in 50-~1 aliquots onto a TSK- 3000SW column (7.5 X 300 mm) eauilibrated with 10 mM sodium phosphate, pH 6.8, containing 156 rn& NaCl and 0.2% digitonin. The column was eluted with the equilibration buffer at 24 ml/h. Aliquots of the eluates were radioiodinated by the chloramine-T method (Hunter and Greenwood, 1962) for the SDS-PAGE analysis or en- zyme digestion studies.

Binding Assay-[3H]DPCPX binding assays were performed as described previously (Nakata, 1989a) for 10 h at 0 “C in a total volume of 0.25 ml. For the assay of the highly purified receptor preparations, i.e. the eluates from either the first XAC-affinity chromatography, hydroxylapatite chromatography, second XAC-affinity chromatog- raphy, or gel permeation chromatography, a small aliquot of pass- through fractions of the first XAC-affinity chromatography which had been heated at 80 “C for 3 min, centrifuged to remove the precipitate, filtered through with 0.22-c(m membranes, and desalted on Sephadex G-50 columns equilibrated with 50 mM Tris acetate (pH 7.2), 100 mM NaCl, 1 mM EDTA, 5 mM MgCl,, 0.1% digitonin, was added to the assay mixture at the protein concentration of about 20 rg/ml. The heat-treated pass-through fraction had no [3H]DPCPX binding activity under the assay conditions employed. Nonspecific binding was determined by the addition of 1 wM XAC. The reaction was terminated by filtration through Whatman GF/B glass fiber filters pretreated with 0.3% polyethyleneimine (Bruns et al., 1983) and the filters were washed three times with 5 ml of cold 50 mM Tris- acetate buffer, oH 7.2, using a cell harvester (M-48, Brandel). The filters were counted in Aquas01 by a liquid scintillation counter.

Affinity Labeling of A, Adenosine Receptor-The affinity labeling of A1 adenosine receptors of testes was performed essentially as described previously (Stiles and Jacobson, 1988). The solubilized or purified receptor preparations were desalted into 50 mM HEPES buffer, pH 8.1, containing 100 mM NaCl and 0.1% digitonin. The desalted preparations were incubated with or without 1 I.&M XAC for 30 min at 0°C. [‘H]p-DITC-XAC (20 nM) was then added and the incubation continued for 30 min at room temperature. The incubated mixture was desalted into buffer A and lyophilized. Labeled receptors were analyzed by SDS-PAGE followed by fluorography.

SDS-PAGE-The discontinuous buffer system of Laemmli (1970) was used for SDS-PAGE with 10,12, or 14% polyacrylamide separat-

ing gel. Samples were dissolved in SDS-sample buffer containing 3% SDS and 3% P-mercaptoethanol, and heated to 95 “C for 3 min unless otherwise indicated and subjected to SDS-PAGE. Gels containing radioiodinated samples were dried after the electrophoresis and ex- posed to Kodak X-Omat AR films with intensifying screens for 2-3 days at -85 “C. Gels containing tritium were fixed in 10% trichloro- acetic acid and then immersed in ENHANCE (Du Pont-New England Nuclear). The gels were dried and exposed to Kodak X-OrnaT AR film at -85 “C for l-2 weeks.

Enzymatic Digestion-Aliquots of ‘Z51-labeled A1 adenosine recep- tors purified from rat testis and brain membranes were digested with S. aureus V8 protease or bovine trypsin for 1 h at room temperature. The radioiodinated receptor preparations were also treated with Flavobacterium meningosepticum endoglycosidase F for 12 h at room temperature. SDS sample buffer was added after the incubation, and the digests were electrophoresed on 12 or 14% polyacrylamide gels.

Protein Determination-Protein concentrations were determined by the modified Lowry method (Peterson, 1977) or Amido Schwarz method (Schaffner and Weisman, 1973) using bovine serum albumin as a standard.

Data Analysis of Binding Assays-Saturation and displacement curves were analyzed by computer programs EBDA-LIGAND (Elsev- ier-Biosoft) and Grauh PAD (ISI Software). resuectivelv. Disulace- - . ment curves were analyzed using a cooperative (logistic) model as described by Bruns et al. (1986): B = & - B. x [L]“/([L]” + [I&,]“) where B is counts/min bound, B, is counts/min total binding without displacers, B. is counts/min specific binding without displacers, [L] is the concentration of displacers, and n is a cooperativity (pseudo- Hill) coefficient.

RESULTS

Solubilization and Purification of A1 Adenosine Receptor from Testis Membranes-The A1 adenosine receptor of testis membranes were solubilized by digitonin/cholate. Approxi- mately 30% of the binding activity and 40% of membrane proteins were released in a soluble form with no increase in the specific activity (see Table I). When the solubilized prep- aration was applied to the XAC-agarose column, most of the proteins passed through while about 90% of the [3H]DPCPX binding activity remained in the column. After the column was washed extensively, it was eluted specifically with a potent Ai adenosine receptor antagonist, CPT. More than 40% of the binding activity applied on the column were recovered. This affinity chromatography step resulted in a 2000-fold purification over the solubilized preparation. A typ- ical chromatography profile was shown in Fig. 1.

Adsorption of the [3H]DPCPX binding activity to the XAC- agarose was inhibited by preincubation of the solubilized preparation with CPT or XAC and to a lesser extent by CPA, (R)-PIA or NECA. CPA and CPT were the most effective agents in eluting the bound receptor from XAC-agarose. The less active agonists or antagonists such as (S)-PIA, CV-1808, IBMX, or theophylline were not effective in either experi- ments (data not shown).

The final purification to homogeneity of the receptor was achieved by gel permeation chromatography on TSK- 3000SW. Fig. 2 shows a typical elution profile of [3H]DPCPX binding activity from the TSK-3000SW column. The protein concentration of the eluate was too low to be determined and the specific binding activity of the final receptor preparation could not be determined in this study. The active fractions from the TSK-3000SW column were radioiodinated and sub- jected to SDS-PAGE. The autoradiography pattern of the peak fractions revealed a single broad M, 41,000 protein (Fig. 2, top inset).

The results of a typical purification are summarized in Table I. The receptor was purified approximately 24,000-fold after the second affinity chromatography. An overall yield of the [3H]DPCPX binding activity after the gel permeation chromatography was approximately 2.5% of the initial [3H]

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TABLE I PurJ~catwrz of Al ndenosme receptors from rat tortes

All the data in this table are from the same purlficatlon experiment vchlch is representative of three experunents.

673

step Total Total SpWltiC step\\ 1% 01 ernll protem actl\lty” act1\ It? Ydd purlficatlon purllicatlon

w pl7101 pmoi/m~~ r< -/h/d

Membranes 3ilh 122 0.33 100 1 1 Solublllzed 14P 40.5 0.28 33 0.8 0.8 1st XAC-agarose 0.026’ 17.8 685 14 2,4g50 1,960 Hydroxylapatlte 0.009~ 11.7 1,300 9.6 1.9 3,710 2nd XAC-agarose 0.001’ 8.5 8,500 7.0 6.3 24,300 TSK .1OOOSW ND” 3.0 2.5

’ Determmed as described under “Exnerimental Procedures” usme 1.3 nM I’HIDPCPX. “Determined by the modified Lowrymethod by Peterson (1977). ~’ . ’

Determined by Amido Schwarz method (Schaffner and Weissman, 1973). ” Not determined.

0 5 10 15 20 25 FRACTIONS (10 ml)

FIG. 1. XAC-agarose chromatography of digitonin-solubi- lized rat testis A, adenosine receptors. Starting material (75 ml of digitonin-solubilized preparation) was passed through a XAC- agarose column (2 x 4.8 cm) at a flow rate of 15 ml/h. The column was then washed with buffer A (50 mM Tris acetate, 100 mM NaCl, 1 mM EDTA, 0.1% digitonin, pH 7.2) at a flow rate of 15 ml/h. Receptor activity was eluted biospecifically with 100 pM CPT in buffer A at a flow rate of 15 ml/h. Aliquots of eluted fractions were desalted on Sephadex G-50 columns equilibrated with 50 mM Tris acetate buffer, pH 7.2, containing 100 mM NaCl, 1 mM EDTA, 5 mM MgCI,, and 0.1% digitonin and assayed for [“HIDPCPX binding activity using 1.3 nM [lH]DPCPX as described under “Experimental Procedures.” The protein in the pass-through and wash fractions was determined by the modified Lowry method (Peterson, 1977) and the protein in the eluates was measured by the Amino Schwarz method (Schaffner and Weissman, 1973). The presumed structure of XAC- agarose was shown in the inset.

DPCPX binding activity in intact membranes. Purity and Identity of the Receptor Protein-The SDS-

PAGE of the purified A, adenosine receptor under reducing conditions revealed a single band of M, 41,000 (Fig. 3, lane A). The same mobility of the band in SDS-PAGE was also obtained under nonreducing conditions (Fig. 7). SDS-PAGE of the receptor preparation after the second affinity chroma- tography showed minor bands of M, 65,000,30,000, and 28,000 in addition to a major band of M, 41,000 (data not shown). Lanes B-E of Fig. 3 show the results of affinity labeling of the purified and solubilized A, adenosine receptor prepara- tions with [‘H]p-DITC-XAC. The M, 41,000 peptide of the

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18-

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18 19 20 21 22

FRACTIONS

+41 kD;

FRACTION NUMBER (0.4 ml) FIG. 2. Gel permeation chromatography of testis A, adeno-

sine receptor on TSK-3000SW column. The eluate of the second XAC-agarose chromatography was concentrated by Centricon 10 and an aliquot (50 ~1) which contained 2 pmol of [ ‘HIDPCPX binding activity was loaded on a TSK-3000SW column (7.5 x 300 mm). The column was eluted with 10 mM sodium phosphate buffer, pH 6.8, containing 150 mM NaCl and 0.2% dlgitonin at a flow rate of 24 ml/ h at 2-4 “C. Fractions of 0.4 ml were collected, and ahquots (100 ~1) of each fraction were assayed for [ ‘HIDPCPX binding as described under “Experimental Procedures.” The elution positlons of standard protein were indicated by the arrows. I, thyroglobulin; 2, y-globulm; 3, ovalbumin; 4, myoglobin. Aliquots of the eluates were also radiol- odmated by chloramme-T method and subjected to SDS-PAGE. An autoradiogram of the resulting gel is shown with the fraction numbers (top Inset). Prestained protein standards (Bethesda Research Labo- ratories) were used for the molecular weight calibration.

specific labeling reagent (Fig. 3, lanes B and C), although a distinct peptide which corresponds to A, adenosine receptor was not detected for the crude solubilized preparation (Fig. 3, lanes D and E). These results indicate that the nurified A, purified preparation was labeled specifically with this A,-

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ABC DE FIN. :3. SDS-PAGE analyses of testis A, adenosine receptor.

Lane A, autoradiogram of the purified A, adenosine receptor radioi- odinated by chloramine-T method. Lanes B-E, fluorography of the purified (lanes R and C) or solubilized (lanes D and E) A, adenosine receptors labeled with [“H]p-DlTC-XAC in the absence (lanes B and D) or in the presence of 1 pM XAC (lanes C and E). The SDS-PAGE was performed in 12% polyacrylamide gels as described under “Ex- perimental Procedures” under reducing conditions. Prestained pro- tein standards (Bethesda Research Laboratories) were used for the molecular weight calibration.

15 r- Specific binding

2 4 6 FREE ?HIDPCPX InM)

1

FIG. 4. Saturation isotherm for [3H]DPCPX binding with purified testis A, adenosine receptor. The receptor binding assay was performed as described under “Experimental Procedures.” The A, adenosine receptor purified through the second XAC-affinity chromatography (6 ng of protein) was assayed with various concen- trations of [“HIDPCPX. The KI, and B,,, values were 1.4 k 0.1 nM and 16 f 3 nmol/mg of protein, respectively (n = 3). Inset, Scatchard plots of the same data.

adenosine receptor from rat testis membranes consists of a M, 41,000 polypeptide chain. It was also shown that the appearance of the M, 41,000 peptide in the SDS-PAGE co- incided with that of the [“HIDPCPX binding activity in the gel permeation chromatography (Fig. 2) supporting that the M, 41,000 peptide contains the ligand binding site.

Binding Characteristics of the Purified A1 Adenosine Recep- tor-Because of the limited amount of the completely purified receptor preparations, most of the ligand binding experiments were performed with the receptor preparations purified through the second affinity chromatography. The binding of [“HIDPCPX to the highly purified receptor preparations was very low when the assays were performed as described under “Experimental Procedures” without the addition of the pass- through fractions of the first affinity chromatography. How- ever, the addition of a small amount of heat-treated pass-

TABLE II Inhibition of t’H]DPCPX binding to A, adenosine receptors by

adenosine agonists and antagonists For each adenosine ligand, competitive inhibition experiments

were performed as described under “Experimental Procedures” using l-l.4 nM [“HIDPCPX. I&,, and Hill coefficient (n) values were estimated from nonlinear curve-fitting to a logistic model (Bruns et al., 1986) using a computer program Graph Pad, and the ICSO values were converted to K, values by the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The values are the means of two to three experi- ments.

Solubilized Purified Agents

K n K, R

llM RM

Agonists CPA 3.3 k 0.1 0.83 f 0.06 23 +- 5 0.76 -c 0.05 (R)-PIA 4.9 f 0.4 0.97 f 0.02 28 f 5 0.88 + 0.06 NECA 30 f 1.6 0.82 f 0.03 230 + 20 0.78 f 0.03 (S)-PIA 230 +40 0.85 f 0.04 1200 + 200 0.83 + 0.05

Antagonists DPCPX 0.9 -c 0.6 0.97 + 0.03 1.1 f 0.3 1.1 rfr 0.05 CPT 7.8 + 3.0 0.91 f 0.03 8.5 + 1.0 0.97 f 0.03 IBMX 3700 f 1000 1.00 + 0.10 4900 f 100 0.93 f 0.05

6

0 -7 -6 -5 -4 OoLW -a

log [GPP(NHIP] (M) log IGPPINHIPI (MI

FIG. 5. Effects of Gpp(NH)p on [3H]DPCPX binding activity of A, adenosine receptors. Solubilized (80 pg of protein) (A) or affinity-purified (40 ng of protein) (B) preparations of testis A, adenosine receptor were incubated with 1.5 nM [“HIDPCPX and various concentrations of Gpp(NH)p in the presence or absence of 1 mM NEM for 10 h at 0 “C. The reaction was terminated by filtration through the polyethyleneimine-treated GF/B filters as described un- der “Experimental Procedures.” Specific [“HIDPCPX binding activ- ity of control (100%) in solubilized and affinity-purified receptors represented 5400 and 5800 dpm, respectively.

through fractions of the first affinity chromatography in- creased the [3H]DPCPX binding activity to approximately 5- fold. The similar effect was observed with bovine serum albumin or calmodulin, although the increase was slightly low (data not shown). This activation was not seen when the crude solubilized preparation was used as a receptor sauce.

Scatchard plots of the saturation isotherm of [“HIDPCPX binding with the purified receptor preparations (Fig. 4, inset) identified a single set of sites with a binding capacity (B,,,,J of approximately 16 + 3 nmol/mg of protein (n = 3) and an apparent equilibrium dissociation constant (&) of 1.4 f 0.1 nM (n = 3). The KI, value is similar to that obtained in either completely purified receptor preparations through the TSK- 3000SW column chromatography (Ku = 1.3 nM, n = 1, data not shown) or crude soluble receptor preparations (Kn = 1.0 + 0.3, n = 3, Fig. 6A). The specificity of the receptor was investigated by competition binding studies with various

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BOUND I?HIDPCPX (pmol mg protein)

-__ B. Affinity-purified I

0 200 400 600 BOUND 13HIDPCPX (pmolimg protein)

13~:. 6 Scatchard plots of [“HIDPCPX binding to the solu- bilked (A) and affinity-purified (B) testis A1 adenosine recep- tors in the presence or absence of Gpp(NH)p. The solubilized preparation (100 pg of protein) or the affinity-purified preparation (0.1 pg of protelni were mcubated with 0.2-20 nM [ ‘HIDPCPX in the presence or absence 01 100 FM Gpp(NH)p as described under “Ex- perunental Procedure< ” The parameter estimates for the solubilized preparation5 were as follows: K,,. 1.0 (control) and 0.9.5 nM (+Gpp(NH)p, H,,,,,,, 0 31 (control) and 1.X pmol/mg of protein (+(;pp(NH)p). The parameter estimates for the affinity-purified preparatlone were as follo\f s: K,,, 1.3 n&t (control) and 1.0 nM (+(:pp(NH)p): H ,,,‘,\, 70.5 pmol/mg of protein (control) and ‘i20 pmol/ mg of protein (+Gpp(NH)p). These values are the mean of two to three heparate experiments.

adenosine analogues. The purified receptor showed a typical A, adenosine recept,or pharmacological specificity similar to that of the solubilized preparation (Table II). The affinity of the purified receptor for adenosine agonists was found to be lower than that of the crude solubilizedpreparations, although there were no significant differences in the affinity for the antagonists between the purified and crude solubilized recep- tor preparations.

Modulation of r’H]DPCPX Binding by Guanine Nucleo- tide--Stable guanine nucleotide, Gpp(NH)p, increased [“HI DPCPX binding of the solubilized receptor preparation by 2.5.fold as shown in Fig. 5A. In addition, it was shown that the sulfhydryl alkylating agent NEM activates [“HIDPCPX binding activity of the solubilized preparation to the similar extent as Gpp(NH)p. However these effects were not additive because Gpp(NH)p failed to activate the binding activity in the presence of NEM as shown in Fig. 5A. In contrast, the activation by Gpp(NH)p or NEM was not observed with the purified receptor preparations as shown in Fig. 5B. Fig. 6 shows Scatchard plots of [“HIDPCPX binding with the solu- bilized and purified receptor preparations in the presence and absence of 100 pM Gpp(NH)p. These plots indicate that the

kDa 94- 67- m

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144-

BPB-

+ME -ME FIG. 7. SDS-PAGE autoradiography of ‘““I-iodinated puri-

fied A1 adenosine receptors from rat testis and brain mem- branes. The purified receptor preparations were radiolodinated by chloramine-T method and subjected to SDS-PAGE. SDS-PAGE was performed m 10% polyacrylamide gel under either reducing (ex- pressed as +ME) or nonreducing (expressed as -ME) conditions. ME, 2-mercaptoethanol; BPR, bromphenol blue.

activation of [“HIDPCPX binding with the solubilized recep- tor preparations by Gpp(NH)p was due mainly to the increase of B,“,,, and there were no significant effects of Gpp( NH)p on [“HIDPCPX binding with the purified receptor.

Comparison of Subunit Structure of Purified Testis A, Aden- osine Receptor with Purified Brain A, Adenosine Receptor- Fig. 7 shows the autoradiograms of SDS gels of radioiodinated A, adenosine receptors purified from rat testis and brain membranes. There was a significant difference in the molec- ular weight between the two receptors, i.e. the apparent mo- lecular weights for the testis and brain A, adenosine receptors are estimated to be 41,000 and 34,000, respectively. Aliquots of the ““I-labeled purified receptor preparations were sub- jected to partial proteolytic degradation with trypsin or V8 protease followed by SDS-PAGE as shown in Fig. 8, A and B. The profiles of peptides generated by the proteolysis of the two receptors are similar but not the same. After the trypsin treatment, both receptors generated two major small peptides at M, = 15,000 and 11,000 in addition to peptides of M, = 31,000 and 27,000 for testis and brain receptors, respectively. Treatment of the receptors with V8 protease generated two major peptides at M, = 29,800 and 13,300 for the testis receptor and also two major peptides at M, = 26,000 and 13,300 for the brain receptor. To examine the contribution of the N-linked glycosyl moieties to the molecular size, testicular and brain A, adenosine receptors were treated with endogly- cosidase F. Endoglycosidase F caused a reduction in apparent

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Std A B C D E F Std

Brain Testis

Std A B C D E F

Brain Testis

FIN. 8. SDS-PAGE of purified A, adenosine receptors from rat testis and brain membranes treated by bovine trypsin and S. aureus VS protease. The purified receptors were radioiodinated by the chloramine-T method and were subjected to the enzyme digestion studies. A, effect of bovine trypsin on A, adenosine recep- tors. Purified receptors (10 ~1) from brain (lanes A-C) and testis (Innes 1)-F) were proteolyzed using 0 pg (lanes A and D), 0.1 fig (lams R and E), or 0.2 pg (lanes C and F) or trypsin for 1 h at room temperature. The samples were then subjected for SDS-PAGE as described under “Experimental Procedures” on 14% polyacrylamide gel. R, effect of S. aureus V8 protease on A, adenosine receptors. The purified recept,ors (10 ~1) from brain (lanes A-C) and testis (lanes D- F) were partially proteolyzed using 0 pg (lanes A and D), 1 pg (lanes H and E), or 2 pg (lanes C and F) of V8 protease for 1 h at room temperature. The samples were then prepared for SDS-PAGE and subjected for electrophoresis on 14% polyacrylamide gel. The auto- radiogram of the dried SDS gel was obtained as described under “Experimental Procedures.” Std, radioiodinated standard proteins, i.e. bovine serum albumin, ovalbumin, carbonic anhydrase, and soy- bean trypsin inhibitor.

molecular weight in both receptors as shown in Fig. 9. The molecular weight of the testis A, adenosine receptor shifted to 30,000 from 41,000. The peptide of the purified brain A, adenosine receptor was also shifted to 30,000 from 34,000 after the endoglycosidase F treatment. Several low M, pep- tides were also observed after the endoglycosidase F treat- ment. They are probably due to the action of contaminated proteases in the endoglycosidase F preparations used in this study.

DISCUSSION

Although adenosine receptors in rat testicular tissue have been well established, their physiological function is still largely unknown. Monaco and Conti (1986) suggested the possibility that purines might serve as local modulators in the testis. They have provided evidence that adenosine and its analogs modulate the hormonal response of Sertoli cell-en- riched cultures by interacting with A1 adenosine receptors (Monaco et al., 1984; Conti et al., 1985; Monaco et al., 1988; Conti et al., 1988). Whatever the functions are, the molecular characterization of components for the adenosine transduc- tion system, especially of A, adenosine receptor itself, is essential for the further advancement of the studies men-

67- c

BPB - II,- ..:;,:!; ,‘.&+

Std A B C D E F

Testis Brain

FIG. 9. Effect of endoglycosidase F treatment on purified A, adenosine receptors. The radioiodinated purified receptors (10 ~1) from testis (lanes A-C) and brain (lanes D-F) were treated with 0 unit (lanes A and D), 0.05 unit (lanes B and E), or 0.1 unit (lanes C and F) of endoglycosidase F for 12 h at room temperature. The samules were then subiected to SDS-PAGE on 120/O uolvacrvlamide gel As described under “Experimental Procedures.“‘Tie a;toradi- ograms of the dried gels were shown. Std, standard proteins radioio- dinated by chloramine-T method (bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor); BPB, brom- phenol blue.

tioned above. In this study, purification of the A, adenosine receptor of rat testicular membranes has been performed.

The most effective step in the purification was affinity chromatography using XAC as an immobilized ligand. The interaction of the solubilized receptors with the immobilized ligand appears to be solely on the basis of biospecific affinity interactions. The purified A, adenosine receptor migrated as a single broad band on SDS-PAGE with an apparent molec- ular weight of about 41,000, either in the absence or presence of reducing agent (Fig. 7). Affinity labeling with [“H]p-DITC- XAC, a specific acylating agent for brain A, adenosine recep- tors (Stiles and Jacobson, 1988), demonstrated that the M, 41,000 protein contains the adenosine binding sites (Fig. 3). The affinity labeling of the crude soluble preparation did not show a specifically labeled band in SDS gels probably because a significant amount of nonspecific incorporation masked the specific labeling of the receptor (Fig. 3). It has been reported that a peptide of M, 42,000 was labeled in testicular mem- branes by an A, photoaffinity labeling reagent (Stiles et al., 1986). The specific binding activity of the receptor prepara- tion purified through the second affinity chromatography was 16 nmol/mg of protein on the basis of Amido Schwarz protein assay (Schaffner and Weissman, 1973). If we assume a M, 41,000 for the receptor protein and one binding site per molecule, the theoretical binding capacity for a pure receptor is 24.4 nmol/mg protein. Based on such a model, the purified receptor through the second affinity chromatography is about 66% pure.

During preliminary studies, the purification of the testis A, adenosine receptor has been hampered by a very low recovery of the binding activity after the XAC-affinity chromatogra- phy. However, it was found that the addition of the heat- treated pass-through fractions of the first XAC-affinity chro- matography into the assay mixture can restore the binding activity to about s-fold. Because the similar activation was observed by the addition of other proteins, it is likely that a small amount of protein prevents the ligand-bound receptors from adsorbing to the assay tubes or to the filtration appara- tus.

The competition studies with adenosine receptor agonists and antagonists showed that the purified receptor retained the characteristic pharmacology of A, adenosine receptors

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Testicular A1 Adenosine Receptor 677

(Table II). Thus, the following potency series for agonists and antagonists were obtained: CPA > (R) - PIA > NECA > (S) - PIA for agonists and DPCPX > CPT >> IBMX for antag- onists. These pharmacological properties of the purified recep- tor were similar to those found in the solubilized preparation (Table II) as well as in membrane preparations (Murphy and Snyder, 1981; Cushing et al., 1988), although the K, values for every agonist were increased about B-fold after the purifica- tion (Table II). The similar decrease in affinities for agonists after the purification was observed with rat brain A1 adenosine receptors (Nakata, 1989a). These results indicate that the purified testis Ai adenosine receptor exists as a low agonist- high antagonist affinity state. This is probably due to sepa- ration of G protein(s) responsible for the high affinity agonist binding from the receptor during the purification.

As shown in Fig. 5A, the binding of [3H]DPCPX, a specific A1 adenosine antagonist, with the solubilized receptor prepa- rations was enhanced by Gpp(NH)p in a similar fashion to the enhancement of [3H]DPCPX binding with the solubilized brain preparations by GTP or Gpp(NH)p (Nakata, 1989a). This activation was not observed if the receptor preparations had been activated with NEM suggesting the mechanism of activation by Gpp(NH)p and NEM is similar. From the Scatchard plots of [3H]DPCPX binding in the presence or absence of Gpp(NH)p, the activation by Gpp(NH)p was ex- plained by the increase of B,,, (Fig. 6A). On the contrary, there was no significant effect on [3H]DPCPX binding by either Gpp(NH)p or NEM with purified receptor preparations (Fig. 5B). Similar activation effects by guanine nucleotides were reported (Yeung and Green, 1983; Ramkumar and Stiles, 1988; Stiles, 1988). NEM treatment of the solubilized A1 adenosine receptor mimicked the effect of Gpp(NH)p in that it increased the binding of [3H]DPCPX and coaddition with Gpp(NH)p did not increase the binding more than the level obtained by the Gpp(NH)p alone as shown in Fig. 5A. It is known that the modification of sulfhydryl groups by NEM inactivates Gi, because it leads to a shift of different receptor types to a low affinity state. The present results are similar to those of Yeung and Green (1983) who reported that both Gpp(NH)p and NEM increased the binding of adenosine antagonist [3H]diethylphenylxanthine to rat hippocampus membranes, but these effects were not additive. Taken to- gether, the influence of guanine nucleotides on [3H]DPCPX binding with the solubilized adenosine receptor is probably mediated via G proteins which is known to be coupled with A1 receptor even in the solubilized form (Stiles, 1985), al- though the precise mechanism of the activation is not known yet. The results that there is no change in [3H]DPCPX binding for the purified A1 adenosine receptor by treatment with NEM indicate that no essential sulfhydryl groups for the antagonist binding exist in the testis Ai adenosine receptor.

A direct comparison of the purified receptors by SDS- PAGE demonstrated that the molecular weight of the rat testis A, adenosine receptor (41,000) is significantly larger than the rat brain A1 adenosine receptor (34,000) as shown in Fig. 7. In order to compare the structure of these adenosine receptor peptides and also to examine the possibility that the difference in the molecular weight is due to the different carbohydrate moiety attached to these receptors, the radioi- odinated receptors were treated with two proteases and en- doglycosidase F. As shown in Fig. 8, A and B, purified A, adenosine receptors from testis and brain membranes were

degraded by trypsin and S. aureus V8 protease and produced several peptides which may be useful for sequence analysis. The patterns of peptide fragments generated from the two Ai adenosine receptors are similar but not the same. The differ- ence in molecular weight of some peptides, i.e. a 31,000 peptide of the testis receptor and a 27,000 peptide of the brain receptor generated by trypsin shown in Fig. 8A and a 29,800 peptide of the testis receptor and a 26,000 peptide of the brain receptor generated by V8 protease shown in Fig. 8B could be due to the difference of the carbohydrate moiety present in those peptides as discussed below. It should be noted that the peptide mapping performed in this study was based on the visualization of ‘251-labeled peptides by autoradiography. Thus, unlabeled peptides which lack iodinated tyrosines are not visualized. The lz51-labeled purified A, adenosine recep- tors were also deglycosylated with endoglycosidase F which can remove both N-linked high mannose- and complex-type carbohydrate chains from proteins (Elder and Alexander, 1982). Deglycosylation of the receptors led to a peptide of M, 30,000 in both brain and testis adenosine as shown in Fig. 8. Taken together, it is likely that the core primary structures of these two adenosine receptors are similar and N-linked glycosyl moieties are responsible for the differences in the apparent molecular size observed in SDS-PAGE.

In summary, this study presents the first purification of a peripheral Ai adenosine receptor. The ability to purify the receptor should allow the detailed molecular characterization of this receptor as well as the production of specific anti- receptor antibodies, although it is necessary to improve the yields in the various purification steps in addition to scale up the purification method to obtain sufficient amount of puri- fied A, receptor for these purposes.

Acknowledgments-I wish to thank Dr. David M. Jacobowitz for his support and encouragement. I also wish to acknowledge the excellent technical assistance of Robert P. McDevitt and the expert secretarial assistance of Lois Brown.

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H Nakatacharacterization.

A1 adenosine receptor of rat testis membranes. Purification and partial

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