Proc. Nati. Acad. Sci. USA
Vol. 76, No. 5, pp. 2263-2267, May 1979 Biochemistry
Antibodies raised against purified fl-adrenergic receptors specifically bind f3-adrenergic ligands (agarose-alprenolol/affinity chromatography/catecholamines/immunogen/model receptor binding site)
MARC G. CARON*t4, YOGAMBAL SRINIVASANt§, RALPH SNYDERMANt11 , AND ROBERT J. LEFKOWITZtt§ §Howard Hughes Medical Institute Laboratory and Departments of tMedicine, tBiochemistry, IMicrobiology, and tIlmmunology, Duke University Medical Center, Durham, North Carolina 27710
Communicated by James B. Wyngaarden, March 8, 1979
Antibodies raised against purified jl-adrenergic ABSTRACT receptors themselves specifically bind 1&-adrenergic ligands. Digitonin-solubilized frog (Rana pipiens) erythrocyte #-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (±)isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the P-adrenergic antagonist [3H]dihydroalprenolol with high affinity (Kd = 1 nM). [3H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [3H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the B-adrenergic receptor, i.e., (-)isoproterenol > (-)epinephrine > (-)norepinephrine; alprenolol propranolol >> phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the binding antibodies could be specifically 13H]dihydroalprenolol adsorbed onto purified frog erythrocyte membranes, whereas Xenopus and human erythrocyte membranes, both of which are almost devoid of f3-adrenergic receptors, were ineffective in adsorbing [3H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a fl-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.
implications for studies of hormone and drug receptor structure and function are described in this communication.
The adenylate cyclase-coupled f3-adrenergic receptors of frog erythrocyte plasma membranes have been extensively characterized (1). In addition to being identified initially by direct ligand-binding techniques using the f3-adrenergic antagonist [3H]dihydroalprenolol ([3H]DHA) (2), these receptors have been solubilized with detergents (3) and more recently purified by affinity chromatography (4). In order to further characterize this receptor it was of interest to raise an antibody to the partially purified receptors. Antibodies against hormone receptors have previously been raised (5-7) or found in the circulation of patients with specific diseases (8-10). Circulating antibodies against thyrotropin receptors, insulin receptors, and the cholinergic receptors have been implicated in the mechanisms of such diseases as hyperthyroidism, the syndrome of insulin resistance with acanthosis nigricans, and myasthenia gravis. In the course of immunizing rabbits with affinity chromatography-purified preparations of the frog erythrocyte 3-adrenergic receptors, we obtained antibodies that unexpectedly were found to bind adrenergic agonists and antagonists with a specificity resembling that of the f3-adrenergic receptor. Moreover, a portion of the antibodies capable of binding f3-adrenergic ligands could be adsorbed specifically to frog erythrocyte membranes. The properties of these unique antibodies and their
MATERIALS AND METHODS Materials. (±)-Alprenolol was a gift from Hassle Pharmaceutical (Molndal, Sweden). Freund's adjuvant was from Difco. [3H]DHA was from New England Nuclear. All other drugs and chemicals were from sources described before (2-4) and were of the highest grade available. Preparation of the Immunogen. Soluble f3-adrenergic receptor preparations from frog (Rana pipiens) erythrocyte membranes were obtained by digitonin treatment and were purified 100- to 200-fold in a single step on a Sepharose 6Balprenolol affinity gel as described (4). Briefly, 40-50 ml of soluble receptor preparation was chromatographed on 5-8 ml of Sepharose 6B-alprenolol gel. About 80-90% of the receptor activity was routinely bound, whereas as low as 3-5% of total protein was retained by the column. After the column was washed, receptor activity was eluted specifically by addition of 1-100 mM (+)-isoproterenol to the equilibration buffer (0.2% digitonin/50 ,uM dithiothreitol/100 mM NaCl/10 mM TrisHCI, pH 7.4). The eluate was then concentrated to 6-7 ml by lyophilizing and then chromatographing on Sephadex G-50. This chromatography removed most of the free isoproterenol so that it was possible to assay [3H]DHA binding without interference by using saturating concentrations of the radioligand. In separate experiments in which [3H]isoproterenol was used we documented that after Sephadex G-50 chromatography of affinity column eluates the residual isoproterenol concentration was about 0.1 uM or lower. Soluble receptor preparations obtained in this way were routinely purified 100- to 200-fold. Immunization. Routinely, 5-10 pmol of receptor in 7 ml of 0.2% digitonin/100 mM NaCl/10 mM Tris-HCl, pH 7.4, was emulsified with 7 ml of complete Freund's adjuvant. One milliliter was injected intradermally in 10-12 sites on the back and 1 ml intramuscularly in the subscapular muscle region to each of six New Zealand White rabbits. Injections were repeated at intervals of 2-3 weeks. Immune Sera and Immunoglobulin Fractions. Twelve days after the fourth booster injection and periodically thereafter animals were bled from the ear artery. Blood was allowed to coagulate for 2 hr at room temperature and serum was removed and stored in aliquots at -20°C. An immunoglobulin-rich fraction was prepared from some sera by precipitation with ammonium sulfate to 50% saturation. After resuspension of the precipitate in 0.01 M potassium phosphate at pH 8.0, dialysis, and centrifugation, the material was stored frozen. The con-
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
Abbreviation: DHA, dihydroalprenolol. * To whom correspondence and reprint requests should be addressed at: Box 3287, Duke University Medical Center, Durham, NC
this fact.
27710.
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Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Caron et al.
2264
centration of immunoglobulin was measured by absorbance at 280 nm with bovine gamma globulin as the standard. Detection of Antibody Activity. The ability of the immune sera to interact with adrenergic drugs was assessed by direct ligand binding, using the 3-adrenergic antagonist [3H]DHA. As with particulate f-adrenergic receptor preparations, it was found that [3H]DHA bound to immune sera could be separated from free ligand by rapid vacuum filtration on glass fiber filters. We further documented that [3H]DHA binding to immune sera could also be assessed by filtration on Millipore filters, by Sephadex G-50 chromatography (3), or by ammonium sulfate precipitation. These methods appeared to be qualitatively equivalent, and the glass fiber filtration assay was routinely used for the results reported here. [3H]DHA specifically bound was assessed (2, 3) by defining the nonspecific binding as that which was not competed for by 10 ,tM unlabeled (±)-alprenolol or 100 ,M unlabeled (-)-isoproterenol. Most assays were performed with whole antiserum at a final dilution of 1:500 unless otherwise stated. Under these conditions specific [3H]DHA binding represented 95-98% of total binding. RESULTS Characteristics of binding of ,B-adrenergic agents to antisera Initially, immune sera were tested for their ability to interact with the 0-adrenergic receptor by examining their effects on the binding of [3H]DHA to purified or solubilized frog erythrocyte membranes by using the rapid filtration or the Sephadex G-50 chromatography technique. It was then realized that immune sera themselves were capable of binding the ligand [3H]DHA. Of the six animals immunized, all developed detectable [3H]DHA binding activity in their serum. The general binding properties of these six antisera were similar, though differences in detailed binding specificity were found. The binding data presented below were obtained with the antiserum from one animal (no. 349) unless otherwise noted. [3H]DHA binding to these immune serum sites was rapid and rapidly reversible (Fig. 1). At 23°C the rates of association and dissociation were too fast to be determined with any accuracy; that is, within 1 min the whole process was complete. However, at -4oC the rate constant of association ki was found to be 9.5 X 107 min-1 M-1, whereas the rate constant of dissociation k2
was 0.054 min-1. The ratio k2/kl provides an estimate of the value of the dissociation constant (Kd) of these sites for [3H]DHA, 0.6 nM. Scatchard analysis of equilibrium binding data for [3H]DHA to these sites at 230C indicated a saturable binding process and yielded a monophasic curve (Fig. 2). From these data a value of Kd for [3H]DHA binding of 2-4 nM was obtained. The Kd value obtained by kinetic analysis was in close agreement with the value of 0.7 nM obtained for (-)-alprenolol by binding competition (Table 1). The Kd value derived by equilibrium binding (2-4 nM) was determined on an earlier bleed of the immune serum and correlated well with the potency of (-)alprenolol binding in that immune serum. The specificity of binding of [3H]DHA to these sites was examined by measuring the ability of agonists and antagonists to compete for [3H]DHA binding. As shown in Fig. 3, fadrenergic agonists were potent competitors of binding, with a potency order (-)-isoproterenol > (-)-epinephrine > (-)norepinephrine that is identical to the order of potency for competition of binding to the 3-adrenergic receptor. Binding also displayed the stereoselectivity that characterizes betaadrenergic receptor interactions, because (-)-isorpoterenol competed 10-fold more effectively than (+)-isoproterenol. ,B-Adrenergic antagonists such as (-)-alprenolol and (-)-propranolol (not shown) were also very potent competitors and the (-) isomers of these drugs were also preferentially recognized by these binding sites over their (+) isomers (Fig. 3, Table 1). The detailed specificity of the interaction of several adrenergic agents with immune serum 349 as assessed by competition for [3H]DHA binding is shown in Table 1. Table 1 also compares the Kd values obtained for the antiserum binding sites with those previously obtained for the interaction of these drugs with the membrane-bound 0-adrenergic receptors of frog erythrocytes (12). It can be seen that striking similarities exist between the pharmacological properties of both binding sites. Dopamine, the dopaminergic antagonist haloperidol, and the a0.12
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FIG. 1. Association of [3H]DHA with and dissociation of [;HJDHA from antiserum 349 as a function of time at 0C. Dissociation of bound [3H]DHA was assessed after the addition of 10 ,1M
(±)-alprenolol to incubation mixtures at equilibrium. The data are shown as fmol of specific binding per 50 jul of 1:500 dilution of the antiserum. The association and dissociation rate constants k1 and k2 were calculated as described (4). Results shown are representative of two or three experiments.
0
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FIG. 2. Scatchard plot of [3HJDHA binding to antiserum 349. Antiserum at 1:500 final dilution was incubated with increasing concentrations of [3H]DHA (0.3-50 nM) for 30 min at 23°C. Nonspecific binding, which ranged from 5% to 15% of total binding, was determined in parallel incubations in the presence of 10 IM(i)alprenolol. Binding was assessed by filtration. Results are representative of two experiments.
Biochemistry:
Caron et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Trable 1. Dissociation constants (Kd) of various adrenergic agents obtained by binding competition of [3HJDHA
Kd,,UM Frog erythrocyte membrane 3-adrenergic receptors
Antiserum 349
Compounds Antagonists (-)-Alprenolol
0.0034 0.150 0.0046 0.286 0.57
0.0007 0.084 0.001 0.031 0.002
(+)-Alprenolol (-)-Propranolol (+)-Propranolol
(W)-Dichlorisoproterenol
(W)-Practolol (W)-Phentolamine
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Agonists (-)-Isoproterenol (+)-Isoproterenol (-)-Epinephrine (-)-Norepinephrine (-)-Soterenol
0.40 183 4.6 49 0.7 0.06
0.04 0.4 9.3 280 0.006 7.7
(±)-Hydroxybenzyl
isoproterenol Dopamine Carbachol [3H]DHA (8-10 nM) binding assessed by the filtration technique was determined on 50-yl aliquots of 1:100-1:500 diluted antiserum 349 in the presence and absence of five to seven concentrations of each of the drugs shown. Kd values for each drug were calculated from the concentration of the drug that competed for 50% of specific [3H]DHA binding according to the method of Cheng and Prusoff (11), using a dissociation constant of 3.7 nM obtained by Scatchard analysis for [3HIDHA binding to the antiserum. No effect of the drug at concentrations of 100 ,M or effects too slight to permit calculation of a Kd value. Results shown are from one to three experiments performed in duplicate for each drug. Results on the specificity of [3H]DHA binding to erythrocyte membranes were taken from previously published work (12). -,
adrenergic antagonist phentolamine did not interact with the antibody sites at concentrations as high as 10-100 ,M; this is similar to their lack of effect on binding to the fl-adrenergic
2265
receptor. The catechol analogs dihydroxphenylalanine and dihydroxymandelic acid were also without effect at 100 AM. Despite these similarities to the specificity of the 13-receptor, detailed studies revealed a number of striking differences. Thus the 3-antagonists (+)-dichlorisoproterenol and (±)-practolol were, respectively, 300- and 3000-fold more potent in competing for the antibody than for the receptor binding sites, and the agonist (+)-hydroxybenzylisoproterenol was only 1/100th as potent at the antibody sites. Moreover, whereas all antisera [3H]DHA binding activities clearly possessed a fl-adrenergic specificity, each differed from the other on detailed examination (data not shown). Immunoglobulin nature of the [3H]DHA binding sites in immune sera In order to document that [3H]DHA binding activity in the immune sera was in fact due to the presence of immunoglobulin, the following tests were performed. First, [3H]DHA binding activity in the immune sera was quantitatively precipitable by 50% saturation with ammonium sulfate. Second, [3HJDHA binding activity was stable to heating at 560C for 30 min. Third, [3H]DHA binding activity could be fully precipitated by goat antiserum to rabbit IgG (Fig. 4). Fourth, the [3H]DHA binding activity was stable to a reduction of the pH to 3.5 for 10 min. Finally, the binding activity was found to cochromatograph with authentic IgG on Sephadex G-200. None of six preimmune sera contained any [3H]DHA binding activity. Further, several hyperimmune sera obtained in response to other antigens failed to show any binding activity. Similarly, sera from rabbits immunized with isoproterenolcontaining digitonin solutions as well as isoproterenol-containing soluble preparations depleted of f3-adrenergic receptors by affinity chromatography failed to show any [3H]DHA binding activity. These results indicate that the [3H]DHA binding activity observed in these immune sera is due to an immunoglobulin fraction raised in response to the partially purified ,B-adrenergic receptor preparations used as the immunogen. 100 C C
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FIG. 3. Ability of agonists, antagonists, and other compounds to compete for [3HJDHA binding to antiserum 349. Antiserum 349 (50 ,ul of a 1:100 dilution) was incubated with 8 nM [3H]DHA (final volume 500 1l) in the presence and absence of various concentrations of the different drugs shown. The results, expressed as % of total binding (100% control binding was 0.52-0.57 pmol) are from an experiment in duplicate and are representative of three such experiments. ALP,
alprenolol; ISO, isoproterenol; EPI, epinephrine; NE, norepinephrine; DHMA, dihydroxymandelic acid.
01 0
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FIG. 4. Immunoprecipitation of [3HJDHA binding activity in immune serum 349 by goat antiserum to rabbit IgG. A 1:10 dilution of the immune serum was incubated in 25 mM Tris-HCl/2 mM MgCl2, pH 7.4, at 25°C for 24 hr with various concentrations of the anti-rabbit IgG. The precipitate was removed by centrifugation and the supernatant was assayed for [3H]DHA binding (12 nM). Results shown are from an experiment performed in duplicate.
2266
Biochemistry: Caron et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
alo
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00
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FI(.. 5. Interaction of antiserum 349 with various erythrocyte membrane preparations. Frog, Xenopus, and human erythrocyte membranes were all prepared as described (3,. 4) for the frog erythrocyte membranes. Membranes (1.1-1.4 mg of protein per ml) were incubated at 250C for 11/2 hr in 25 mM Tris-HCl/5 mM MgCI2, pH 7.4, with a 1:50 final dilution of whole antiserum 349 (hatched bars) or normal rabbit serum (empty bars) preheated at 560C for 30 min to inactivate complement. Membranes were washed twice with 5-ml samples of the incubation buffer and [3H]DHA binding was measured on the resuspended membranes by filtration on GF/C glass fiber filters. Membrane preparations incubated with normal rabbit serum showed the same [3H]DHA binding activity as untreated preparations. Results ± SEM shown are the means of 3 different experiments performed in duplicate. These experiments are representative of 8-10 similar experiments.
Interaction of the antibodies with erythrocyte membranes The evidence described thus far indicates that the antibodies raised in response to partially purified preparations of frog erythrocyte f3-adrenergic receptor possess specificity toward catecholamines and related f-adrenergic drugs. Studies were next performed to elucidate whether these immune sera also possessed affinity for components of erythrocyte membranes. As shown in Fig. 5, a portion of the [3H]DHA binding activity present in immune sera was specifically adsorbed by frog erythrocyte membranes, whereas Xenopus erythrocyte membranes as well as human erythrocyte membranes, both of which are essentially devoid of f3-adrenergic receptors, were almost ineffective in adsorbing [3H]DHA binding activity from the antisera. DISCUSSION In this communication we report that antibodies raised in rabbits against partially purified preparations of the frog erythrocyte f-adrenergic receptors appear to recognize adrenergic catecholamines as well as components of the erythrocyte membrane. As evidenced by the results obtained with the specific f-adrenergic radioligand [3H]DHA, it is clear that these antisera binding sites display specificity that bears striking similarities to that of the physiological f3-adrenergic receptors. In addition, the data indicate that the [3H]DHA binding activity in these sera is due to immunoglobulins, because binding was stable to exposure to 560C for 30 min and to acidic conditions (pH 3.5), was precipitated by 50% saturated ammonium sulfate, and was quantitatively immunoprecipitated by goat antiserum to rabbit IgG. None of these properties is shared by either membrane-bound or solubilized f-adrenfi-
ergic
receptors.
The properties of the antibodies described in this communication appear to be unique. Although antibodies have previously been raised to several hormone receptors, such as those for insulin (7), prolactin (6), and acetylcholine (5), in none of these cases did the antibodies themselves mimic the binding specificity of the receptor. In one case an antibody that mimicked the effects of insulin was obtained (13), but this antibody was raised against an anti-insulin antibody. Attempts to raise antibodies to catecholamines (14, 15) or to adrenergic antagonists (16, 17) have to date met with very limited success. This is not surprising in view of the chemical nature of these agents, which are small molecules with molecular weights of only about 300. Thus, in the few cases in which an antibody has been successfully raised against an adrenergic drug or antagonist this has required covalent linkage of the agent to a protein carrier in order to form the immunogen. In addition, the antibodies obtained have possessed very restricted specificities. Antibodies raised to catecholamine analogues have had very low affinities for the native catecholamines (14, 15). In no case have any of these antibodies displayed a specificity even vaguely reminiscent of an adrenergic receptor. What, then, is the explanation for the unique antibodies obtained in the present studies? Several explanations will be considered, though a firm conclusion may not be possible at this time. One possibility is that two separate immunogens have been involved, the receptors and free isoproterenol in the receptor preparations, not removed by the Sephadex G-50 chromatographic step used prior to injection of partially purified receptors. This seems unlikely on several grounds. First, as noted above, the free catecholamine would certainly not be expected to be antigenic by itself. Second, if two totally distinct immunogenic stimuli were involved, the antibodies that can be specifically adsorbed on the erythrocyte membrane and that are presumably directed against a component of the membrane would not be expected to bind [3HJDHA too; the data in Fig. 5 did demonstrate such binding. Another possibility is that the antibodies studied have been raised in response to partially purified receptor alone. This also seems somewhat unlikely. There would be no reason to anticipate that such an antibody, even if directed at the active binding site of the receptor, would itself display a binding specificity that so closely mimics that of the receptor. In fact, a somewhat different or complementary specificity might be
predicted. A third possiblity is that antibodies formed against the receptor have in turn served as antigens leading to the formation of "anti-idiotypic" antibodies, the binding specificity of which now resembles that of the receptor. In this case too, however, the antibodies that can be adsorbed by the erythrocyte membranes would not be expected to be the same as those that can bind DHA. An additional possibility is that the immune sera contain soluble anti-receptor antibody-receptor complexes and that binding found in such sera is due to the presence of receptors. This is unlikely because, in contrast to the antibody [3H]DHA binding sites, soluble fl-adrenergic receptor binding activity is completely destroyed by heating to 560C for 30 min, or by decreasing the pH to 3.5. At low pH, putative antibody-receptor complexes would be dissociated and the receptors then inactivated. Upon chromatography of antiserum 349 on Sephadex G-200, the [3H]DHA binding activity was found to cochromatograph with authentic rabbit IgG. The presence of antibody-receptor complexes would have produced a peak of binding activity of larger apparently molecular size. In addition, the specificity of the antiserum is readily distinguishable from that of the receptors as noted above.
Biochemistry:
Caron et al.
Perhaps the most attractive hypothesis is that the antibodies studied have been formed in response to a complex of isoproterenol bound to the purified receptors. Viewed in this light, the receptor-bound isoproterenol might be immunologically equivalent to a hapten conjugated to a carrier and hence be antigenic (18). That some of the antibodies to the hapten (isoproterenol) also recognize determinants on the carrier (receptor) would thus not be surprising. That the specificity for binding catecholamines and related drugs so closely resembles that of the 3-adrenergic receptor in terms of rank order of potencies, stereospecificity, etc. might reflect the particular conformation in which isoproterenol was held by the receptors. Regardless of the exact mechanism leading to the generation of these unusual antibodies, their further characterization should provide important new insights into f3-adrenergic receptor structure and function. Among the potentially interesting avenues which could be explored are: (i) The possible biological activity of the antibodies. (ii) The use of the antibodies as a tool for purification of the receptors. (iii) The use of the antibodies to probe the structure of the specific adrenergic drug binding site. Because the antibodies are available in much larger quantities than the P-receptors and bind drugs with specificities and affinities similar to those of the receptor, purification and characterization of the antibody binding sites might help shed light on the nature of the structural features that contribute to the specificity of the receptor. (iv) Development of anti-catecholamine antibodies for radioimmunoassay. Another point raised by these studies that deserves mention is that immunization with drugs noncovalently bound to their receptor may be a means of raising antibodies to otherwise nonimmunogenic biologically active small molecules. The expert technical assistance of Miss Laurie Card is greatly appreciated. This work was supported by U.S. Public Health Service. Grants HL16037 and HL20339 and a grant-in-aid from the American Heart Association.
Proc. Natl. Acad. Sci. USA 76 (1979)
2267
1. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C. & Caron, M. G. (1976) Biochim. Biophys. Acta 457, 1-39. 2. Mukherjee, C., Caron, M. G., Coverstone, M. & Lefkowitz, R.
J. (1975) J. Biol. Chem. 250,4869-4876. 3. Caron, M. G. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251, 2374-2384. 4. Caron, M. G., Srinivasan, Y., Pitha, J., Kociolek, K. & Lefkowitz, R. J. (1979) J. Biol. Chem. 254, 2923-2927. 5. Patrick, J. & Lindstrom, J. (1973) Science 180, 871-872. 6. Shiu, R. P. C. & Friesen, H. G. (1976) Science 192,259-261. 7. Jacobs, S., Chang, K.-J. & Cuatrecasas, P. (1978) Science 200, 1283-1284. 8. Flier, S. J., Kahn, C. R., Roth, J. & Bar, R. S. (1975) Science 190, 63-65. 9. Hall, R., Smith, B. R. & Mukhtar, E. D. (1975) Clin. Endocrinol. 4,213-230. 10. Lindstrom, J. M., Seybold, M. E., Lennon, V. A., Whittingham, S. & Duane, D. D. (1976) Neurology 26, 1054-1059. 11. Cheng, Y. & Prusoff, W. H. (1973) Biochem. Pharmacol. 2g, 3099-3108. 12. Mukherjee, C., Caron, M. G., Mullikin, D. & Lefkowitz, R. J. (1976) Mol. Pharmacol. 12, 16-31. 13. Sege, K. & Petersen, P. A. (1978) Proc. Nati. Acad. Sci. USA 75, 2443-2447. 14. Spector, S., Dalton, C. & Felix, A. M. (1973) in Frontiers in Catecholamine Research, eds. Usdin, E. & Snyder, S. H. (Pergamon, New York), pp. 345-349. 15. Grota, L. J. & Brown, G. M. (1976) Endocrinology 98, 613622. 16. Kawashima, K., Levy, A. & Spector, S. (1976) J. Pharmacol. Exp. Ther. 196,517-523. 17. Hoebeke, J., Vauquelin, G. & Strosberg, A. D. (1978) Biochfm. Pharmacol. 27, 1527-1532. 18. Green, I., William, E. P. & Benacerraf, B. (1966) J. Exp. Med. 123, 859-879.
Vol. 76, No. 5, pp. 2263-2267, May 1979 Biochemistry
Antibodies raised against purified fl-adrenergic receptors specifically bind f3-adrenergic ligands (agarose-alprenolol/affinity chromatography/catecholamines/immunogen/model receptor binding site)
MARC G. CARON*t4, YOGAMBAL SRINIVASANt§, RALPH SNYDERMANt11 , AND ROBERT J. LEFKOWITZtt§ §Howard Hughes Medical Institute Laboratory and Departments of tMedicine, tBiochemistry, IMicrobiology, and tIlmmunology, Duke University Medical Center, Durham, North Carolina 27710
Communicated by James B. Wyngaarden, March 8, 1979
Antibodies raised against purified jl-adrenergic ABSTRACT receptors themselves specifically bind 1&-adrenergic ligands. Digitonin-solubilized frog (Rana pipiens) erythrocyte #-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (±)isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the P-adrenergic antagonist [3H]dihydroalprenolol with high affinity (Kd = 1 nM). [3H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [3H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the B-adrenergic receptor, i.e., (-)isoproterenol > (-)epinephrine > (-)norepinephrine; alprenolol propranolol >> phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the binding antibodies could be specifically 13H]dihydroalprenolol adsorbed onto purified frog erythrocyte membranes, whereas Xenopus and human erythrocyte membranes, both of which are almost devoid of f3-adrenergic receptors, were ineffective in adsorbing [3H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a fl-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.
implications for studies of hormone and drug receptor structure and function are described in this communication.
The adenylate cyclase-coupled f3-adrenergic receptors of frog erythrocyte plasma membranes have been extensively characterized (1). In addition to being identified initially by direct ligand-binding techniques using the f3-adrenergic antagonist [3H]dihydroalprenolol ([3H]DHA) (2), these receptors have been solubilized with detergents (3) and more recently purified by affinity chromatography (4). In order to further characterize this receptor it was of interest to raise an antibody to the partially purified receptors. Antibodies against hormone receptors have previously been raised (5-7) or found in the circulation of patients with specific diseases (8-10). Circulating antibodies against thyrotropin receptors, insulin receptors, and the cholinergic receptors have been implicated in the mechanisms of such diseases as hyperthyroidism, the syndrome of insulin resistance with acanthosis nigricans, and myasthenia gravis. In the course of immunizing rabbits with affinity chromatography-purified preparations of the frog erythrocyte 3-adrenergic receptors, we obtained antibodies that unexpectedly were found to bind adrenergic agonists and antagonists with a specificity resembling that of the f3-adrenergic receptor. Moreover, a portion of the antibodies capable of binding f3-adrenergic ligands could be adsorbed specifically to frog erythrocyte membranes. The properties of these unique antibodies and their
MATERIALS AND METHODS Materials. (±)-Alprenolol was a gift from Hassle Pharmaceutical (Molndal, Sweden). Freund's adjuvant was from Difco. [3H]DHA was from New England Nuclear. All other drugs and chemicals were from sources described before (2-4) and were of the highest grade available. Preparation of the Immunogen. Soluble f3-adrenergic receptor preparations from frog (Rana pipiens) erythrocyte membranes were obtained by digitonin treatment and were purified 100- to 200-fold in a single step on a Sepharose 6Balprenolol affinity gel as described (4). Briefly, 40-50 ml of soluble receptor preparation was chromatographed on 5-8 ml of Sepharose 6B-alprenolol gel. About 80-90% of the receptor activity was routinely bound, whereas as low as 3-5% of total protein was retained by the column. After the column was washed, receptor activity was eluted specifically by addition of 1-100 mM (+)-isoproterenol to the equilibration buffer (0.2% digitonin/50 ,uM dithiothreitol/100 mM NaCl/10 mM TrisHCI, pH 7.4). The eluate was then concentrated to 6-7 ml by lyophilizing and then chromatographing on Sephadex G-50. This chromatography removed most of the free isoproterenol so that it was possible to assay [3H]DHA binding without interference by using saturating concentrations of the radioligand. In separate experiments in which [3H]isoproterenol was used we documented that after Sephadex G-50 chromatography of affinity column eluates the residual isoproterenol concentration was about 0.1 uM or lower. Soluble receptor preparations obtained in this way were routinely purified 100- to 200-fold. Immunization. Routinely, 5-10 pmol of receptor in 7 ml of 0.2% digitonin/100 mM NaCl/10 mM Tris-HCl, pH 7.4, was emulsified with 7 ml of complete Freund's adjuvant. One milliliter was injected intradermally in 10-12 sites on the back and 1 ml intramuscularly in the subscapular muscle region to each of six New Zealand White rabbits. Injections were repeated at intervals of 2-3 weeks. Immune Sera and Immunoglobulin Fractions. Twelve days after the fourth booster injection and periodically thereafter animals were bled from the ear artery. Blood was allowed to coagulate for 2 hr at room temperature and serum was removed and stored in aliquots at -20°C. An immunoglobulin-rich fraction was prepared from some sera by precipitation with ammonium sulfate to 50% saturation. After resuspension of the precipitate in 0.01 M potassium phosphate at pH 8.0, dialysis, and centrifugation, the material was stored frozen. The con-
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
Abbreviation: DHA, dihydroalprenolol. * To whom correspondence and reprint requests should be addressed at: Box 3287, Duke University Medical Center, Durham, NC
this fact.
27710.
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Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Caron et al.
2264
centration of immunoglobulin was measured by absorbance at 280 nm with bovine gamma globulin as the standard. Detection of Antibody Activity. The ability of the immune sera to interact with adrenergic drugs was assessed by direct ligand binding, using the 3-adrenergic antagonist [3H]DHA. As with particulate f-adrenergic receptor preparations, it was found that [3H]DHA bound to immune sera could be separated from free ligand by rapid vacuum filtration on glass fiber filters. We further documented that [3H]DHA binding to immune sera could also be assessed by filtration on Millipore filters, by Sephadex G-50 chromatography (3), or by ammonium sulfate precipitation. These methods appeared to be qualitatively equivalent, and the glass fiber filtration assay was routinely used for the results reported here. [3H]DHA specifically bound was assessed (2, 3) by defining the nonspecific binding as that which was not competed for by 10 ,tM unlabeled (±)-alprenolol or 100 ,M unlabeled (-)-isoproterenol. Most assays were performed with whole antiserum at a final dilution of 1:500 unless otherwise stated. Under these conditions specific [3H]DHA binding represented 95-98% of total binding. RESULTS Characteristics of binding of ,B-adrenergic agents to antisera Initially, immune sera were tested for their ability to interact with the 0-adrenergic receptor by examining their effects on the binding of [3H]DHA to purified or solubilized frog erythrocyte membranes by using the rapid filtration or the Sephadex G-50 chromatography technique. It was then realized that immune sera themselves were capable of binding the ligand [3H]DHA. Of the six animals immunized, all developed detectable [3H]DHA binding activity in their serum. The general binding properties of these six antisera were similar, though differences in detailed binding specificity were found. The binding data presented below were obtained with the antiserum from one animal (no. 349) unless otherwise noted. [3H]DHA binding to these immune serum sites was rapid and rapidly reversible (Fig. 1). At 23°C the rates of association and dissociation were too fast to be determined with any accuracy; that is, within 1 min the whole process was complete. However, at -4oC the rate constant of association ki was found to be 9.5 X 107 min-1 M-1, whereas the rate constant of dissociation k2
was 0.054 min-1. The ratio k2/kl provides an estimate of the value of the dissociation constant (Kd) of these sites for [3H]DHA, 0.6 nM. Scatchard analysis of equilibrium binding data for [3H]DHA to these sites at 230C indicated a saturable binding process and yielded a monophasic curve (Fig. 2). From these data a value of Kd for [3H]DHA binding of 2-4 nM was obtained. The Kd value obtained by kinetic analysis was in close agreement with the value of 0.7 nM obtained for (-)-alprenolol by binding competition (Table 1). The Kd value derived by equilibrium binding (2-4 nM) was determined on an earlier bleed of the immune serum and correlated well with the potency of (-)alprenolol binding in that immune serum. The specificity of binding of [3H]DHA to these sites was examined by measuring the ability of agonists and antagonists to compete for [3H]DHA binding. As shown in Fig. 3, fadrenergic agonists were potent competitors of binding, with a potency order (-)-isoproterenol > (-)-epinephrine > (-)norepinephrine that is identical to the order of potency for competition of binding to the 3-adrenergic receptor. Binding also displayed the stereoselectivity that characterizes betaadrenergic receptor interactions, because (-)-isorpoterenol competed 10-fold more effectively than (+)-isoproterenol. ,B-Adrenergic antagonists such as (-)-alprenolol and (-)-propranolol (not shown) were also very potent competitors and the (-) isomers of these drugs were also preferentially recognized by these binding sites over their (+) isomers (Fig. 3, Table 1). The detailed specificity of the interaction of several adrenergic agents with immune serum 349 as assessed by competition for [3H]DHA binding is shown in Table 1. Table 1 also compares the Kd values obtained for the antiserum binding sites with those previously obtained for the interaction of these drugs with the membrane-bound 0-adrenergic receptors of frog erythrocytes (12). It can be seen that striking similarities exist between the pharmacological properties of both binding sites. Dopamine, the dopaminergic antagonist haloperidol, and the a0.12
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40
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FIG. 1. Association of [3H]DHA with and dissociation of [;HJDHA from antiserum 349 as a function of time at 0C. Dissociation of bound [3H]DHA was assessed after the addition of 10 ,1M
(±)-alprenolol to incubation mixtures at equilibrium. The data are shown as fmol of specific binding per 50 jul of 1:500 dilution of the antiserum. The association and dissociation rate constants k1 and k2 were calculated as described (4). Results shown are representative of two or three experiments.
0
0.1
0.2
0.3
0.4
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(3HIDHA bound, nM
FIG. 2. Scatchard plot of [3HJDHA binding to antiserum 349. Antiserum at 1:500 final dilution was incubated with increasing concentrations of [3H]DHA (0.3-50 nM) for 30 min at 23°C. Nonspecific binding, which ranged from 5% to 15% of total binding, was determined in parallel incubations in the presence of 10 IM(i)alprenolol. Binding was assessed by filtration. Results are representative of two experiments.
Biochemistry:
Caron et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Trable 1. Dissociation constants (Kd) of various adrenergic agents obtained by binding competition of [3HJDHA
Kd,,UM Frog erythrocyte membrane 3-adrenergic receptors
Antiserum 349
Compounds Antagonists (-)-Alprenolol
0.0034 0.150 0.0046 0.286 0.57
0.0007 0.084 0.001 0.031 0.002
(+)-Alprenolol (-)-Propranolol (+)-Propranolol
(W)-Dichlorisoproterenol
(W)-Practolol (W)-Phentolamine
20.9
0.007 -
Agonists (-)-Isoproterenol (+)-Isoproterenol (-)-Epinephrine (-)-Norepinephrine (-)-Soterenol
0.40 183 4.6 49 0.7 0.06
0.04 0.4 9.3 280 0.006 7.7
(±)-Hydroxybenzyl
isoproterenol Dopamine Carbachol [3H]DHA (8-10 nM) binding assessed by the filtration technique was determined on 50-yl aliquots of 1:100-1:500 diluted antiserum 349 in the presence and absence of five to seven concentrations of each of the drugs shown. Kd values for each drug were calculated from the concentration of the drug that competed for 50% of specific [3H]DHA binding according to the method of Cheng and Prusoff (11), using a dissociation constant of 3.7 nM obtained by Scatchard analysis for [3HIDHA binding to the antiserum. No effect of the drug at concentrations of 100 ,M or effects too slight to permit calculation of a Kd value. Results shown are from one to three experiments performed in duplicate for each drug. Results on the specificity of [3H]DHA binding to erythrocyte membranes were taken from previously published work (12). -,
adrenergic antagonist phentolamine did not interact with the antibody sites at concentrations as high as 10-100 ,M; this is similar to their lack of effect on binding to the fl-adrenergic
2265
receptor. The catechol analogs dihydroxphenylalanine and dihydroxymandelic acid were also without effect at 100 AM. Despite these similarities to the specificity of the 13-receptor, detailed studies revealed a number of striking differences. Thus the 3-antagonists (+)-dichlorisoproterenol and (±)-practolol were, respectively, 300- and 3000-fold more potent in competing for the antibody than for the receptor binding sites, and the agonist (+)-hydroxybenzylisoproterenol was only 1/100th as potent at the antibody sites. Moreover, whereas all antisera [3H]DHA binding activities clearly possessed a fl-adrenergic specificity, each differed from the other on detailed examination (data not shown). Immunoglobulin nature of the [3H]DHA binding sites in immune sera In order to document that [3H]DHA binding activity in the immune sera was in fact due to the presence of immunoglobulin, the following tests were performed. First, [3H]DHA binding activity in the immune sera was quantitatively precipitable by 50% saturation with ammonium sulfate. Second, [3HJDHA binding activity was stable to heating at 560C for 30 min. Third, [3H]DHA binding activity could be fully precipitated by goat antiserum to rabbit IgG (Fig. 4). Fourth, the [3H]DHA binding activity was stable to a reduction of the pH to 3.5 for 10 min. Finally, the binding activity was found to cochromatograph with authentic IgG on Sephadex G-200. None of six preimmune sera contained any [3H]DHA binding activity. Further, several hyperimmune sera obtained in response to other antigens failed to show any binding activity. Similarly, sera from rabbits immunized with isoproterenolcontaining digitonin solutions as well as isoproterenol-containing soluble preparations depleted of f3-adrenergic receptors by affinity chromatography failed to show any [3H]DHA binding activity. These results indicate that the [3H]DHA binding activity observed in these immune sera is due to an immunoglobulin fraction raised in response to the partially purified ,B-adrenergic receptor preparations used as the immunogen. 100 C C
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FIG. 3. Ability of agonists, antagonists, and other compounds to compete for [3HJDHA binding to antiserum 349. Antiserum 349 (50 ,ul of a 1:100 dilution) was incubated with 8 nM [3H]DHA (final volume 500 1l) in the presence and absence of various concentrations of the different drugs shown. The results, expressed as % of total binding (100% control binding was 0.52-0.57 pmol) are from an experiment in duplicate and are representative of three such experiments. ALP,
alprenolol; ISO, isoproterenol; EPI, epinephrine; NE, norepinephrine; DHMA, dihydroxymandelic acid.
01 0
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FIG. 4. Immunoprecipitation of [3HJDHA binding activity in immune serum 349 by goat antiserum to rabbit IgG. A 1:10 dilution of the immune serum was incubated in 25 mM Tris-HCl/2 mM MgCl2, pH 7.4, at 25°C for 24 hr with various concentrations of the anti-rabbit IgG. The precipitate was removed by centrifugation and the supernatant was assayed for [3H]DHA binding (12 nM). Results shown are from an experiment performed in duplicate.
2266
Biochemistry: Caron et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
alo
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00
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Xenopus
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FI(.. 5. Interaction of antiserum 349 with various erythrocyte membrane preparations. Frog, Xenopus, and human erythrocyte membranes were all prepared as described (3,. 4) for the frog erythrocyte membranes. Membranes (1.1-1.4 mg of protein per ml) were incubated at 250C for 11/2 hr in 25 mM Tris-HCl/5 mM MgCI2, pH 7.4, with a 1:50 final dilution of whole antiserum 349 (hatched bars) or normal rabbit serum (empty bars) preheated at 560C for 30 min to inactivate complement. Membranes were washed twice with 5-ml samples of the incubation buffer and [3H]DHA binding was measured on the resuspended membranes by filtration on GF/C glass fiber filters. Membrane preparations incubated with normal rabbit serum showed the same [3H]DHA binding activity as untreated preparations. Results ± SEM shown are the means of 3 different experiments performed in duplicate. These experiments are representative of 8-10 similar experiments.
Interaction of the antibodies with erythrocyte membranes The evidence described thus far indicates that the antibodies raised in response to partially purified preparations of frog erythrocyte f3-adrenergic receptor possess specificity toward catecholamines and related f-adrenergic drugs. Studies were next performed to elucidate whether these immune sera also possessed affinity for components of erythrocyte membranes. As shown in Fig. 5, a portion of the [3H]DHA binding activity present in immune sera was specifically adsorbed by frog erythrocyte membranes, whereas Xenopus erythrocyte membranes as well as human erythrocyte membranes, both of which are essentially devoid of f3-adrenergic receptors, were almost ineffective in adsorbing [3H]DHA binding activity from the antisera. DISCUSSION In this communication we report that antibodies raised in rabbits against partially purified preparations of the frog erythrocyte f-adrenergic receptors appear to recognize adrenergic catecholamines as well as components of the erythrocyte membrane. As evidenced by the results obtained with the specific f-adrenergic radioligand [3H]DHA, it is clear that these antisera binding sites display specificity that bears striking similarities to that of the physiological f3-adrenergic receptors. In addition, the data indicate that the [3H]DHA binding activity in these sera is due to immunoglobulins, because binding was stable to exposure to 560C for 30 min and to acidic conditions (pH 3.5), was precipitated by 50% saturated ammonium sulfate, and was quantitatively immunoprecipitated by goat antiserum to rabbit IgG. None of these properties is shared by either membrane-bound or solubilized f-adrenfi-
ergic
receptors.
The properties of the antibodies described in this communication appear to be unique. Although antibodies have previously been raised to several hormone receptors, such as those for insulin (7), prolactin (6), and acetylcholine (5), in none of these cases did the antibodies themselves mimic the binding specificity of the receptor. In one case an antibody that mimicked the effects of insulin was obtained (13), but this antibody was raised against an anti-insulin antibody. Attempts to raise antibodies to catecholamines (14, 15) or to adrenergic antagonists (16, 17) have to date met with very limited success. This is not surprising in view of the chemical nature of these agents, which are small molecules with molecular weights of only about 300. Thus, in the few cases in which an antibody has been successfully raised against an adrenergic drug or antagonist this has required covalent linkage of the agent to a protein carrier in order to form the immunogen. In addition, the antibodies obtained have possessed very restricted specificities. Antibodies raised to catecholamine analogues have had very low affinities for the native catecholamines (14, 15). In no case have any of these antibodies displayed a specificity even vaguely reminiscent of an adrenergic receptor. What, then, is the explanation for the unique antibodies obtained in the present studies? Several explanations will be considered, though a firm conclusion may not be possible at this time. One possibility is that two separate immunogens have been involved, the receptors and free isoproterenol in the receptor preparations, not removed by the Sephadex G-50 chromatographic step used prior to injection of partially purified receptors. This seems unlikely on several grounds. First, as noted above, the free catecholamine would certainly not be expected to be antigenic by itself. Second, if two totally distinct immunogenic stimuli were involved, the antibodies that can be specifically adsorbed on the erythrocyte membrane and that are presumably directed against a component of the membrane would not be expected to bind [3HJDHA too; the data in Fig. 5 did demonstrate such binding. Another possibility is that the antibodies studied have been raised in response to partially purified receptor alone. This also seems somewhat unlikely. There would be no reason to anticipate that such an antibody, even if directed at the active binding site of the receptor, would itself display a binding specificity that so closely mimics that of the receptor. In fact, a somewhat different or complementary specificity might be
predicted. A third possiblity is that antibodies formed against the receptor have in turn served as antigens leading to the formation of "anti-idiotypic" antibodies, the binding specificity of which now resembles that of the receptor. In this case too, however, the antibodies that can be adsorbed by the erythrocyte membranes would not be expected to be the same as those that can bind DHA. An additional possibility is that the immune sera contain soluble anti-receptor antibody-receptor complexes and that binding found in such sera is due to the presence of receptors. This is unlikely because, in contrast to the antibody [3H]DHA binding sites, soluble fl-adrenergic receptor binding activity is completely destroyed by heating to 560C for 30 min, or by decreasing the pH to 3.5. At low pH, putative antibody-receptor complexes would be dissociated and the receptors then inactivated. Upon chromatography of antiserum 349 on Sephadex G-200, the [3H]DHA binding activity was found to cochromatograph with authentic rabbit IgG. The presence of antibody-receptor complexes would have produced a peak of binding activity of larger apparently molecular size. In addition, the specificity of the antiserum is readily distinguishable from that of the receptors as noted above.
Biochemistry:
Caron et al.
Perhaps the most attractive hypothesis is that the antibodies studied have been formed in response to a complex of isoproterenol bound to the purified receptors. Viewed in this light, the receptor-bound isoproterenol might be immunologically equivalent to a hapten conjugated to a carrier and hence be antigenic (18). That some of the antibodies to the hapten (isoproterenol) also recognize determinants on the carrier (receptor) would thus not be surprising. That the specificity for binding catecholamines and related drugs so closely resembles that of the 3-adrenergic receptor in terms of rank order of potencies, stereospecificity, etc. might reflect the particular conformation in which isoproterenol was held by the receptors. Regardless of the exact mechanism leading to the generation of these unusual antibodies, their further characterization should provide important new insights into f3-adrenergic receptor structure and function. Among the potentially interesting avenues which could be explored are: (i) The possible biological activity of the antibodies. (ii) The use of the antibodies as a tool for purification of the receptors. (iii) The use of the antibodies to probe the structure of the specific adrenergic drug binding site. Because the antibodies are available in much larger quantities than the P-receptors and bind drugs with specificities and affinities similar to those of the receptor, purification and characterization of the antibody binding sites might help shed light on the nature of the structural features that contribute to the specificity of the receptor. (iv) Development of anti-catecholamine antibodies for radioimmunoassay. Another point raised by these studies that deserves mention is that immunization with drugs noncovalently bound to their receptor may be a means of raising antibodies to otherwise nonimmunogenic biologically active small molecules. The expert technical assistance of Miss Laurie Card is greatly appreciated. This work was supported by U.S. Public Health Service. Grants HL16037 and HL20339 and a grant-in-aid from the American Heart Association.
Proc. Natl. Acad. Sci. USA 76 (1979)
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1. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C. & Caron, M. G. (1976) Biochim. Biophys. Acta 457, 1-39. 2. Mukherjee, C., Caron, M. G., Coverstone, M. & Lefkowitz, R.
J. (1975) J. Biol. Chem. 250,4869-4876. 3. Caron, M. G. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251, 2374-2384. 4. Caron, M. G., Srinivasan, Y., Pitha, J., Kociolek, K. & Lefkowitz, R. J. (1979) J. Biol. Chem. 254, 2923-2927. 5. Patrick, J. & Lindstrom, J. (1973) Science 180, 871-872. 6. Shiu, R. P. C. & Friesen, H. G. (1976) Science 192,259-261. 7. Jacobs, S., Chang, K.-J. & Cuatrecasas, P. (1978) Science 200, 1283-1284. 8. Flier, S. J., Kahn, C. R., Roth, J. & Bar, R. S. (1975) Science 190, 63-65. 9. Hall, R., Smith, B. R. & Mukhtar, E. D. (1975) Clin. Endocrinol. 4,213-230. 10. Lindstrom, J. M., Seybold, M. E., Lennon, V. A., Whittingham, S. & Duane, D. D. (1976) Neurology 26, 1054-1059. 11. Cheng, Y. & Prusoff, W. H. (1973) Biochem. Pharmacol. 2g, 3099-3108. 12. Mukherjee, C., Caron, M. G., Mullikin, D. & Lefkowitz, R. J. (1976) Mol. Pharmacol. 12, 16-31. 13. Sege, K. & Petersen, P. A. (1978) Proc. Nati. Acad. Sci. USA 75, 2443-2447. 14. Spector, S., Dalton, C. & Felix, A. M. (1973) in Frontiers in Catecholamine Research, eds. Usdin, E. & Snyder, S. H. (Pergamon, New York), pp. 345-349. 15. Grota, L. J. & Brown, G. M. (1976) Endocrinology 98, 613622. 16. Kawashima, K., Levy, A. & Spector, S. (1976) J. Pharmacol. Exp. Ther. 196,517-523. 17. Hoebeke, J., Vauquelin, G. & Strosberg, A. D. (1978) Biochfm. Pharmacol. 27, 1527-1532. 18. Green, I., William, E. P. & Benacerraf, B. (1966) J. Exp. Med. 123, 859-879.
