SOLUBILIZATION OF EPINEPHRINE-SPECIFIC a2-ADRENERGIC RECEPTORS FROM ADRENOCORTICAL CARCINOMA
Ponnal NAMBI, Nambi V. AIYAR and Rameshwar K. SHARMA Department of Biochemistry, Laboratory of Basic Hormone Research, University of Tennessee, Center for the Health Sciences, Memphis, TN 38163, USA
Received 18 December 1981
1. Introduction
Normal isolated adrenal cells [1,2] are markedly and specifically stimulated by a singular polypeptide hormone, ACTH, in the production of corticosterone, indicating that these cells contain only one type of plasma membrane receptors that are coupled to the steroid metabolism. Adrenocortical carcinoma 494 [3] cells [4], in contrast, possess two additional epinephrine-binding receptors, @-adrenergic [5] and ß-adrenergic [6]. While the biological function of these ectopic catecholamine-sensitive receptors is unknown, there is evidence that ß-adrenergic recep- tors are coupled to adenylate cyclase [7] and @-adren- ergic receptors to guanylate cyclase [8]. Thus the model system of neoplastic adrenal cells not only provides the opportunity to assess the relationship of these epinephrine-sensitive receptors in the endocrine control of neoplasia, it also enables the studies designed to elucidate the molecular mechanism of their hormonal regulation and transduction of the biological signal. Such studies could be greatly facili- tated if these individual receptors could be isolated and purified. CHAPS, a zwitterionic detergent, has been originally used for the solubilization of opiate receptors [9]. Here, this detergent was successfully utilized in the selective solubilization of a-receptors. The results indicate that these receptors are of exclu- sively @2-subtype. While the only biological function of azreceptors has been ascribed to the inhibition of adenylate cyclase [10,11], these results together with [8], indicate that another major function of these
Abbreviations: [‘H]DHE, [‘H]dihydroergocryptin; [3H]- DHA, [3H]dihydroalprenolol; CHAPS, 3-[(3-cholamidopro- pyl)dimethylammonio]-1-propane sulfonate
receptors may be the mediation of epinephrine- induced transmembrane signal via the activation of guanylate cyclase. Such a hormonal pathway would rationalize the mediatory role of cyclic GMP in the transduction of membrane signal.
2. Materials and methods
Adrenocortical carcinoma 494, a spontaneously occurring tumor discovered in [3] and maintained in our laboratory [4] was used for the membrane prepa- ration and binding studies.
[3H]DHE (spec. act. 39.9 Ci/mmol), and [3H]- DHA (spec. act. 43.0 Ci/mmol) and 125I-ACTH (spec. act. 30 uCi/ug) were obtained from New England Nuclear; (-)-epinephrine bitartrate, (-)-norepineph- rine hydrochloride, yohimbine hydrochloride, ergot- amine tartrate, «-ergocryptine, (-)-isoproterenol hydrochloride, and (+)-propranolol hydrochloride were from Sigma. ACTH1-39 was from USP Cortico- tropin Reference Standard (Bethesda MA); phentol- amine hydrochloride, prazosin hydrochloride and QUSO G-32 were gifts from Ciba-Geigy, Pfizer Inc. and Philadelphia Quartz Co., respectively. PD-10 Col- umns were purchased from Pharmacia Fine Chemicals.
2.1. Membrane preparation
Adrenocortical carcinoma membranes were pre- pared as in [12] with minor modifications. The fresh tumor tissue was collected into 10 mM Tris (pH 7.5) maintained on ice. Necrotic cells were removed, sur- rounding fibrous tissues were dissected away, and viable tissue was homogenized in 6 vol. ice cold buffer (0.25 M sucrose, 1 mM MgCl2, 5 mM Tris-HCl (pH 7.5) for 4 X 30 s periods in a Brinkman Polytron
at a setting of 6. After filtration through a double layer of gauze, the homogenate was centrifuged at 400 X g for 10 min at 4℃ and the supernatant recentrifuged at 28 000 X g for 10 min at 4℃. The resulting pellet was washed twice in ice cold incuba- tion buffer (10 mM MgCl2, 50 mM Tris-HCI (pH 7.5)) by resuspension and centrifugation at 28 000 X g for 10 min. The final pellet resuspended in incubation buffer was used for the binding assays. Protein was determined by the Bradford method using bovine serum albumin as a standard [13].
2.2. Solubilization of receptors
Adrenocortical carcinoma membranes were solubil- ized as in [9]. To the membrane suspension (~4 mg/ ml) was added 50 µg/ml lima bean trypsin inhibitor, 0.01% dimethyl sulfoxide and 10 mM CHAPS (final conc.). This was stirred vigorously on ice for 1 h, then centrifuged at 105 000 X g for 60 min. The clear, slightly yellow supernatant was used for the binding studies.
2.3. Binding assays
2.3.1. Membranes
The binding assays were performed as indicated below unless otherwise stated. [3H]DHE (8 nM) and adrenocortical carcinoma membranes (~400 µg/assay) were incubated at 37℃ for 20 min in a total volume of 1.0 ml incubation buffer (10 mM MgCl2, 50 mM Tris-HCI (pH 7.5)). Incubation was terminated by diluting the incubation mixture with 5 ml buffer fol- lowed by immediate filtration through Whatman GF/A glass fiber filters which were washed with 4 × 5 ml incubation buffer. This procedure signifi- cantly reduced the non-specific binding of the ligand. After drying, filters were counted for radioactivity in Omniflor/toluene scintillation mixture. Non-specific binding was measured in the presence of 0.1 mM phentolamine, a potent «-adrenergic antagonist.
2.3.2. Solubilized fractions
Aliquots of the solubilized receptors were incu- bated in 1.0 ml final vol. for 20 min at 37℃ in a mix- ture containing 50 mM Tris, 10 mM MgCl2 (pH 7.5) and 8 nM [3H]DHE. The reaction was stopped by adding 1.5 ml 10 mM Tris and 0.32 M sucrose (pH 7.5). After cooling on ice, the mixture was applied to a Sephadex G-25 column (PD-10), pre-equilibrated with 0.32 M sucrose, 10 mM Tris-HCI (pH 7.5). The initial 2.5 ml effluent was discarded. The high M,
fraction, eluted in the next 3.5 ml was collected and counted for radioactivity with 12 ml Scintiverse. Non-specifically bound radioactivity was determined in the presence of 0.1 mM phentolamine in the incu- bation mixture. All the experiments were done in triplicate and repeated at least 2 times. The specific binding was between 15-30% of the total counts bound.
3. Results
The zwitterionic detergent CHAPS is very selective in solubilizing a-adrenergic receptors. Adrenocortical carcinoma tissue contains @- and ß-adrenergic as well as ACTH receptors. When binding studies were done for all the 3 receptors, it was found that only @-adrener- gic receptors were solubilized (table 1). The recovery of protein in the solubilized fraction was ~30-45% with the specific activity of [3H]DHE binding almost the same as that for the membrane receptors. The solubilized receptors exhibited identical binding char- acteristics as the membrane receptors. Fig.1A shows the binding of various concentrations of [3H]DHE to the solubilized receptors. The saturation curve revealed
| Particulate (fmol/mg | protein) Solubilized | |
|---|---|---|
| «-Adrenergic receptors [3H]DHE binding | 50.7 ± 7.2 | 62.8 ± 1.9 |
| B-Adrenergic receptors [3H]DHA binding | 29.7 ± 3.0 | Non-detectable |
| ACTH receptors 125 I-ACTH binding | 43.0 ± 3.2 | Non-detectable |
a- and ß-Adrenergic receptors were quantitated both in the membrane and solubilized fractions as in section 2. The pro- cedure for ß-adrenergic receptor binding was the same as that of a-adrenergic receptors except the radioligand used was [$H]DHA and non-specific binding was measured in the pres- ence of 0.1 mM isoproterenol. ACTH binding was done on ice for 10 min using 125I-ACTH and non-specific binding was measured in the presence of 1000-fold excess of ACTH1 -39. The total assay volume was 0.5 ml and the reaction was stopped by diluting the reaction mixture to 1.5 ml with incu- bation buffer (50 mM Tris-HCI (pH 7.5)). To this was added 10 mg QUSO and let stand for 10 min on ice. The bound 125 I-ACTH was separated from the free by centrifugation and the supernatant was counted for radioactivity in a y-counter
[3H]-DHE BOUND, fmol/mg PROTEIN
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PHI-DHE BOUND, fmol/mg PROTEIN
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a Ka of ~4 nM and maximum binding of 35 fmol/mg protein. The binding of [3H]DHE at 37℃ and 23℃ for various time intervals is shown in fig.1B. The bind- ing is rapid reaching maximum in 20 min at 37℃ and in 10 min at 23℃. The potency of various adrenergic ligands to displace [3H]DHE binding in membranes was retained in the solubilized fraction. Fig.2 shows the displacement of [3H]DHE binding from the receptor by various adrenergic agonists and antagonists. Half-maximal inhibition of [3H]DHE binding was exhibited at 1 × 10-6 M, 8 X 10-7 M and 2 × 10-4 M for epinephrine, norepinephrine and isoproterenol, respectively. Among the antagonists yohimbine was potent in displacing the bound [3H] DHE. Prazosin (@1-antagonist) was without any effect on the displace- ment of [3H]DHE from the binding sites. In addition, the membrane receptors bound [3H]yohimbine which can be displaced >90% by non-radioactive yohimbine and 65-70% by phentolamine and epi- nephrine (not shown). This confirms [14] the obser- vation made in the adrenocortical carcinoma mem- branes that these a-adrenergic receptors belong to @2-subclassification. Propranolol (3-antagonist) did not have any effect on [3H]DHE binding.
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AGONIST, M
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4. Discussion
The native adrenocortical carcinoma cell mem- branes contain ACTH-, «-adrenergic and ß-adrenergic receptors [5,6,15]. The treatment of the crude mem- branes with CHAPS selectively solubilizes receptors having characteristics of true œ-adrenergic receptors. These bind [3H]DHE rapidly with a high affinity (Kd 4 × 10-9 M), in a saturable fashion (35 fmol/mg pro- tein) and the potency of a-agonists and antagonists to displace [3H]DHE is higher than that of ß-adrenergic agonists and antagonists.
That the a-adrenergic binding activity is indeed soluble was established, since it neither sediments at 105 000 X g for 1 h nor does it show any membrane characteristics upon electron microscopic examina-
tion (not shown). Since the presence of ACTH- or ß-adrenergic receptors was not detected in the solubil- ized preparation, these studies demonstrate that the adrenocortical carcinoma a-adrenergic receptors are not only solubilized by the CHAPS treatment, but they also retain the original @-adrenergic characteris- tics of the native membranes.
Based on the binding studies of receptors with spe- cific radioactive ligands, in many tissues the @-adren- ergic receptors have been classified into @1 and @2 subtypes [16-18]. @2-Adrenergic receptors show higher affinity for yohimbine and little affinity for @1-antagonist prazosin and WB-4101. However, the reverse is the case for @1-adrenergic receptors. Based on these criteria the solubilized adrenocortical carci- noma membranes are exclusively of @2-subtype, since they have a high affinity for yohimbine (EC50 10-7 M) and no affinity for prazosin. It is generally believed that @2-receptors predominantly reside on presynap- tic sites on the nerve terminals and their function is the inhibition of norepinephrine release by feedback mechanism [17,19]. However, these receptors coexist with @1-receptors in rat heart membranes [20], rat brain [21] and hamster adipocytes [22], and predomi- nantly [23] or exclusively in human platelets [24-27].
During the course of this investigation, solubiliza- tion of the human platelet @2-adrenergic receptors by digitonin treatment was reported [25,27]. There is, however, one important difference between the charac- teristics of the solubilized receptors of platelets and adrenocortical carcinoma membranes. The solubilized platelet receptors show a reduced affinity for agonists as compared to the native receptors [25,27]; in con- trast, the @2-agonist and antagonist affinity for the native and solubilized carcinoma membrane receptors remains unchanged. Such a difference between the native and solubilized platelet @2-receptor has led to the interpretation that the digitonin treatment results in the loss of a receptor component protein that is essential for the interaction of the @2-receptor with the GTP-binding protein [25,27]. Such an interpreta- tion is obviously not necessary in the case of the solu- bilized adrenocortical carcinoma membrane receptors prepared by the CHAPS treatment.
The molecular mechanism by which @2-adrenergic receptors exhibit their biological activity is not known. Since these receptors in many tissues exam- ined inhibit adenylate cyclase [10,11], it has been proposed that @2-receptors are coupled to the adenyl- ate cyclase in an inhibitory manner. In such a hypo-
thesis, both the ß-adrenergic and @2-adrenergic agonists will exhibit their biological activity by interacting with the adenylate cyclase, the ß-adrenergic agonist stimu- lating it and the @2-adrenergic agonist inhibiting it.
The results with intact isolated adrenocortical car- cinoma cells indicate that the epinephrine-activated rise of cyclic GMP is mediated by the a-adrenergic receptors [8]. The demonstration that these a-receptors are exclusively of the @2-subtype in native membranes indicates that the epinephrine-activated rise of cyclic GMP in intact cells is mediated by the @2-adrenergic receptors. This then indicates that the @2-adrenergic receptors are coupled to guanylate cyclase and the transmembrane signal induced by the @2-agonist occurs by its interaction with the @2-adrenergic recep- tors which in turn results in the rise of cyclic GMP.
All these results indicate that the @2-adrenergic coupled guanylate cyclase system might be one of the means of transmitting the catecholamine-sensitive bio- logical response in the adrenocortical carcinoma cell.
Acknowledgements
This work was supported by the grants from National Science Foundation (PCM 7800860) and National Cancer Institute (CA-16091).
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