Beta-Adrenergic agonists cause adenylate cyclase activation via a conformational change of their receptor. This was earlier revealed by the use of N-ethylmaleimide as a structural probe: agonist-bound receptors are rapidly inactivated by 0.1 mM N-ethylmaleimide while free and antagonist-bound receptors remain unaffected. Furthermore, the beta-adrenergic receptors only change conformation when coupled to the guanine nucleotide regulatory component of the adenylate cyclase system. It is shown in this report that treatment of turkey erythrocyte membranes with the sulphydryl-specific agent 2,2'-dinitro-5,5'-dithiodibenzoic acid and elevated concentrations of N-ethylmaleimide (1-10 mM) do not affect the total receptor number, but prevent the ability of beta-adrenergic agonists to mediate conformational changes. This effect is mimicked by GTP. These three compounds cause also a two- to four-fold decrease in agonist affinity. Both phenomena may be explained by the ability of the reagents to prevent, and of GTP to reverse, the functional coupling between the receptors and the regulatory component of the adenylate cyclase system. Removal of Mg2+ from the incubation medium (i.e. presence of 1 mM EDTA) produces a similar decrease in agonist affinity, but does not impair the ability of agonist/N-ethylmaleimide to inactivate the receptors. This suggests that Mg2+ increases the agonist affinity for the receptor-regulatory component complex, but is not required for its formation.

Muscarinic acetylcholine receptors were identified in the microsomal P fraction of rat forebrain by the specific binding of the radiolabeled antagonist [3H]dexetimide. Binding occurred to a single class of noncooperative sites (3.25 mumol/mg protein) with an equilibrium dissociation constant of 1.1 nM. Agonist/[3H]-dexetimide competition binding experiments allowed the distinction between two major muscarinic receptor subpopulations, having respectively high affinity (20% of the total receptor population) and low affinity for agonists, but with the same affinity for antagonists. A 610-fold difference in affinity was calculated for carbamoyl-choline, the agonist extensively investigated in this study. The alkylating reagent N-ethylmaleimide did not affect the total receptor number, antagonist binding to the high-affinity and low-affinity sites, nor agonist binding to the high-affinity sites. The reagent, however, caused a net increase in agonist affinity for the low-affinity sites. This process was dependent on time and dose of N-ethylmaleimide, until a maximal increase in affinity (fourfold increase for carbamoylcholine) was attained. This suggests a quantal conversion of the low-affinity sites by the reagent into an alkylated form, which possesses a higher affinity for agonists but an unchanged affinity for antagonists. The rate of alkylation was enhanced by the presence of agonists but not of antagonists, which is indicative for the ability of agonists to mediate a conformational change of these sites. The close correlation between the N-ethylmaleimide-mediated increase in drug affinity for the low-affinity sites and the ability of the drugs to enhance alkylation of these sites by N-ethylmaleimide can be explained by the ability of (a) muscarinic drugs to interact with the low-affinity sites according to the Monod-Wyman-Changeux 'Plausible Model' and (b) N-ethylmaleimide to freeze these sites in the 'active' conformation by alkylation.

The radiolabelled β-adrenergic antagonist (−)-[3H]dihydroalprenolol bound to two independent classes of sites on intact turkey erythrocytes. About 1300 high affinity and 18,000 low affinity sites per cell were observed. The low affinity sites (Kd = 210 nM) were different from the functional β-adrenergic receptors since the binding of (−)-[3H]dihydroalprenolol could be displaced by β-adrenergic antagonists but not by agonists and since, in marked contrast with the β-adrenergic receptors in membranes, the sites were sensitive to the alkylating agent N-ethylmaleimide and resistant to the reducing agent dithiothreitol.

The biochemical characterization of the catecholamine β-adrenergic receptor from turkey erythrocyte membranes is rapidly progressing. The complex relationship with the adenylate cyclase and other membrane components which intervene in the hormonal stimulation of the cell is discussed in view of the effect of various ligands on the membrane-bound and affinity-purified receptor.

Rabbit antibodies induced against alprenolol, a potent P-adrenergic antagonist, bind to other antagonists and, with less avidity, to catecholamine agonists. Anti-idiotypic antibodies were raised against the anti-alprenolol immunoglobulins. The anti-idiotypic antibodies specifically bind and agglutinate turkey erythrocytes; this is not observed for human or sheep erythrocytes, which are devoid of fl-adrenergic receptors. The anti-idiotypic antibodies compete with (-)[3Hdihydroalprenolol for binding on the fl-adrenergic receptors on purified turkey erythrocyte membranes. The binding to the membrane-bound receptors is prevented by preincubation of the anti-idiotypic antibodies with their immunogen, the antialprenolol immunoglobulins. The binding to the receptor is not merely passive: the anti-idiotypic antibodies stimulate basal adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and enhance adenylate cyclase activation by catecholamine. These observations support the notion that antiidiotypic antibodies may constitute an "internal image" of the original antigen and may mimic its biological effects. To better understand the interaction between f3-adrenergic ligands and their specific membrane receptors, we have undertaken to isolate and purify the various components intervening in the catecholamine-mediated activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. For this purpose we have developed an affinity chromatography method that allows physical separation of the functional cyclase and f3-adrenergic receptors from turkey erythrocyte membranes (1). Various biochemical and pharmacological properties of the receptors were described (2). The minute amounts of protein prepared by this method and the risk of losing essential components of the receptor cyclase complex during the membrane purification and solubilization steps prompted us to develop a second line of investigation based on immunological methods. Anti-receptor antibodies can be obtained by immunization of rabbits and mice with purified ,B-adrenergic receptor, but most of the antibodies react with parts of the molecule other than the binding site (ref. 3; unpublished data). This is in agreement with observations made in other systems: animals do not make anti-receptor binding site antibodies upon immunization with insulin, acetylcholine, or thyrotropin receptors despite the fact that in pathological conditions such as autoimmune diabetes (4), myasthenia gravis (5), Graves disease (6), and 02-adrenergic hyporesponsiveness in allergic rhinitis (7) anti-binding site antibodies probably constitute an important basis for the etiopathogeny. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 7385 Recently a report appeared in which anti-f3-adrenergic ligand antibodies were obtained by immunization with purified receptor (8). These immunoglobulins are analogous in their binding properties to the antibodies obtained by immunization with alprenolol chemically linked to a carrier protein (9). Whereas anti-alprenolol antibodies were not useful for the isolation of receptor-alprenolol complexes, it occurred to us that anti-idiotypic antibodies (10-12) against the anti-alprenolol immunoglobulins could possibly mimic in some way the properties of ,B-adrenergic ligands. We report here that anti-alprenolol anti-idiotypic antibodies indeed do recognize specifically f3-adrenergic receptors and modulate the catecholamine-sensitive adenylate cyclase. MATERIALS AND METHODS Materials. The following were obtained as kind gifts: (±)alprenolol hydrochloride from CIBA-Geigy, (-)-propranolol from Imperial Chemical (Macclesfield, England), (-)-isoproterenol bitartrate and (-)-epinephrine bitartrate from Sterling Winthrop (New York), (-)-norepinephrine tartrate from Sigma, and butoxamine hydrochloride from Burroughs Wellcome (Research Triangle Park, NC), (-)-[3H]dihydroalprenolol hydrochloride [(-)-[3H]DHA] (33 Ci/mmol), cyclic [8-3H]AMP (13 Ci/mmol), and [a-32P]ATP (20-30 Ci/mmol), were obtained from New England Nuclear (1 Ci = 3.7 X 1010 becquerels). Bovine gamma globulin (BGG) was from Miles. Cyclic AMP, ATP, creatine kinase, creatine phosphate, and sodium dodecyl sulfate were from Sigma. Turkey erythrocytes and erythrocyte membranes were prepared as described (13). Immune Sera and Immunoglobulin Fractions. Flemish giant rabbits were immunized with a covalent alprenolol-bovine serum albumin conjugate as described (9). The IgG fraction from the immune sera was purified by ammonium sulfate precipitation (33% saturation) followed by DEAE-cellulose ion-exchange chromatography and extensive dialysis against phosphate-buffered saline (Pi/NaCl). Allotype matched (al a2 b4 b4) rabbits were immunized with 1 mg of the anti-alprenolol IgG fraction dissolved in Pi/NaCl and emulsified with 1 ml of complete Freund's adjuvant. The emulsion was injected intramuscularly. One, two, and three weeks later the rabbits were reinjected intramuscularly with 1 mg of the anti-alprenolol IgG emulsified with 1 ml of incomplete Freund's adjuvant. Animals were bled weekly from the ear vein. IgG was prepared from the sera, concentrated by vacuum dialysis, and stored frozen in aliquots. Binding of /3-adrenergic ligands to anti-alprenolol IgG was assayed by inhibition of binding of (-)-[3H]DHA by means of Abbreviations: (-)-[3HJDHA, (-)-[3Hjdihydroalprenolol; BGG, bovine gamma globulin; Pi/NaCl, phosphate-buffered saline. 7386 Immunology: Schreiber et al. an ammonium sulfate precipitation assay (Farr technique) using 0.5 mg of BGG as carrier protein as described (9). Nonspecific binding was determined in the presence of a 100-fold excess of unlabeled alprenolol and did not exceed 4% of the specific binding. Anti-idiotypic IgG was detected by inhibition of (-)[3H]DHA binding to anti-alprenolol IgG, using the Farr assay as above. Preimmune IgG and normal rabbit IgG were used as controls. F(ab')2 fragments prepared from purified '25I-labeled sheep anti-rabbit IgG antibodies were a kind gift of J. Urbain; the specific activity was 2500 cpm/ng of protein. Binding Assays. (i) Hemagglutination assay. Turkey, human, or sheep erythrocytes (1 X 106) were incubated for 1 hr at 370C with dilutions of anti-idiotypic IgG in Pi/NaCI/BGG (0.5%) in a total volume of 50 Al in hemagglutination wells. (ii) Radiolmmunoassay on intact cells. Turkey, human, or sheep erythrocytes (2 X 106) were incubated for 1 hr at 37'C with dilutions of anti-idiotypic IgG in Pi/NaCI/BGG (0.5%) in a final volume of 200 ul in Eppendorf tubes. Cells were washed three times with buffer and further incubated for 1 hr with 10 nM F(ab')2 fragments of '25I-labeled sheep anti-rabbit Ig antibodies (corresponding to 250,000 total cpm). Cells were washed six times, and the bottoms of the tubes were sliced out and their radioactivities were measured. (iii) (-)-[3H]DHA binding to turkey erythrocyte membranes. Membranes were preincubated with dilutions of antiidiotypic IgG for 1 hr at 30'C in 75 mM Tris-HCI buffer, pH 7.4/25 mM MgCI2/0.5% BGG. Membranes were washed twice by centrifugation and assayed at a final protein concentration of 2 mg/ml for (-)-[3H]DHA binding after 10-min incubation at 30°C and vacuum filtration on glass fiber filters as described (1). Nonspecific binding was determined in the presence of a 1000-fold excess of propranolol and did not exceed 10% of the specific binding. Results are expressed as pmol of (-)-[3H]DHA specifically bound per mg of protein. Preimmune IgG and normal rabbit IgG were used throughout as controls. Adenylate Cyclase Assay. Turkey erythrocyte membranes were preincubated with dilutions of anti-idiotypic IgG for 90 min at 0°C. After washing, 70 ,g of membrane was assayed for adenylate cyclase activity by conversion of [a-32P]ATP into cyclic [32P]AMP. The incubation medium contained 50 mM Tris-HCl, pH 7.4/1 mM cyclic AMP/10 M [a-32P]ATP/7 mM MgCl2/1 mM EDTA/1 mg of creatine kinase per ml/10 mM creatine phosphate in a final volume of 80 p3. The reaction was allowed to proceed for 20 min at 30°C; it was stopped by addition of 0.5 ml of 50 mM Tris.HCI, pH 7.4/0.5 mM cyclic AMP/0.S mM ATP/2% (wt/vol) sodium dodecyl sulfate. Cyclic [3H]AMP (5000-7000 cpm) was then added to monitor cyclic [32P]AMP recovery. Cyclic [32P]AMP was separated from labeled ATP by the technique of Salomon et al. (14). Results are expressed as pmol of cyclic AMP produced per mg of membrane protein per 20 min.