May 2, 1983 - MARC G. CARON*t, AND ROBERT J. LEFKOWITZ*t. Howard Hughes Medical ...... Stadel, J. M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron,.
Proc. NatL. Acad. Sci. USA Vol. 80, pp. 4899-4903, August 1983 Biochemistry
Reconstitution of j3-adrenergic receptors in lipid vesicles: Affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system (octyl glucoside/Sepharose-alprenolol)
RICHARD A. CERIONE0, BERTA STRULOVICI*, JEFFREY L. BENOVICt, CATHERINE D. STRADER*, MARC G. CARON*t, AND ROBERT J. LEFKOWITZ*t Howard Hughes Medical Institute, Departments of *Medicine (Cardiology), tBiochemistry, and tPhysiology, Duke University Medical Center, Durham, North Carolina 27710
Communicated by Henry A. Lardy, May 2, 1983
Membrane-bound hormone and drug receptors perform two essential functions in transducing extracellular messages into intracellular signals. First, they specifically bind agonist drugs or hormones at the cell surface. Second, they activate specific effectors such as enzymes and ion channels. The binding function of receptors has been probed by radioligand binding techniques (1, 2), which have facilitated the purification of the binding macromolecules of the receptors (3-5). However, the "activating" function of such purified receptors has not generally been amenable to direct study. Receptors coupled with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] have been widely investigated because of their ubiquity and physiological importance. Such receptors provide attractive models because of their close coupling to a well-defined biochemical effector, the adenylate cyclase. The 0-adrenergic receptors (BARs), which mediate stimulation of the enzyme by catecholamines, have been the most thoroughly studied and best characterized of this class of receptors (6).
In recent years the fusion of cells or crude soluble preparations containing BARs with receptor-deficient cells has been reported (7-9). These studies established that receptors from one cell could couple with the adenylate cyclase moiety of another cell, thus indicating that these entities are functionally distinct. Subsequently, unpurified soluble receptor was combined with the purified guanine nucleotide regulatory component of the adenylate cyclase system (10) in lipid vesicles and the ability of catecholamines to activate the nucleotide regulatory component via the receptors was documented. This activation was assessed by the ability of the regulatory protein to increase adenylate cyclase activity in cyc- S49 lymphoma cell membranes (10). Thus these approaches, utilizing crude receptor preparations, have served to establish the functional independence of the receptors and the cyclase and to confirm that the nucleotide regulatory protein is the proximal effector of receptor action. However, to date no experimental approach for investigating the direct coupling of purified receptors to an adenylate cyclase system has been available. The detergents (deoxycholate) and procedures utilized previously (9, 10) have led to loss of the binding function of the receptors, thus precluding receptor purification. An approach to reconstitution of purified receptor preparations that both maintains receptor binding and permits functional coupling to an adenylate cyclase system is necessary if the structure of the recently purified receptors is ultimately to be related to their biological functions. With this aim we set out to develop procedures that would permit the coupling of a purified BAR to a previously unresponsive adenylate cyclase system, thus rendering it sensitive to catecholamine stimulation. Three specific accomplishments were necessary to achieve this goal: first, development of methods for the insertion of solubilized, purified BARs into carrier lipid vesicles; second, selection of a suitable acceptor cell containing an adenylate cyclase system (hence presumably both the catalytic moiety of the enzyme and the nucleotide regulatory protein) but lacking BARs; third, adaptation of previously published technology (9, 11-13) to the task of fusing the receptor-containing lipid vesicles with the acceptor cells. In this communication we report the successful development of such a system, utilizing BARs purified by affinity chromatography from frog erythrocyte membranes and rat lung membranes and the adenylate cyclase system of the Xenopus laevis erythrocyte as the acceptor. These developments open the way for direct
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Abbreviations: BAR, -adrenergic receptor; PtdCho, phosphatidyleholine; Myr2-PtdCho, dimyristoyl PtdCho; OcGlc, octyl -D-glucopyranoside; '25I-CYP, '25I-labeled cyanopindolol; PGE1, prostaglandin E1.
ABSTRACT The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified f8-adrenergic receptor preparations. Digitonin-solubilized J3-adrenergic receptors from frog erythrocytes or rat lung were purified -100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of 8-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted fi-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin El-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few ,3-adrenergic receptors and little or no catecholaminesensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the p3-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted fi-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the 8-adrenergic receptor prior to reconstitution. The successful coupling of a- purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure-function relationships.
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study of the structure of adenylate cyclase-coupled receptors in relation to their biological activating function.
MATERIALS AND METHODS Materials. Dimyristoyl phosphatidylcholine (Myr2-PtdCho), soybean phosphatidylcholine (PtdCho), sodium deoxycholate, fatty acid-free bovine serum albumin, and (-)-isoproterenol were obtained from Sigma. Octyl (3-D-glucopyranoside (octyl glucoside, OcGlc) was obtained from Calbiochem, (±)-alprenolol was purchased from Hassle Pharmaceutical (Mblndal, Sweden), and digitonin was from Gallard Schlesinger (Carle Place, NY). "2I-Labeled cyanopindolol ('"I-CYP) and [3H]dihydroalprenolol were from New England Nuclear. Bio-Beads SM-2 adsorbent was from Bio-Rad and was washed with methanol and water (14) prior to use. Southern grass frogs (Rana pipiens) and Xenopus laevis were obtained from Nasco (Fort Atkinson, WI). Preparation of BAR. Purified frog erythrocyte membranes were prepared as described (15). The BAR was solubilized from erythrocyte plasma membranes (specific activity 1 pmol/mg of protein) and purified =100-fold by affinity chromatography on a Sepharose-alprenolol gel as described (4) but with some minor modifications (final specific activity =100 pmol/mg of protein). BAR activity was typically eluted with 0.025% digitonin/100 mM NaCl/10 mM Tris HCI, pH 7.4, containing 40100 ,uM (±)-alprenolol rather than with a buffer containing 0.1% digitonin and 40 ,uM (±)-alprenolol. In some cases, more highly purified preparations (500- to 750-fold) were obtained by first washing the Sepharose-alprenolol gel with 0.5% digitonin/500 mM NaCl/50 mM Tris HCl, pH 7.4 (cf. Table 2, entry 4). The BAR was solubilized from rat lung membranes and purified (-5,000 fold) by affinity chromatography (unpublished data). BAR eluates were concentrated by using an Amicon concentration cell with a PM 30 membrane to a final concentration of 20-60 pmol/ml, as assayed by [3H]dihydroalprenolol binding (15). The concentrated receptor preparations were stored frozen at -90°C. Unless otherwise specified the receptor used in reconstitution and fusion experiments was that purified from frog erythrocytes to a specific activity of -100 pmol/mg. Reconstitution with BARs. Three different types of lipid preparations were used in the reconstitution experiments. These were Myr2-PtdCho, soybean PtdCho, and a light membrane vesicle fraction of frog erythrocyte lipids. The light membrane vesicle fraction of lipids was prepared as earlier reported (13, 16), essentially by centrifuging the 40,000 X g supernatant derived from a lysate of frog erythrocytes at 158,000 X g for 1 hr. The resulting pellet, which contains negligible quantities of plasma membrane markers and BARs (13) and =0.05 ,umol of lipid per mg of protein is hereafter referred to as "control vesicles." Concentrations of lipids in crude PtdCho and control vesicles are expressed in terms of lipid phosphate as determined by the method of Ames and Dubin (17), using Myr2PtdCho as a standard. Prior to use in reconstitution experiments all lipid preparations were sonicated 5-15 min in 100 mM NaCl/10 mM Tris-HCI, pH 7.4, with a bath-type sonicator (Laboratory Supply, Hicksville, NY). Several methods were examined in attempting to insert the affinity-purified receptor into lipid vesicles. In general, the receptor (1-10 pmol) was incubated with sonicated lipids alone, or with sonicated lipids and a detergent (OcGlc or deoxycholate), in 90 mM NaCl/9 mM Tris HCl, pH 7.4, for 20-60 min at 0°C. The detergent was then removed either by chromatography on Sephadex G-50 columns (0.5 x 13 cm or 1.5 x 16 cm) or by addition of Bio-Beads SM-2 adsorbent (0.2-0.4 g wet weight). The Sephadex G-50 columns were typically equilibrated in 100 mM NaCI/10 mM Tris HCl, pH 7.4/1-4 mg of bovine serum albumin (fatty acid-free) per ml. After removal
Proc. Natl. Acad. Sci. USA 80 (1983)
of detergent, the reconstituted BAR was isolated by centrifugation (usually 250,000 x g for 1.5 hr in a Beckman Ti 50.2 rotor or 250,000-300,000 X g for 1 hr in a Sorvall Ti 75 rotor at 4°C). The specific reconstitution procedure that was used to provide the donor receptor-lipid fraction in fusion experiments was as follows: first, affinity-purified BAR was incubated with fatty acid-free bovine serum albumin (1-2 mg/ml), control vesicles (0.1 mM) or PtdCho (0.4-2.6 mM), Myr2-PtdCho (0.5 mM), OcGlc (0.8-0.9%), 90 mM NaCl, and 9 mM Tris1HCI, at pH 7.4 for 20-45 min on ice. The Bio-Beads SM-2 adsorbent was added and the incubation mixture was stirred end-over-end for 45-60 min at 4°C. The adsorbent was removed by centrifugation for 7 min at 200 x g. The supernatant was then diluted about 1:20 to 1:40 with cold buffer (100 mM NaCl/10 mM Tris HCI, pH 7.4) and centrifuged at 250,000 X g for 1.5 hr. The resultant pellets were either used directly in fusion experiments or resuspended in 1 ml of 100 mM NaCl/10 mM Tris HCI, pH 7.4, and assayed for receptor binding. The fusion of the reconstituted BAR with Xenopus erythrocytes was performed as described in ref. 13. First, the pellets containing reconstituted BARs were mixed with 2 X 107 packed Xenopus erythrocytes. This mixture was then incubated with phospholipids (200 ,ug of PtdCho, 10 ,tg of lyso-PtdCho) for 5 min (10°C) and then with MgCl2 (10 mM) for an additional 5 min. Fusion was then performed at 30°C with polyethylene glycol, essentially as described (9, 11, 12), except using a fusion buffer containing 95 mM NaCl, 5 mM KC1, 4.8 mM MgCl2, 10 mM Tris, 5 mM glucose, and 2 mM ATP (pH 7.5, adjusted at 30°C) (13). After fusion, membranes of the resulting hybrids were prepared as described (18) to assess adenylate cyclase activity. Radioligand Binding Assays. The binding of 125I-CYP to reconstituted BARs was assayed by removing unbound ligand by chromatography on Sephadex G-50 columns (0.5 x 13 cm) equilibrated in 100(mM NaCl/10 mM Tris HCI, pH 7.4/0.05% digitonin (15). Specific binding was defined as the amount of 125I-CYP binding competed for by 200 uM isoproterenol and was typically greater than 90%. Protein Determinations. Protein was determined by the method of Lowry et al. (19). Adenylate Cyclase Assays. Adenylate cyclase assays were performed as described (20, 21) in the presence of 0.1 mM GTP and 0.1 mM ATP. All assay data are reported as pmol of cyclic AMP generated in a 30-min period at 30°C. RESULTS The present work proceeded in two distinct phases. In the initial phase, procedures were developed for the insertion of purified BARs into phospholipid vesicles in a form that would still bind ligands. In the second stage of the work, procedures were developed for the fusion of these vesicles with Xenopus erythrocytes to recouple the BARs with the adenylate cyclase effector. Constraints were placed on the possible reconstitution approaches by the fact that digitonin is the only detergent that solubilizes BARs in a form that binds ligands and that therefore permits purification. The low critical micelle concentration of digitonin makes removal of this detergent, during reconstitution, difficult to achieve. Even small amounts of residual digitonin have been shown to uncouple hormone-stimulated adenylate cyclase. It was also found that high concentrations of phospholipids (>5 mM) strongly inhibited ligand binding to the receptor. The general reconstitution approaches that we used involved incubating purified receptor (in digitonin) with phospholipids alone or with lipids and an additional detergent, rapid removal of the detergents to preserve receptor functionality, and isolation of the receptor-lipid fraction by centrifugation. The ini-
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Proc. Natl. Acad. Sci. USA 80 (1983)
Cerione et aL
tial choice of phospholipids in the reconstitution experiments was Myr2-PtdCho because it was reported that BARs in crude preparations could be inserted into these lipids (10). However, methods entailing simple incubations of receptor with Myr2PtdCho alone or Myr2-PtdCho and detergents, followed by chromatography on Sephadex G-50 gels or Extracti-gel (Pierce) or treatment with Bio-Beads SM-2 to remove detergent, all proved unsuccessful (Table 1, entries 1-6). Although attempts at exchanging digitonin in the receptor preparations with relatively low concentrations of dispersed phospholipids did result in a significant increase in the efficiency of reconstitution (Table 1, entry 7), it has not been possible to successfully couple this approach to a cell fusion procedure. A drawback with this approach is that in order to reconstitute sufficient BAR binding in the lipid fraction it was necessary to use amounts of lipid that Table 1. Comparison of procedures for reconstitution with affinity chromatography-purified BAR Procedure for Efficiency Components of receptor detergent of reconstireconstitution incubation* Detergent removalt tutiont % Lipid 1 1. Myr2-PtdCho None G-50 (5 mM) SM-2
