Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin.

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.

Exposure of the a-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the a-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad Sei U. S. A. 78,4026-4030). The soluble a-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the a-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32P]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify a-receptor-mediated inhibition of adenylate cyclase or interaction of the a-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated M, = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-areceptor complex. These data suggest that the GTPbinding protein that mediates GTP activation of adenylate cyclase i n the human platelet membrane is distinct from the GTP-binding protein that modulates aadrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.
Hormonal systems coupled to inhibition of adenylate cyclase activity bear certain phenomenological similarities to systems coupled to activation of the enzyme. Thus, a requirement for GTP is demonstrated for both inhibition and activation of adenylate cyclase in broken cell preparations (1). In addition, GTP decreases affinity for agonists, but not antagonists, at receptors coupled to either activation or inhibition of catalytic activity (1). The similar function of guanine nucleotides in both stimulatory and inhibitory adenylate cyclase * This research was supported by National Institutes of Health Grant HL 25182 and a grant-in-aid from the Tennessee Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of an American Association of University Women Marie Curie Fellowship Award.
5 Recipient of National Institutes of Health Research Career De-systems suggests that a common pool of GTP-binding proteins could be shared by hormonal systems coupled to activation and inhibition of the enzyme.
Differential sensitivity to proteases (2, 3), Mn2+ (4, 5), sulfhydryl-directed reagents (4), and radiation inactivation (6), as well as the 10-fold greater GTP concentrations typically required to elicit half-maximal regulatory effects in inhibitory systems than in simulatory systems (l), has suggested that distinct GTP-binding proteins may be involved in activation and attenuation of adenylate cyclase. Alternatively, these data might simply indicate that discrete domains of a single GTPbinding protein responsible for communicating opposing signals to the catalytic moiety are differentially sensitive to the above perturbants.
In an attempt to resolve whether or not a single population of GTP-binding proteins participates in both stimulatory and inhibitory adenylate cyclase systems, we investigated the aadrenergic system of human platelets. This receptor system is coupled to inhibition of basal and PGEl'-stimulated adenylate cyclase and induction of platelet aggregation and serotonin release (10). The GTP-binding protein coupled to activation of adenylate cyclase was labeled with ["P]ADP-ribose using cholera toxin (7)(8)(9). Incorporation of [""PIADP-ribose into a M , = 42,000 peptide was paralleled by enhancement of GTPsensitive catalytic activity in the platelet membranes.
We assessed whether this "'P-labeled GTP-binding protein might associate with the human platelet a-adrenergic receptor in a manner analogous to its association with the agonist-occupied &adrenergic receptor coupled to activation of adenylate cyclase in other target membranes (11,12).
The data demonstrate that cholera toxin treatment of human platelet membranes does not modify a-receptor-mediated inhibition of adenylate cyclase. Furthermore, the ["PIADPribosylated M, = 42,000 subunit of the stimulatory GTPbinding protein does not appear to associate with the agonista-receptor complex which is known to contain the GTP-binding protein that modulates receptor affinity for agonists. Thus, these studies provide further evidence that the GTP-binding moieties involved in hormonal inhibition of adenylate cyclase are distinct from those mediating hormonal stimulation of the enzyme.

Correlation of Cholera Toxin-catalyzed [R'P]ADP Ribosylation and Changes in Human Platelet Adenylate Cyclase
Activity-In the studies shown in Fig. 1, the effects of cholera toxin on human platelet membranes were varied by changing the concentration of the toxin co-substrate, [:'"PINAD'. The autoradiograms of SDS-polyacrylamide gels in Fig. 1A demonstrate the peptides labeled in the absence ( 4 1 3 or presence (+CT) of cholera toxin. Fig. 1B demonstrates that, in parallel incubations of the same membrane preparation, incubation with cholera toxin in the presence of 0-20 p~ [:'"PINAD+ resulted in a concentration-dependent increase in GTP-sensitive adenylate cyclase activity. This increment in adenylate cyclase activity is paralleled by increased [""PIADP ribosylation of a M, = 42,000 peptide, presumably the M, = 42,000 subunit of the GTP-binding protein responsible for activation of adenylate cyclase (7-9, 13). In separate experiments where the concentration of NAD' (not radiolabeled) was increased as high as 500 p~, the enhancement of GTP-sensitive activity is observed to plateau at approximately 100% NaF-stimulated activity and has never been observed to increase beyond 140% of NaF-stimulated catalytic activity (data not shown).
Although cholera toxin enhanced the activation of adenylate cyclase by GTP, it did not alter the extent of inhibition of PGEl-stimulated adenylate cyclase by epinephrine. Phentolamine prevented epinephrine inhibition of PGEI-stimulated adenylate cyclase activity, indicating that this attenuation by (-)-epinephrine was mediated through a-adrenergic receptors.
Effects of Cholera Toxin Incubation on a-Adrenergic Receptor-Agonist Interactions and Their Modulation by Guanine Nucleotides-To assess whether or not ADP ribosylation of the M, = 42,000 protein in human platelet membranes alters the ability of exogenous guanine nucleotides to modulate a-receptor-agonist interactions, competition binding studies were undertaken. Competition of (-)-epinephrine with the radiolabeled antagonist [:'H]yohimbine was virtually the same in control or toxin-treated human platelet membranes (Fig.  2). Thus, the ECrfl for (-)-epinephrine in the absence of added guanine nucleotides was 0.20 2 0.07 p~ (n = 8) in control membranes and 0.28 f 0.07 p~ (n = 8) in membranes treated with cholera toxin. In the presence of 0.1 mM GTP, the ECm for (-)-epinephrine was increased to 2.06 & 0.46 p~ (n = 4) in control membranes and to 1.63 f 0.14 p~ (n = 4) in toxintreated membranes. In addition, the inclusion of GTP in the incubation converted the "shallow" competition curve observed in the presence of (-)-epinephrine alone to a curve of more nearly "normal steepness,":' as has been reported previously (15, 16).
Effects ofAgonist Occupancy of the a-Adrenergic Receptor on the Sedimentation Properties of Solubilized [:' 2P]ADPribosylated Proteins-The data in Figs. 1 and 2 were consistent with the hypothesis that distinct GTP-binding proteins were involved in activation and attenuation of adenylate check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
.' The term "normal steepness" refers to the shape of a competition curve that proceeds from 90-10% competition over an 81-fold concentration range of competitor. This is the behavior expected of a competitor that interacts with the receptor through a reversible, bimolecular reaction obeying simple mass-action law and that competes for a radioligand that also meets the same restrictions (14). "Shallow" curves extend over a greater than SI-fold concentration range of competitor and are consistent with negatively cooperative interactions or receptor heterogeneity. cyclase. However, it was also possible that the same GTPbinding protein, or protein complex, communicated both activating and attenuating signals to adenylate cyclase but that covalent modification of this component did not alter the expression of a GTP-binding site responsible for communicating inhibitory signals to the catalytic moiety. Thus, we utilized sucrose gradient centrifugation to assess whether cholera toxin-catalyzed ["*P]ADP-ribosylated proteins might be physically associated with the agonist-occupied a-receptor in a manner analogous to the association of these ADP-ribosylated proteins with agonist-occupied P-adrenergic receptors coupled to activation of adenylate cyclase (11, 12).
Preliminary studies indicated that the ratio of ["PIADPribosylated M, = 42,000 proteins (femtomoles/mg of membrane protein) to agonist-occupied a-receptors (ferntomoles/ mg of membrane protein) in platelet membranes was 27-331. Because this stoichiometric excess could obscure detection of a-receptor-associated ADP-ribosylated proteins, we attempted to resolve GTP-binding proteins associated with the agonist-occupied a-receptors from the large excess of GTPbinding proteins in human platelet preparations using WGA-Sepharose, which should selectively interact with carbohy- Human platelet membranes were incubated with ["Hlepinephrine or ['Hlyohimbine and solubilized as described under "Experimental Procedures" (see Miniprint). The amount of liganded receptor or ["HI Gpp(NH)p-binding (cf. "Experimental Procedures") was determined prior to lectin exposure (starting material) in the supernatant subsequent to 2-h exposure to WGA-Sepharose at 6-10 "C (not absorbed), in the multiple washes with digitonin-containing buffer, and following exposure of the WGA-Sepharose resin to 0.25 M N-acetyl-D-glucosamine using Sephadex G-50 chromatography as described under "Experimental Procedures" (see Miniprint). The amount of binding drate-containing cell surface components. Fig. 3A demonstrates that 71% of digitonin-solubilized a-adrenergic receptors were adsorbed to the WGA-Sepharose whereas only 12% of [3H]Gpp(NH)p binding proteins were adsorbed. Extensive washing of the resin to remove trapped and loosely associated material did not desorb the a-adrenergic receptors, whereas greater than 80% of the guanine nucleotide binding proteins initially adsorbed were removed upon extensive washing of the resin with detergent plus excess Tris-HC1 (Fig. 3B). Adsorption and elution profiles for ["2P]ADP-ribosylated proteins and for ["H]Gpp(NH)p-binding sites were similar (data not shown). As shown in Fig. 3C Human platelet a-receptors desorbed from WGA-Sepharose by incubation with 0.25 M N-acetyl-D-glucosamine retained the properties of the receptor uniquely associated with agonist occupancy. Thus, ['Hlepinephrine-a-adrenergic receptor complexes retained their sensitivity to guanine nucleotides, as assessed by the ability of Gpp(NH)p to facilitate dissociation of the radiolabeled agonist from the receptor (Fig. 4). 4 The It should be noted that, in digitonin-solubilized preparations (Fig.   4B) and eluates of WGA-Sepharose containing digitonin and N-acetyl-D-glucosamine (Fig. 4C) . The "solubilized preparation" was exposed to WGA-Sepharose for 2 h at 10 "C, the resin was washed extensively, and the adsorbed a-receptors were desorbed with 0.25 M N-acetyl-D-ghcosamine. Dissociation of the ["Hlepinephrine a-receptor complex was monitored at 15 "C in the absence and presence of 0.1 mM Gpp(NH)p and 10 PM phentolamine by transferring 0.5-ml aliquots at the times indicated to columns (0.6 X 14 cm) of Sephadex G-50 as described under "Experimental Procedures" (see Miniprint). The binding of [JHIepinephrine was 1260 cpm/ml in the desorbed preparation. The data are from one experiment representative of three separate experiments. In all experiments (A, B, C ) , the effect of guanine nucleotides was not mimicked by 0.1 mM adenyl-5'yl imidodiphosphate.
was considerably slower than observed for intact membrane preparations (Fig, 44). This effect is presumably due to constraints imposed by digitonin in the solubilized preparations. Thus, in studies not shown here, the K,, for [.'H]yohimbine is virtually identical in membrane and digitonin-solubilized preparations; however, the rates of radioligand association and dissociation are both slowed in a manner comparable to the results for [,"H]epinephrine dissociation in digitonin-solubilized preparations (17). " faster sedimentation in sucrose gradients of the agonist-occupied receptor than unoccupied or antagonist-occupied receptors (Fig. 5 ) was also retained in the eluted preparations. Consequently, the material desorbed from WGA-Sepharose appeared to provide the appropriate starting material for investigation of the molecular components associated with the agonist-occupied a-receptor.
To determine whether or not the faster sedimenting, guanine nucleotide-sensitive agonist-a-receptor complex contained ["'PIADP-ribosylated cholera toxin substrates, we compared the sedimentation profie of ["'PIADP-ribosylated proteins solubilized from membranes incubated with either agonist or antagonist and desorbed from WGA-Sepharose 6MB. As shown in Fig. 6, a similar sedimentation profiie was observed for ['2P]ADP-ribosylated proteins derived from membranes exposed to either a-adrenergic agonists (Fig. 6B) or antagonists (Fig. 6A). However, the possibility existed that a small fraction of the ["'PIADP-ribosylated material might be physically associated with the agonist-occupied receptor but obscured in the gradient profiie in Fig. 6. T o make more rigorous quantitative comparisons between agonist-and antagonist-liganded preparations, gradient fractions corresponding to a portion of the peak region of the agonist-a-receptor complex were pooled from three separate gradients (Fig. 7, left) and resedimented on a second gradient (Fig. 7 , right). The peak height and sedimentation position of [:'2P]ADPribosylated proteins were virtually identical regardless of whether or not the fractions for resedimentation were obtained from gradients containing agonist-or antagonist-a-receptor complexes. This is in distinct contrast to what would have been anticipated if agonist occupancy of the a-receptor resulted in a unique association of the a-receptor with the ["'PIADP-ribosylated GTP-binding protein, thus accounting for the guanine nucleotide sensitivity and larger molecular size of the agonist-receptor complex when compared with the antagonist-receptor complex. Since cholera toxin had increased GTP-sensitive adenylate cyclase activity to 100% of NaF-stimulated activity in the starting material for the experiment shown in Fig. 7, it is not unreasonable to assume that a major fraction of the M, = 42,000 subunits coupled to activation of adenylate cyclase had incorporated ["'PIADPribose.5 Hence, if the agonist-a-receptor complex had included the M, = 42,000 [3'P]ADP-ribosylated subunit in a 1:l molar ratio with the receptor, then the height of the ["ZPIADPribose-containing peak obtained by resedimentation of agonist-receptor complexes should have been approximately 12 fmol greater than that obtained by resedimenting the corresponding fractions from antagonist-receptor-containing gradients. The absence of any quantitative change in the ['"PI ADP-ribose-containing peaks in agonist-receptor versus antagonist-receptor gradients suggests that the faster sediment-' Comparison of the ratio of GTPand NaF-stimulated adenylate cyclase activities has been used to roughly estimate the degree of modification of the M , = 42,000 GTP-binding protein in pigeon (7,8) and rat reticulocyte membranes (12). In the present studies, we observed that cholera toxin-modified GTP-sensitive and NaF-stimulated activities converged as NAD' concentrations were increased (Fig. 1) and that increasing NAD+ to as high as 500 pM only slightly enhanced activity further (to 5140%> of NaF-stimulated activity). Thus, for human platelet preparations, the fraction of NaF-stimulated activity attained by GTP following cholera toxin treatment is a Preparations were then either exchanged into 0.1% digitonincontaining buffers, concentrated, and directly applied to 7.5-208 sucrose gradients (left) or exposed to WGA-Sepharose for 2 h at 10 "C, washed 10 times with 10 ml of digitonin-containing buffer, and  I  I  I  I  I  I  I  I  I  I  I  I  Centriflo cones and applied to 7.5-20% sucrose gradients as described under "Experimental Procedures" (see Miniprint). GTP-sensitive adenylate cyclase activity was stimulated to 70% of 10 mM NaF activity by exposure to cholera toxin. ["PINAD+ was present at 46 Ci/mmol, which is equivalent to 91 cpm/fmol, after accounting for scintillation counter efficiency. Fractions 10-20 of the gradients shown and of two additional gradients each for agonist-receptor and antagonist-receptor complexes were poolcd, concentrated as described under "Experimental Procedures" (see Miniprint) and applied to a second sucrose gradient for recentrifugtltiorl. The data shown at the right correspond to the resetlimentation profiles. The data shown are from one experiment characteristic of data obtained in four similar experiments.  Fig. 7 were run on IO%, polyacrylamide gels containing 0.1% SDS. Identical volumes of agonist-and antagonist-liganded preparations were applied to the SDS-polyacrylamide gels. Gels were run and autoradiograms developed as described previously (12) sylated M, = 42,000 subunits was obtained from preparations nucleotide-sensitive agonist-a-receptor complex does n o t conderived from agonist-liganded human platelet membranes as tain the [:'"P]ADP-ribosylated M , = 42,000 subunit of the from antagonist-liganded membranes. T h u s , the findings in GTP-binding protein coupled to activation of adenylate cy-Figs. 7 and 8 provide substantial evidence that the guanine clase.

DISCUSSION
The present studies indicate that the molecular component(s) mediating the regulatory effects of guanine nucleotides in hormonal systems coupled to activation and inhibition of adenylate cyclase appear to be distinct. Thus, incubation of human platelet membranes with cholera toxin and increasing concentrations of [3'P]NAD+ resulted in ihcreased incorporation of ["'PIADP-ribose into a M , = 42,000 membrane substrate and yielded dose-dependent increases in GTP-sensitive adenylate cyclase activity. However, over the same concentrations of [""PINAD+, cholera toxin had no effect on the ability of (-)-epinephrine to attenuate basal (data not shown) or PGEl-stimulated catalytic activity (Fig. 1). Similarly, cholera toxin treatment did not modify (-)-epinephrine interactions with the a-receptor, or the ability of GTP to decrease receptor affinity for the agonist. In contrast, cholera toxin treatment has been demonstrated to alter receptoragonist interactions a t &adrenergic receptors coupled to activation of adenylate cyclase (18) and, in other studies, to enhance the sensitivity of the glucagon receptor to GTP (19). In a manner completely analogous with ,&adrenergic receptors coupled to activation of adenylate cyclase (11,12), agonist occupancy of human platelet a-adrenergic receptors promoted an increase in receptor size, manifested by a faster rate of sedimentation in sucrose gradients (Ref. 15;. Earlier observations for the P-adrenergic receptor demonstrated that the agonist-receptor complex of larger molecular size was uniquely associated with the cholera toxin-catalyzed ["'P] ADP-ribosylated protein responsible for cyclase stimulation (12). In contrast, the present studies demonstrate that the quantity of ["'PIADP-ribosylated proteins is not enriched in sucrose gradient fractions containing the ["]epinephrine agonist-a-receptor complex when compared to gradients containing antagonist-occupied receptors. T o enhance our ability to analyze the composition of the agonist-promoted a-adrenergic-receptor complex, we developed a method using WGA-Sepharose which successfully resolved cell surface proteins, including the a-adrenergic receptor, from the vast majority of GTP-binding proteins in human platelet preparations. Autoradiographic data demonstrated that the quantity of M,. = 42,000 ["'PIADP-ribosylated proteins isolated by WGA-Sepharose from preparations solubilized from agonist-liganded membranes was virtually identical with that isolated from preparations solubilized from human platelet membranes liganded with the a-adrenergic antagonist ['Hjyohimbine (Fig. 8). This observation is in distinct contrast to observations for /3-adrenergic receptors coupled to activation of adenylate cyclase (20), wherein adsorption of agonist-occupied /3-adrenergic receptors to WGA-Sepharose resulted in retention and subsequent desorption of a greater quantity of ["2P]ADPribosylated M, = 42,000 proteins than observed for preparations in which receptors were unoccupied or filled with antagonist at the time of solubilization (20). Thus, the present data provide strong evidence that the faster sedimenting agonista-receptor complex does not contain the M , = 42,000 GTPbinding protein recognized by cholera toxin for ["'PIADP ribosylation. However, the faster sedimenting agonist-a-receptor complex presumably does contain a GTP-binding site, since guanine nucleotides facilitate the rate of [3H]epinephrine dissociation from this complex (Fig. 4). Thus, it is likely that the GTP-binding protein that modulates a-receptor affinity for agonist agents is distinct from that which mediates GTP activation of adenylate cyclase in human platelet membranes.
The observation that cholera toxin modification of GTPstimulated adenylate cyclase activity is not correlated with alterations in the ability of epinephrine to attenuate %E,stimulated adenylate cyclase activity (Fig. 1) is consistent with earlier reports that cholera toxin does not modify inhibition of GTP-stimulated adenylate cyclase by muscarinic agents in rabbit cardiac membranes (21) or by a-adrenergic agents in human platelet membranes (22). Similarly, an absence of cholera toxin effects on attenuating systems has been reported for adenosine-induced inhibition of lipolysis and CAMP accumulation in hamster adipocytes (23) and for opiate and a-adrenergic-mediated inhibition of basal and PGE,-stimulated adenylate cyclase in neuroblastoma-glioma hybrid cells (24). However, interpretation of these earlier studies was limited by the omission of data describing the dose-related effects of cholera toxin in the target membranes. Hence, the extent to which the total fraction of adenylate cyclase-coupled GTP-binding proteins was covalently modified under their experimental conditions could not be estimated. In contrast, in the present studies, the effects of cholera toxin on both receptor and catalytic functions of the human platelet aadrenergic system were always evaluated under conditions in which cholera toxin had enhanced GTP-sensitive adenylate cyclase activity to levels approximately equal to or greater than NaF-stimulated activity.
Future studies will hopefully elucidate the biochemical nature of the GTP-binding protein that modulates affinity for agonists at receptors coupled to inhibition of adenylate cyclase as well as determine the relationship, if any, between this protein and the GTP-binding protein that conveys that inhibitory signals to the catalytic moiety of the human platelet adenylate cyclase system.