Regulation of Agonist-Receptor Binding by G Proteins and Divalent Cations in Spermatozoa Solubilized A1 Adenosine Receptors

MOLECULAR GENETICS AND METABOLISM ARTICLE NO.

63, 183–190 (1998)

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Regulation of Agonist-Receptor Binding by G Proteins and Divalent Cations in Spermatozoa Solubilized A1 Adenosine Receptors Alba Minelli, Cinzia Allegrucci, Roberto Rosati, and Isabella Mezzasoma Dipartimento di Biologia Cellulare e Molecolare, Universita` di Perugia, Perugia, Italia Received November 24, 1997

Receptors on the cell surface regulate G protein function by catalyzing the release of GDP from the a-subunit of the G protein, allowing GTP to bind and activate the G protein. GTP hydrolysis has two important consequences: it constitutes the basic mechanism by which deactivation occurs and makes the overall kinetics of the cycle those of a steadystate system. G protein activation is thought to be concomitant with a subunit dissociation from bg subunits. Activated G protein a and bg subunits interact with effector proteins to modulate intracellular secondmessenger metabolism. G protein deactivation is mediated by a-subunit GTPase activity. The deactivation rate, which may be limited by the rate of phosphate released from a-GDP-Pi , is catalyzed by membrane-bound GTPase accelerating proteins. Upon deactivation, a- and bg-subunits reassociate (2). It is known that guanine nucleotides selectively decrease the affinity of agonists for A1 receptors (3). Sodium also decreases the affinity of agonists for histamine H1, opiate, and a2 adrenergic receptors (4–6). In contrast, in several instances (7–9), receptor binding for agonists is enhanced by divalent cations which do not influence antagonist interactions with receptors. The inhibitory effect of the nonhydrolyzable guanine nucleotide Gpp(NH)p is partially reversed by Mg2/ which, in turn, can totally reverse the decrease in GTP-induced agonist binding (3). This effect suggests that divalent cation-dependent hydrolysis of GTP is the molecular basis of the reversal of GTP-induced inhibition of agonist binding by Mg2/ and that this hydrolysis is essential for receptor-mediated inhibition of activity (10). Hydrolysis of bound GTP terminates the active form of G pro-

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with Gb-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as a-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2/, Ca2/, and Mg2/, potentiated the agonist-specific binding: Mn2/ had the highest apparent affinity with half-maximal effect at 50 mM. Binding assays, performed in the presence of 100 mM Mn2/, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2/, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2/ and 100 mM Mn2/ enhanced GTPase activity, whereas 5 mM Ca2/ resulted in the increase of Vmax values without significant alterations of Km . In the presence of A1-specific agonists, Mn2/ and Mg2/ caused a decrease of Vmax values and an increase of GTP affinity. Other cations, such as Co2/, Cd2/, Cu2/, and Zn2/, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics. q 1998 Academic Press Key Words: solubilized A1 adenosine receptor; G proteins; GTPase activity; divalent cations; spermatozoa A1AR.

G protein-coupled receptors play an important role in biological systems and function as conditional catalysts of the G protein nucleotide-exchange cycle (1). At the moment, their activation mechanisms can be suggested only by thermodynamic and structural models (1,2). 183

1096-7192/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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tein, and GTPase, responsible for this hydrolysis, is an integral function in the a subunit of the G protein, strictly dependent on nanomolar concentrations of Mg2/ (11). Apart from this low-Km GTPase, specifically stimulated by activation of receptors coupled to the inhibition of adenylate cyclase (11,12), plasma membranes also express high-level activities of nucleotide triphosphatases which metabolize GTP and other nucleotides with an affinity in the high micromolar range (13). These high-Km enzymes cause nonspecific GTP hydrolysis which needs to be considered when GTP is used as a modulator of receptor-related events. As Me–GTP complexes are the preferred substrates for such activities, it is not unlikely that specific interaction between divalent cations and GTP in regulating receptor binding could be hindered by these nonspecific activities (13). Recently, the agonist-binding properties of the human A1 receptor, after saponin-permeabilizing treatment, have been characterized (14). Saponin was shown to facilitate the interaction of guanine nucleotides with receptor–G protein complexes, possibly by removing a permeability barrier which prevents the access of GTP to the G protein. It is assumed that if the receptor–GTP-binding protein complex can be kept intact in the solubilized membrane fractions, then the molecular interactions of the proteins can be studied more efficiently. Until now, studies on the modulation of A1 agonist binding by divalent cations have been performed with solubilized receptors and it has been assumed that these receptors were still coupled to the guanine nucleotide-binding protein because, in many cases, they retained almost all the regulatory functions (15–18). In this paper, we provide direct evidence of the existence of the binary complex receptor–G protein in the solubilized A1 receptor. It is known (11) that cycling with GTP requires not only GTP but also the receptor whose role is to facilitate the activation of G proteins. Therefore we used this solubilized preparation, tightly coupled to its G proteins, to investigate the effects of divalent cations on A1-agonist binding modulation and on GTP hydrolysis in an attempt to establish whether the modulatory mechanism, responsible for the different responses observed, is independent of coupling of the receptor to its G proteins. MATERIAL AND METHODS DTT, Mops, benzamidine, App(NH)p, CPA, digitonin, aprotinin, leupeptin, soybean trypsin inhibitor,

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monomeric serum albumin, alcohol dehydrogenase, catalase, apoferritin, BSA, and GTP were obtained from Sigma (St. Louis, MO, USA). Adenosine deaminase, alkaline phosphatase from calf intestine, and Escherichia coli b-galactosidase were obtained from Boehringer Mannheim. Acrylamide/Bis 40% (w/v) solution was obtained from Ambion Inc. Islet-activating protein (IAP; Bordetella pertussis toxin) was obtained from Research Biochemicals International (RBI). [3H]R-PIA (37 Ci/mmol) was obtained from Amersham. Gb (M-14) rabbit polyclonal IgG was obtained from Santa Cruz Biotechnology, Inc. All other chemicals were of analytical grade. Membrane Preparation Bovine caudal epidydimis was perfused with 10 mM Mops, 120 mM NaCl, 5 mM KCl, 10 mM MgSO4 , 1 mM benzamidine, and 0.5 mM DTT, pH 7.4. Spermatozoa were washed twice by centrifugation at 800g for 10 min at 47C, suspended at a concentration of 150 1 106 cells/ml in the same buffer, and kept at 207C until use. Spermatozoa number and motility were determined and their intactness was judged by eosine vital-dye and by measurements of LDH activity (19). In accordance with Shen et al. (20) and Kops et al. (21), spermatozoa were suspended in 10 mM Mops, 1 mM DTT, 5 mM benzamidine, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 10 mg/ml soybean trypsin inhibitor, pH 7.4. After hypotonic lysis on ice for 1 h, sperm cells were frozen at 0207C and, after thawing, sonicated with six bursts of 30 s (standby interval of 15 s). The preparation, added to a buffer containing 10 mM Mops, 250 mM mannitol, 30 mM sucrose, 1 mg/ ml aprotinin, 1 mg/ml leupeptin, and 10 mg/ml soybean trypsin inhibitor, pH 7.4, was centrifuged at 800g for 10 min at 47C. The supernatant was again centrifuged at 12,000g for 40 min, and the pellet, containing the membrane preparation, was resuspended in 50 mM Tris–HCl, 1 mM DTT, 5 mM benzamidine, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 10 mg/ml soybean trypsin inhibitor, pH 7.4, for radioligand-binding assay. Treatment of Spermatozoa with Pertussis Toxin Pertussis toxin was activated at 327C for 30 min in 50 mM Tris–HCl, pH 7.4, and 25 mM DTT immediately before use. Spermatozoa, suspended in 10 mM Mops, 120 mM NaCl, 5 mM KCl, 10 mM MgSO4 , 0.5 mM DTT, 5

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mM benzamidine, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 10 mg/ml soybean trypsin inhibitor, pH 7.4, were treated with 1 mg/ml PTX for 24 h at 47C (22,23). Membranes were prepared and the receptors solubilized. Receptor Solubilization According to Stiles (17), membranes were solubilized by suspension in 1% digitonin, 20 mM Tris– HCl, 10 mM MgCl2 , 5 mM EDTA, 100 mM NaCl, pH 7.4, for 40 min at 257C. Membrane suspension was first homogenized with Ultraturrax T-25 (Ika Labortechnik) by six strokes on ice and then sonicated with four bursts of 15 s (standby interval of 15 s). The suspension was centrifuged at 105,000g for 40 min at 47C. The supernatant was chromatographed on a Sephadex G-50 column, previously equilibrated with 20 mM Tris–HCl, 10 mM MgCl2 , 1 mM EDTA, 0.05% digitonin, pH 7.4, and eluted with the same buffer. The eluted fractions were assayed for [3H]R-PIA binding and the fractions containing the A1 soluble receptors were collected and used as the soluble preparation. Soluble Binding Radioligand binding to adenosine receptors was assayed with [3H]R-PIA, as reported (24). Soluble A1 receptor binding was performed in an assay volume of 600 ml consisting of 30–40 ml of soluble preparation containing 70 mg of protein in 0.05% digitonin, 20 mM Tris–HCl, pH 7.4, 10 mM MgCl2 , 5 mM EDTA, 100 mM NaCl, 440 ml of diluting buffer (50 mM Tris–HCl, pH 7.4, 1% DMSO), 60 ml of 1–40 nM radioligand [3H]R-PIA, and 60 ml of buffer or 100 mM CPA as competing ligand. The incubation was for 3 h at 257C. The reaction was terminated, and bound and free radioligands were separated by filtration on Whatman GF/B glass fiber filters presoaked in 0.3% polyethylenimine (2– 4 h, pH 10). The filter disks were washed and counted as described (24). Sucrose Density Gradient Centrifugation Sucrose density gradient centrifugation was assessed according to Stiles (17) with a slight modification (25). Western Blot Analysis Solubilized spermatozoa A1 receptors, obtained by sucrose gradient, were added to electrophoresis sam-

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ple buffer to yield a final concentration of 8 mg. Samples, electrophoresed with constant current at 35 mA for 2 h on 12% polyacrylamide gels, were transferred onto Western polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) using a constant current of 250 mA for 1 h. Nonspecific sites were blocked by incubating blots overnight at 47C in 1% BSA, 2% milk-blocking buffer. Blots were rinsed with TSB/T buffer (20 mM Tris–HCl, 137 mM NaCl, 0.05 Tween 20, pH 7.6), incubated for 1 h at room temperature in TBS/T containing Ab (Gb M-14), washed for 30 min in TBS/T, and incubated for 1 h with secondary antibody (goat anti-rabbit immunoglobulin–alkaline phosphatase linked) at a dilution of 1:3000. Blots were rinsed for 30 min with TBS/ T and immune complexes were identified by color development (Bio-Rad Immune-Blot assay kit). Protein molecular weight was determined by lowrange prestained standards (Bio-Rad, Hercules, CA, USA). GTPase Activity Assay Specific low-Km GTPase assay was performed by determining the release of inorganic phosphate. Incubations were carried out at 377C in a medium containing 50 mM Tris–HCl, pH 7.4, 0.5 mM GTP, 1 mM App(NH)p, and other additions, as indicated. The reaction, in a total volume of 200 ml, was terminated by adding phosphate dye reagent, according to Itaya and Ui (26). All the experimental values were corrected by subtracting the respective control, i.e., the amount of inorganic phosphate present in parallel assays performed with inactivated enzyme. The assay was linear with protein and time up to 30 min and conditions were chosen so that less than 20% of the substrate was used. One unit was defined as the amount of enzyme which cleaves 0.2 nmol of substrate in 1 min at 377C. Data Analysis of Binding Assays Saturation binding data were transformed into Scatchard plots and were analyzed using a nonlinear least-squares curve-fitting to one- and two-site models by the computer program Ligand (27). The model adopted was that in which the F test gave the highest probability. Displacement data were analyzed according to a four-parameter logistic equation to determine IC50 values with a statistical package allowing determination of statistical differences between curves. IC50 values were converted to Ki values according to the Cheng – Prusoff equation (28).

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M-14, showing the existence of G protein tightly bound to the receptor molecule. Effects of Divalent Cations on [3H]R-PIA Binding to Solubilized A1 Receptor

FIG. 1. Western blot of solubilized A1 adenosine receptor. Lane 1, low molecular weight standards; lane 2, purified bg subunits (gift from Dr. A. De Blasi, Ist. Ricerche Farmacologiche ‘‘M. Negri’’ Sud, Chieti, Italia); lane 3, uncoupled solubilized A1 receptor; lane 4, coupled solubilized A1 receptor.

The inhibition curves were analyzed assuming the Kd values for [3H]R-PIA obtained in the fitted saturation kinetic plots. Data were presented as means { SEM, with n representing the number of independent experiments. Significant differences were assessed by Student’s t test (paired, two-tailed).

[3H]R-PIA is a high-affinity agonist radioligand which binds to the solubilized A1AR from bovine spermatozoa membranes with a Kd of 5.3 { 1.2 nM (n Å 55) and an apparent Bmax value of 460 { 33 fmol/mg protein (24). The same values of Kd and Bmax were obtained in the presence of 1 and 5 mM EDTA, suggesting that the possible background of divalent cations does not affect the binding results (data not shown). The concentration–response curves for the effects of CaCl2 , MgCl2 , and MnCl2 on [3H]R-PIA binding to A1 solubilized receptor are reported in Fig. 2. Each of the three ions potentiated the specific binding, causing as much as a 34% increase in the level of binding compared with the amount of binding in the absence of divalent cations. This finding agrees with data reported for A1 binding under similar experimental conditions (3,30,31). Mn2/ had the highest apparent affinity with halfmaximal effect at 50 mM. Mg2/ and Ca2/ half-maximally enhanced binding at 0.5 and 2.5 mM, respectively. In contrast to the effects of divalent cations on agonist binding, magnesium had no significant effect on the binding of A1AR antagonist CPT (data not shown).

Protein Determination Protein was determined by Bio-Rad protein method using bovine serum albumin as standard (29). RESULTS Western Blot of A1 Solubilized Receptor Western blotting analysis of the A1 solubilized adenosine receptor is shown in Fig. 1. Spermatozoa, treated with 1 mg/ml PTX for 24 h at 47C, were used to obtain digitonin-solubilized A1 receptors. This preparation was applied to a sucrose density gradient. The elution resulted in two peaks of binding activity corresponding to A1 receptor–G protein complex and A1 uncoupled receptor, respectively (24). It can be seen that the peak corresponding to the suggested complex (24) specifically reacts with Gb

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FIG. 2. Dose–effect curves of divalent cations on [3H]R-PIA specific binding to solubilized A1 receptors. The data represent the means of at least four experiments in triplicate. SEM was less than 10% and not shown. h, Ca2/; m, Mg2/; l, Mn2/.

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the agonist. Ca2/ (5 mM) affected the apparent affinity and the total number of sites available, whereas 1 mM Mg2/ altered only the apparent affinity. Therefore any irreversible component of the interaction between ion–agonist and binding sites cannot be considered. Effects of Divalent Cations on Low Km Specific GTPase

FIG. 3. CPA competition curves in solubilized A1 adenosine receptor. Competition of CPA for [3H]R-PIA in presence of increasing concentrations of Cu2/ (n), Zn2/ (s), Cd2/ (l), and Co2/ (j).

The concentration – response curves of the effect of Co2/, Cd2/, Cu2/, and Zn2/ on [3H]R-PIA binding to A1 solubilized receptor showed an inhibitory effect exerted by these cations on the binding capacity (Fig. 3). Computer analyses of the competition curves resulted in the following IC50 values: 0.52 { 0.13 mM for Co2/, 4.6 { 0.85 mM for Cd2/, 3.1 { 0.4 mM for Zn2/, and 84 { 7.6 nM for Cu2/. These results show that Cu2/ is the most powerful inhibitor among the cations tested. Effects of Ca2/, Mg2/, and Mn2/ on [3H]R-PIA Saturation Curves Kinetic analyses of the binding of [3H]R-PIA are reported in Table 1. Results of binding assays performed under standard conditions in the presence of 5 mM CaCl2 , 1 mM MgCl2 , and 100 mM MnCl2 were compared to control values obtained in the absence of these cations. In the absence of ions, [3H]R-PIA saturation data were fitted to a high-affinity site model with Kd Å 5.3 { 1.2 nM and Bmax Å 460 { 33 fmol/mg protein. These data were considered as control values. The presence of 100 mM Mn2/ caused an increase in the apparent affinity of the binding sites but not in the total number of sites available. Hence any competitive nature of the interaction between ion- and agonist-binding sites must be ruled out as the competition would have lowered the apparent affinity for

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It is known that the rate of GTP hydrolysis is slow, with the majority of G protein having turnover numbers (Kcat ) in the range of 2–5 min01 (32,36). However, the high concentration of G proteins in plasma membranes allows the measurement of GTPase activity under the conditions described. In addition, it is possible to measure receptor stimulation of the rate of GTP hydrolysis since the receptors increase the rate of GTP hydrolysis (33). The interaction and activation of G proteins by unoccupied receptors account for the basal GTPase activity which, for the solubilized A1 receptor, is characterized by Km and Vmax values of 4.0 { 0.6 1 1005 M and 5.5 { 0.9 U/mg of protein, respectively (Table 2). Kcat ranges between 45 and 53 min01, values similar to those of retinal (37) and atrial K/ channel (11) G proteins. The kinetic values were calculated in the absence of Na/, which is thought to bind to a regulatory site on the receptor and to attenuate the interaction of the unoccupied receptor with G protein (34). However, the addition of 100 mM NaCl, as suggested (35), did not cause any change in the kinetic parameters and the presence of nanomolar concentrations of Mg2/ altered significantly only Vmax values. With the addition of specific A1 agonists R-PIA and CPA, the rate of GTP hydrolysis increased,

TABLE 1 Effects of Divalent Cations on Binding Parameters of [3H]R-PIA to Solubilized A1 Adenosine Receptor from Bovine Spermatozoa Kd (nM)

Ions None Ca2/ (5 mM) Mg2/ (1 mM) Mn2/ (100 mM)

5.3 5.03 3.8 2.2

{ { { {

1.2 1.3* 0.94* 0.3*

Bmax (fmol/mg protein) 460 533 468 524

{ { { {

33 41* 37 40

Note. Binding assay, performed as described under Materials and Methods, was run in triplicate. Values are the means { SEM (n Å 7). * P õ 0.05.

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TABLE 2 Effect of Divalent Cations on Low Km GTPase Kinetic Parameters Compound None EDTA MgCl2 NaCl CPA R-PIA CPT Mg2/ Ca2/ Mn2/ CPA / CPA / CPA / CPA / Co2/ Cd2/ Cu2/ Zn2/

Mg2/ Ca2/ Mn2/ Na/

Concentration

1 mM 10 nM 100 mM 1 mM 1 mM 1 mM 1 mM 5 mM 100 mM 1 mM, 1 mM 1 mM, 5 mM 1 mM, 100 mM 1 mM, 100 mM 500 mM 1 mM 5 mM 1 mM 100 mM 1 mM 5 mM 1 mM

Km (1005 M) 4.0 4.7 6.2 4.4 5.8 5.3 1.7 2.7 3.8 3.0 1.9 4.1 2.0 1.2 5.5 35 6.5 2.0 3.8 31 3.4 1.8

{ { { { { { { { { { { { { { { { { { { { { {

0.6 1 1.5 0.9 0.8* 0.9* 0.8* 0.4* 0.9 0.5* 0.7* 0.5 0.3* 0.3* 0.6* 0.9* 0.9* 0.3* 0.7 1* 0.9* 0.2*

Vmax (U/mg protein) 5.5 5.8 7.8 6.8 8.9 8.1 4.3 7.5 7.2 7.8 3.6 5.9 3.8 3.4 5.8 3.3 5.5 3.6 5.3 2.9 4.7 2.2

{ { { { { { { { { { { { { { { { { { { { { {

0.9 0.5 1.4* 1.5 0.9* 0.7* 0.5* 0.7* 0.8* 0.6* 0.8* 0.9 0.5* 0.6* 0.7 0.5* 0.7 0.6* 0.4 0.5* 0.8 0.5*

Note. The assay, performed as described under Materials and Methods, was run in triplicate. Values are the means { SEM (n Å 6). * P õ 0.05.

whereas the presence of A1 antagonist CPT lowered GTPase activity. The addition of Mg2/ at 1 mM, i.e., the concentration used in the binding assay, resulted in an activation of GTPase activity and a concomitant increase of affinity. On the other hand, Ca2/ and Mn2/, at concentrations where the effect on the binding is maximal, resulted in the increase of Vmax values and only Mn2/ significantly changed the affinity for GTP. When these cations were tested in the presence of A1 specific agonist, all but Ca2/ caused a decrease in Vmax values accompanied by a substantial increase of affinity for the substrate. This effect is difficult to explain since agonists and cations, tested separately, resulted in a Vmax increase. Other cations such as Co2/, Cd2/, Cu2/, and Zn2/, when used at their respective IC50 concentrations, caused almost no change in GTP hydrolysis rates and only Co2/ and Cd2/ decreased GTPase affinity for GTP. When the same cations were used at 1 mM concentrations, they all significantly lowered the maximal velocity of GTP hydrolysis. Co2/ and Cu2/

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also caused a 10-fold increase of Km values, indicating an interaction with the enzyme molecule. These cations, Co2/ and Cu2/, are those with lesser inhibitory potency. DISCUSSION Studies on agonist-binding properties of A1AR, solubilized from bovine spermatozoa, showed, by competition curve analysis, that CPA recognizes two affinity states of the solubilized receptor (24). Guanine nucleotides shift the equilibrium of the agonist low- and high-affinity states to an almost uniform low-affinity state, whereas NaCl causes only a decrease of the percentage of receptor in the high-affinity state. Thus NaCl appears to facilitate a change in the equilibrium, while Gpp(NH)p appears to decrease the affinity of both states. These results indicated the possible existence of a very tight complex between the solubilized A1 receptor and its G proteins. Moreover, the fact that two different agonistbinding affinities still existed in the presence of guanine nucleotides suggested that the coupling of bovine spermatozoa A1 receptor to Gi is different from the coupling of the inhibitory a2-adrenergic receptor to Gai and from the coupling of the stimulatory badrenergic receptor to Ga (24). In the present study we produce direct evidence of G proteins being cosolubilized with A1 receptor. Therefore this preparation was used as an appropriate model system to investigate whether the modulation of agonist-binding properties by divalent cations depends on receptor conformational state variations or on coupling with the second-messenger system. Different profiles of Scatchard curves of [3H]R-PIA binding to A1 solubilized receptor are produced by Ca2/, Mg2/, and Mn2/ ions, suggesting that they may act by different processes in regulating agonist binding. A1AR agonists, as well as a-adrenergic and muscarinic agonists, inhibit adenylate cyclase by stimulating GTPase activity (10). Moreover, stimulation of low Km GTPase is mechanistically related to inhibition of adenylate cyclase. Opiates with high intrinsic activity stimulate high-affinity GTPase and the extent of GTP hydrolysis in the presence of sodium is reciprocally related to the extent of inhibition of cAMP synthesis. A1AR high-affinity GTPase is not affected by 100 mM NaCl in either the absence or the presence of the specific agonist. This finding, unlike those for opiate (5,36), a-adrenergic (6), and histamine H1 (4)

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receptors, shows that the reported small conversion of the receptor to a low-affinity state (24) is probably due to the interaction of the ion with the receptor molecule. This effect is not selective since lithium and potassium are as effective as sodium (data not shown). EDTA was shown to inhibit the agonist binding to opiate (5) and brain A1 adenosine (3) receptors, suggesting that endogenous divalent cations may regulate interactions with the receptor molecules. In our case, EDTA does not affect agonist binding. This finding was not totally unexpected since the receptor is a solubilized preparation lacking in endogenous cations. a-GTPase activity is not modified by EDTA treatment. The relative potencies of divalent cations were shown to vary at different receptors (3,7). With A1 solubilized AR, Mn2/ is the most powerful, Mg2/ has a moderate activity, and Ca2/ is virtually inactive, as reported for opiate (7) and b-adrenergic (8) receptors. This finding contrasts with results obtained with brain A1AR (3), where Mn2/ was reported as the most potent ion, while Mg2/ and Ca2/ were shown to have similar potencies. If the divalent cation effects reflect influences of endogenous cations, it is conceivable that these ions may be regulators of A1AR. The fact that guanine nucleotides and divalent cations (i.e., Mg2/ and Mn2/) affect the affinity of agonists for A1AR (24) suggests the same action for cations and guanine nucleotides. In addition, these cations have a significant effect on the GTP hydrolysis rate and on the affinity of the low Km GTPase. In the presence of agonists, which greatly affect the enzyme Vmax and Km , these cations significantly reduce GTP hydrolysis and increase its affinity for the enzyme. The limited effect of Mg2/ on agonist binding to A1 solubilized receptor might be due to the lack of adenylate cyclase in the model system used. The profound effect of Mg2/ on the binding of agonists to badrenergic and PGE1 receptors (8) was shown to be closely related to the activation of adenylate cyclase. The receptor affinity for compounds which bind to the receptor but do not activate the enzyme (antagonists) is not at all altered by Mg2/. Therefore, because the metal ion binding site is probably on the adenylate cyclase molecule, the limited influence of this ion on the inhibitory A1 agonist binding seems reasonable. On the other hand, Mg2/ and agonist affect GTPase activity by decreasing GTP hydrolysis which results in a slight increase of agonist binding affinity. Thus, it is difficult to explain why these

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ions, which similarly affect GTPase activity either in the presence of A1 specific agonists or in their absence, have such different effects on agonist binding properties. Moreover, the other cations we tested, i.e., Co2/, Cu2/, Zn2/, and Cd2/, all inhibitory for the agonist binding, do not alter GTP hydrolysis. Therefore, the only effect of these divalent cations on GTPase activity which appears to be related to the effects on agonist binding seems to be the variation of Vmax values even though stimulatory cations, when tested in the presence of A1 specific agonist, cause a significant decrease of Vmax value with an increase of GTP affinity. Indeed, aGTPase activity and its modulation do not seem to be the only elements involved in the A1 adenosine receptor response in which coupling with second-messenger systems other than cAMP may play a paramount role. ACKNOWLEDGMENT The authors are grateful to Dr. Mary Kerrigan, MA (Cantab), for linguistic suggestions.

REFERENCES 1. Fong TM. Mechanistic hypotheses for the activation of Gprotein-coupled receptors. Cell Signal 8:217–224, 1996. 2. Remmers AE, Neubig RR. Partial G protein activation by fluorescent guanine nucleotide analogs. J Biol Chem 271:4791–4797, 1996. 3. Goodman RR, Cooper MJ, Gavish M, Snyder SH. Guanine nucleotide and cation regulation of the binding of 3H-cyclohexyl-adenosine and 3H-diethyl-phenyl-xanthine to adenosine A1 receptors in brain membranes. Mol Pharmacol 21:329–335, 1982. 4. Chang RSL, Snyder SH. Histamine H1-receptor binding site in guinea pig brain membranes: Regulation of agonist interactions by guanine nucleotides and cations. J Neurochem 34:916–922, 1980. 5. Pert CB, Snyder SH. Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol Pharmacol 10:868–879, 1974. 6. Greenberg DA, U’Prichard DC, Sheehan P, Snyder SH. Alpha-noradrenergic receptors in the brain: Differential effects of sodium on binding of 3H-agonists and 3H-antagonists. Brain Res 140:378–384, 1978. 7. Pasternak GW, Snowman AM, Snyder SH. Selective enhancement of 3H-opiate agonist binding by divalent cations. Mol Pharmacol 11:735–744, 1975. 8. Williams LT, Mullikin D, and Lefkowitz RJ. Magnesium dependence of agonist binding to adenylate cyclase-coupled hormone receptors. J Biol Chem 253:2984–2989, 1978. 9. Usdin TB, Creese I, Snyder SH. Regulation by cations of 3H-spiroperidol binding associated with dopamine receptors of rat brain. J Neurochem 34:669–676, 1980.

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