G-Protein-Coupled Receptor Chromatographic Stationary Phases. 2

Oct 30, 2004 - mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized β2-AR retained its ability to specifically bind ...
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Anal. Chem. 2004, 76, 7187-7193

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G-Protein-Coupled Receptor Chromatographic Stationary Phases. 2. Ligand-Induced Conformational Mobility in an Immobilized β2-Adrenergic Receptor Farideh Beigi,† Khalid Chakir,‡ Rui-Ping Xiao,‡ and Irving W. Wainer*,†

Laboratory of Clinical Investigation and Laboratory of Cardiovascular Science, NIA, NIH, Baltimore, Maryland 21224-6825

Membranes from a HEK-293 cell line expressing the β2adrenergic receptor (β2-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 × 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (Kd) of CGP 12177A (552.6 nM) and (S)propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized β2-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile phase increased the retention of the CGP-12177A as did the addition of the agonist fenoterol. These results indicate that the immobilized β2-AR retained its ability to undergo ligandinduced conformational changes. The data from this study suggest that the immobilized β2-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor. The β-adrenergic receptors (AR) are members of a family of seven transmembrane proteins coupled to G-proteins, G-proteincoupled receptors. GPCRs represent the largest superfamily of all receptor types and account for the major proportion of current drug targets.1 β-ARs are involved in mediating the effects of catecholamines, epinephrine, and norepinephrine, and they play a role in heart disease, obesity,2,3 and stress responses. For * Corresponding author. Fax: 410-558-8409. Phone: (410)-558-8498. E-mail: [email protected]. † Laboratory of Clinical Investigation. ‡ Laboratory of Cardiovascular Science. (1) Drews, J. Science 2000, 287, 1960-1964. (2) Davies, C. H.; Ferrara, N.; Harding, S. E. Cardiovasc. Res. 1996, 31, 152156. 10.1021/ac048910c Not subject to U.S. Copyright. Publ. 2004 Am. Chem. Soc.

Published on Web 10/30/2004

example, increased cardiac output, elevated blood glucose, and vasodilation of systemic blood vessels are some of the common responses associated with activation of β-AR in mammals.4,5 β-ARs share a common activation mechanism with GPCRs in which agonists induce conformational changes, which convert the receptor from the resting to the active state.6,7 Recent studies have extended the two-state model of GPCR activation and suggest that the process proceeds through intermediate states.8-10 Ligandinduced conformational changes in β2-ARs have been studied using fluorescence lifetime analysis and have demonstrated the plasticity of the receptor in the presence or absence of ligands.11 Studying the interactions between a drug and receptor at different receptor conformations can provide key insights into the molecular basis for agonist and antagonist properties. However, while a variety of screening techniques exist for the study of ligand-GPCR interactions, few have been used to study ligandinduced conformational changes. One approach that was used to study these changes was based upon an immobilized fluorescence labeled β2-adrenergic receptor, which was immobilized on a glass surfaces using streptavidin-biotin affinity interactions.12 The agonist-induced conformational changes were monitored using real-time fluorescence microscopy.12 Another possible approach is affinity chromatography using immobilized receptor-based stationary phases. Previous studies with an immobilized P-glycoprotein-based stationary phase has demonstrated that ATP-induced conformational changes in the (3) Pereira, A. C.; Floriano, M. S.; Mota, G. F. A.; Cunha, R. S.; Herkenhoff, F. L.; Mill, J. G.; Krieger, J. E. Hypertension 2003, 42, 685-692. (4) Rohrer, D. H. J. Mol. Med. 1998, 76, 764-772. (5) Nickerson, J. G.; Dugan, S. G.; Drouin, G.; Moon, T. W. Eur. J. Biochem. 2001, 268, 6465-6472. (6) Strader, C. D.; Fong, T. M.; Tota, M. R.; Underwood, D.; Dixon, R. A. Annu. Rev. Biochem. 1994, 63, 101-132. (7) Ghanouni, P.; Steenhuis, J. J.; Farrens, D. L.; Kobilka, B. K. P. Natl. Acad. Sci. U.S.A. 2001, 98, 5997-6002. (8) Swaminath, G.; Xiang, Y.; Lee, T. W.; Steenhuis, J.; Parnot, C.; Kobilka, B. K. J. Biol. Chem. 2004, 279, 686-691. (9) Kobilka, B. K. Mol. Pharmacol. 2004, 65, 1060-1062. (10) Kobilka, B. K. J. Pept. Res. 2002, 60, 317-321. (11) Ghanouni, P.; Gryczynski, Z.; Steenhuis, J. J.; Lee, T. W.; Farrens, D. L.; Lakowicz, J. R.; Kobilka, B. K. J. Biol. Chem. 2001, 276, 24433-24436. (12) Neumann L., Wohland T., Whelan R. J., Zare R. N., Kobilka B. K., ChemBioChem 2002, 3, 993-998.

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transporter can be studied by monitoring changes in the retention volumes of specific markers.13 In the first part of this study, we reported the development of GPCR-based liquid chromatography affinity phases based on the µ and κ subtypes of the opioid receptor.14 These phases were used to examine ligand-receptor interactions, but no attempt was made to determine whether ligand-induced conformational changes occurred. This study reports the synthesis and characterization of a second GPCR-based liquid chromatographic stationary phase containing immobilized membranes obtained from a cell line expressing the β2-AR. The resulting phase could be used for the on-line determination of ligand-receptor binding interactions. In addition, antagonist- and agonist-induced conformational changes in the β2-AR were observed and could be reversed. The results suggest that the immobilized β2-AR stationary phase can be used to probe ligand-receptor binding to the resting and activated states of the receptor and to develop molecular descriptions of the agonist-antagonist interactions with the immobilized β2-AR. EXPERIMENTAL SECTION Materials. Egg phosphatidylcholine and phosphatidylserine lipids were from Avanti Polar Lipids (Alabaster, AL). Fetal bovine serum and 1× phosphate-buffered saline (PBS) was from Bioscource International (Camarillo CA). Phenylmethanesulfonyl fluoride (PMSF), benzamidine, leupeptin, pepstatin A, sodium cholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, n-dodecyl-β-D-maltoside, glycerol, MgCl2, EDTA, Trizmahydrochloride (Tris-HCl), and NaCl were obtained from Sigma Aldrich (St. Louis, MO). All salts were of analytical grade. The radioactive compounds (-)-[3H]-CGP-12177 and (R,S)-[4-3H]propranolol hydrochloride and (()[3H]-fenoterol were purchased from Amersham Biosciences (Boston, MA). The racemates of butoxamine, nadolol, and CGP-12177A were obtained from Sigma Aldrich. (R)- and (S)-propranolol were kindly provided by Dr. H. Y. Aboul-Enein (King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia). Immobilized artificial membrane with phosphatidylcholine (IAM-PC) silica beads (12-µm particle size, 300-µm pore size) were from Regis Chemical Co. (Morton Grove, IL). Nitrocellulose dialysis tubing (cutoff at 10 000 Da) was from Pierce Chemical (Rockford, IL), and the chromatographic glass columns HR 5/2 (i.d., mm/length, cm) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Buffers, All the buffers were prepared at room temperature, the pH was adjusted to 7.4 using HCl (4 M), and the buffers were filtered (0.45 µm) and degassed prior to use. 1. Homogenization buffer: MgCl2 (2 mM), PMSF (1 mM), benzamidine (1 mM), leupeptin (0.030 mM), pepstatin A (0.005 mM), and EDTA (1 mM) in 50 mM Tris-HCl. 2. Solubilization buffer: prepared from the homogenization buffer with the addition of CHAPS or sodium cholate detergent in the presence of 10% of glycerol. 3. Dialysis buffer: EDTA (1 mM), MgCl2 (2 mM), NaCl (300 mM), and PMSF (0.2 mM) in 50 mM Tris-HCl. 4. Chromatographic running buffer: EDTA (1 mM) and MgCl2 (2 mM) in 10 mM Tris-HCl at pH 7.4. (13) Lu, L.; Leonessa, F.; Clarke, H.; Wainer, I. W. Mol. Pharmacol. 2001, 59, 62-68. (14) Beigi, F.; Wainer I. W. Anal. Chem. 2003, 75, 4480-4485.

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5. Lysis buffer: NaCl (500 mM) supplemented with 1% Igepal, 1% deoxycholate, and 2-5% SDS detergents in Tris-HCl (10 mM). Cell Culture and Membrane Preparation. 1. β2-AR in HEK 293 Cells. Replication-defective adenovirus encoding human β2AR was constructed and kindly provided by Dr. R. J. Lefkowitz (Departments of biochemistry and medicine, Duke University Medical Center, Durham, NC.). The cDNA encoding human β2AR was inserted into the E1 region of the adenoviral genome by homologous recombination. Standard viral amplification and CsCl purification methods were used to amplify and purify adenoviruses encoding β2-AR. The multiplicity of viral infection was determined by dilution assay in HEK 293 cells purchased from Qbiogene (Carlsbad, CA). The cells were grown in a 150-mm Petri dish to 90% confluent, and 1-2 mL of the high-glucose Dulbecco’s Eagle medium (HGDMEM) containing an appropriate titer of genecarrying adenovirus was added. The full volume of HGDMEM was supplied after culture for another 1-2 h. The cells were harvested after 24 h, suspended in PBS solution, and frozen at -70 °C until use. 2. Membrane Preparation. The cell suspension was thawed and the amount of protein was determined by a micro BCA method. An aliquot of cell pellet suspension corresponding to 5-7 mg of total protein was taken resuspended in 10 mL of homogenization buffer (pH 7.4), and the membranes were prepared as previously described.14 3. Membrane Immobilization. The IAM-PC chromatographic support (180 mg) and lipids such as PC (80 µM) were added to the supernatant containing the solubilized membranes, and the resulting mixture was stirred at room temperature for 3 h, transferred into a (5-cm length) nitrocellulose dialysis membrane, and placed in 1 L of dialysis buffer at 4 °C for 24 h. The dialysis step was repeated twice using fresh buffer. After dialysis, the mixture was centrifuged at 120g for 3 min, the supernatant was discarded, and the pellet of IAM support containing the immobilized receptor-bearing membranes was collected. The pellet was resuspended in 2 mL of chromatographic running buffer, and the suspension was pumped through a HR 5/2 chromatographic glass column at a flow rate of 0.3 mL/min using a peristaltic pump. The end adaptors were assembled, producing a total gel bed volume of 0.4 mL. The column was stored at 4 °C when not in use. Chromatographic Experiments. 1. Chromatographic System and Conditions. The chromatographic system was as previously described in ref 14. The ligands were injected individually through a FPLC injector into the column in frontal and zonal chromatographic mode, and the eluted ligand was detected online by a radioflow detector. Sample volume was either 20 µL in zonal studies or 5-10 mL in frontal studies in the nanomolar range of concentration applied continuously. The immobilized receptor column was equilibrated with ∼80 mL of running buffer at a flow rate of 0.2 mL/min between each sample injection. All chromatographic experiments were carried out at room temperature. 2. Frontal Chromatography. The running buffer contained either a 0.8 nM concentration of [3H]-CGP or a 0.2 nM concentration of (R,S)-propranolol. The concentrations of the displacer compounds applied in a volume of 5-7 mL were as follows: (S)propranolol, 0.2, 5, 20, 80, and 200 nM; CGP, 0.08, 80, 240, 600, 5000, and 50 000 nM.

3. Zonal Chromatography. In the zonal mode, 70 nM of the displacer ligand was added to the running buffer and 20 µL of a 2 nM solution of [3H]-CGP was injected. 4. Analysis of Frontal Chromatographic Data. The data were analyzed to determine the number of binding sites and the dissociation constant using the nonlinear eq 1, which as been previously described.14,15-18

[M](Vi - Vmin) )

P[M] Kd + [M]

(1)

here Vi is the solute elution volume, Vmin is the elution volume at the saturation point, P is the number of available binding sites, [M] is the concentration of the marker ligand, and Kd is the dissociation constant of the ligand. Assay of Immobilized Protein. 1. Protein Extraction from Immobilized IAM Beads. After the chromatographic experiments, the stationary phases were removed from two columns. A total of 0.8-1 mL of the stationary phase was collected and incubated for 1 h at 25 °C in 5 mL of PBS (10 mM, pH 7.5) supplemented with 1% (w/v) Igepal, 2% (w/v) SDS, 1% (w/v) deoxycholate, and 0.5% (w/v) Triton X-100. This suspension was filtered through glass filter funnel under vacuum, the collected filtrate was mixed with an equal volume of 20% aqueous methanol (v/v), and the resulting solution was evaporated under vacuum at elevated temperature in a Savant Speed Vac (sc110, Thermosavant, NY). The final collected volume was 0.5 mL, and the protein content was determined by the micro BCA method and stored at -20 °C. The proteins in this sample were analyzed for β2-AR content by western blot. 2. Western Blot Analysis. Protein bands representing ∼4 µg of protein obtained from lysates of HEK-293 native cells, HEK293 β2-AR transfected cells lysate, and the extracted protein from the immobilized columns were resolved on a 10% polyacrylamide Tris/Tricine gel (VWR International, Bridgeport, NJ) at maximum 150 V fixed. The separated protein bands were transferred onto a pre-wetted poly(vinylidene difluoride) (PVDF) membrane (BioRad, Hercules, CA) for 60 min at 100 V fixed. The PVDF membrane was incubated in blocking agent overnight at 4 °C and then allowed to react 1 h with rabbit anti-β2-AR antibody (Genex Biosciences, Hayward, CA) diluted to 1:2100 with blocking agent. Specific antigen-antibody complexes were visualized by a 1-h incubation in horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted to 1:4500 with blocking agent and incubated 2-3 min in Chemi-Luminol substrate solution (Santa Cruz Biotechnology) prior to exposure to films for 15-40 s. Protein Assay. The total amount of solubilized membrane proteins and proteins stripped from the stationary phase were determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology). The interference and compatibility of detergents (15) Zhang, Y.; Xiao, X.; Kellar, K.; Wainer, I. W. Anal. Biochem. 1998, 264, 22-25. (16) Kasai, K.; Oda, Y.; Nishikata, M.; Ishii, S. J. Chromatogr. 1986, 376, 3347. (17) Brekkan, E.; Lundqvist, A.; Lundahl, P. Biochemistry 1996, 35, 1214112145. (18) Lundqvist, A.; Lundahl, P. J. Biochem. Biophys. Methods 2001, 49, 507521.

and lipids was checked prior to protein determinations. To eliminate detergent incompatibility with protein assays, the solutions obtained from cell membranes or from the stationary phase were diluted 100-fold with the lysis buffer to a final concentration of (5 mM) NaCl, (0.01%) Igepal, (0.01%) deoxycholate, and 0.020.06% SDS in (0.1 mM) Tris-HCl at pH 7.4. A protein standard series was prepared with bovine serum albumin in the concentration range of 0-20 µg/mL in the above-mentioned lysis buffer. The protein content was determined following the instructions provided in the Pierce BCA protein assay kit as described in ref 14. The amount of protein was calculated by using the Microsoft Excel program. RESULTS AND DISCUSSION Solubilization and Immobilization of Cellular Membranes. Of the initial 5-7 mg of HEK293 cell lysate proteins, ∼50% were removed by ultracentrifugation. The remaining proteins (2.5-3.5 mg) were solubilized in one of the four different detergents; sodium cholate, CHAPS, and CHAPS/DDM (0.05:0.5 and 1:0.3 w/v%). When sodium cholate or CHAPS were used as the detergent, 25% of the membrane proteins were solubilized, while addition of DDM to the CHAPS solution increased this amount to 70%. This was an expected result since it has been previously demonstrated that DDM is an effective detergent for the preparation of GPCR-containing cellular membranes.19 The IAM stationary phase was added to each of the four solutions, and the mixtures were dialyzed. The sodium cholate and CHAPS detergents were efficiently removed from the solutions during dialysis, and 80% of the solubilized proteins were immobilized on the solid support. Thus, the process resulted in immobilization of 20% of the protein contained in the initial membrane suspension. The DDM-containing detergents were not removed during the dialysis process, and less than 10% of the solubilized proteins were immobilized on the IAM support. The IAM supports obtained using the sodium cholate and CHAPS detergents were packed into chromatographic columns and tested for the presence of β2-AR activity using zonal chromatographic techniques with [3H]-CGP as the marker ligand. Both columns displayed retention volumes indicative of specific binding to an immobilized receptor (data not shown). However, the columns rapidly degenerated, and the specific retention was lost after 4 days. Based on these results, either sodium cholate or CHAPS detergents can be used in the preparation of the immobilized β2-AR columns. However, direct dialysis of the solubilized membrane solutions did not produce viable columns. Stabilization of the Immobilized Cellular Membranes. In our previous study, the addition of PC lipids (80 µM) increased the stability of the opioid receptor subtypes immobilized on the IAM support.14 This approach was based on the observations that detergent solubilization of membrane proteins removes lipids that are crucial for the maintenance of membrane integrity and that the membranes can be stabilized by the addition of lipids.20-22 Further, it was also observed that addition of mixed PC/PS (90: (19) De Jong, L. A. A.; Grunewald, S.; Franke, J. P.; Uges, D. R. A.; Bischoff, R. Protein Expression Purif. 2004, 33, 176-184. (20) Banerjee, P.; Joo, J. B.; Buse, J. T.; Dawson, G. Chem. Phys. Lipids 1995, 77, 65-78. (21) Lagane, B.; Gaibelet, G.; Meilhoc, E.; Masson, J.-M.; Cezanne, L.; Lopez, A. J. Biol. Chem. 2000, 275, 33197-33200. (22) Bidlack, J. M.; Abood, L. G. Life Sci. 1980, 27, 331-340.

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Figure 1. Detection of membrane proteins. In panel A, Coomassie Blue-stained SDS-PAGE image for HEK293 native cell lysate (HEK) and β2-AR overexpressing cell lysate (B2). In panel B, western blot image of β2-AR, Lanes (A): HEK-β2-AR cell lysate before immobilization; (B) extracted β2-AR after immobilization; (C) HEK-293 native cell lysate.

10 mol %) lipids increased the stability of the opioid receptors immobilized on the IAM support relative to the use of the PC lipids alone (data not shown). In this study, PC alone and two mixtures of PC/PS lipids were added to a 10-mL suspension containing solubilized membranes and IAM beads and membranes were dialyzed. Frontal chromatography experiments were conducted with the resulting chromatographic phases. The best results were obtained using PC lipids alone where the columns were stable for up to 4-6 weeks. Addition of PS lipids diminished the effect produced by PC lipids, and the columns were stable for only 10 days. These results were independent of whether sodium cholate or CHAPS were used as the detergent. The detrimental effect of the addition of the PS lipids may be a result of the differences between the opioids and β2-AR in the net molecular charge associated with their transmembrane helices. The opioid receptor has net positive charges exposed or adjacent to the transmembrane helices,23 and addition of a negatively charged lipid such as PS may enhance the interaction of the lipids with the receptor. β2-AR has net negative charges in this region,24 and the addition of the negatively charged PS lipid may disrupt the stabilization produced by the interaction of PC with the β2AR. Western Blot Detection of Immobilized β2-AR. After the frontal chromatography experiments had established that β2-AR binding activity was present on the immobilized membrane-based columns, the immobilized proteins from two columns were stripped and 0.32 mg of proteins was recovered. Western blot analysis confirmed the presence of β2-AR in the IAM stationary phase Figure 1. The β2-AR band detected in lane A of Figure 1B before immobilization reveals two distinct bands at molecular masses of approximately 65 and 90 kDa, respectively. The 65kDa band is the monomeric form of β2-AR in HEK transfected cells,25,26 and the 90-kDa band is a highly glycosylated form of (23) EMBL Bioinformatic Harvester, Human Protein: Q8IWW4. (24) EMBL Bioinformatic Harvester, Human Protein: Q8NEQ9. (25) Luckow, V. A.; Summers, M. D. Biotechnology 1988, 6, 47-55. (26) Salahpour, A.; Bonin, H.; Bhalla, S.; Petaja-Repo, U.; Bouvier, M. Biol. Chem. 2003, 384, 117-123.

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monomeric β2-AR, which is similar to a heavy chain of an R-adrenergic receptor.27 The extracted β2-AR in lane B is consistent with the 65-kDa protein band. It is worth noting that the same 65-kDa band was also present in HEK293 native cells, lane C, which endogenously produces the β2-AR. The frontal chromatography of [3H]-CGP on a column containing immobilized HEK293 cellular membranes produced a frontal chromatogram that was displaced by the addition of CGP to the running buffer (data not shown). However, the column was saturated after the addition of 80 nM CGP to the running buffer and an apparent Kd value for CGP was not determined. Based upon these results, it appears that the overexpressed β2-AR and the endogenous β2-AR both contribute to the observed results. Characterization of β2-AR Using Frontal Chromatography. The development of experimental procedures for the isolation and immobilization of the β2-AR-containing membranes was initially followed by measuring the total protein levels. Since no attempt was made to purify the β2-AR, the protein levels were only a crude measure of the effectiveness of the process. Once the procedures for the synthesis of a stable stationary phase were established, the β2-AR phases were characterized using binding activity (expressed as dissociation constant, Kd) and the number of active binding sites ([P]). This was accomplished using frontal chromatography with [3H]-CGP and (R,S)-[3H]-propranolol as the marker ligands. Elution profiles containing front and plateau regions were obtained for both markers and representative chromatograms produced by (S)-propranolol displacing (R,S)-[3H]-propranolol are presented in Figure 2A. The displacement data were analyzed using nonlinear regression, eq 1, and were successfully fitted with R2 ) 0.996 (propranolol) and R2 ) 0.998 (CGP) as in Figure 2B,C. The calculated Kd values and number of binding sites [P] are presented in Table 1. The Kd values calculated using eq 1 were higher than the values reported in the literature.28,29 It should be noted that the previously reported Kd values were obtained using a variety of species, cell lines, or tissues. Therefore, it is difficult to assess the significance of these differences. However, the relative difference between the Kd values of the markers, i.e., (S)propranolol < CGP, was the same for both the chromatographic methods and for the data from the literature, indicating that the chromatographic approach provides at least a qualitative assessment of ligand-β2-AR binding interactions. Previous studies of stationary phases based upon cellular membranes immobilized on the IAM support have demonstrated that a significant portion of the chromatographic retention was the result of strong lipophilic interactions between test ligands and the IAM backbone.30 When [3H]-CGP and [3H]-propranolol were chromatographed on a control column that contained only the IAM support and the added PC lipids, the observed frontal retention volumes were 1.8 and 12 mL, respectively (data not shown), which represented ∼60% of the observed retention on the immobilized β2-AR column. Increasing concentrations of both (27) Bjorklof, K.; Lundstrom, K.; Abuin, L.; Greasley, P. J.; Cotecchia, S.; Biochemistry 2002, 41, 4281-4291. (28) Liang, W.; Mills S. J. Anim. Sci. 2001, 79, 877-883. (29) Kaumann, A. J.; Engelhardt, S.; Hein, L.; Molenaar, P.; Lohse, M.; NaunynSchmiedeberg’s Arch. Pharmacol. 2001, 363, 87-93. (30) Moaddel, R.; Bullock, P.; Wainer, I. W. J. Chromatogr., B. 2004, 799, 255263.

Figure 2. Frontal chromatographic elution profile of (S)-propranolol. (A) Concentrations from right to left with elution volumes (mL) given in parentheses: 0.2 (19.2), 5 (17.4), 20 (17), 80 (16.8), and 200 (16.4) nM, respectively. Similarly for (()-CGP, the concentrations and elution volumes are 0.08 (2.51), 80 (2.4), 240 (2.34), 600 (2.3), and 5000 (2.2) nM, respectively. The nonlinear regression equation related to (B) and (C) is Y ) a(X/b) + X, for (S)-propranolol R2 ) 0.996 and for (()-CGP is R2 ) 0.998.

Table 1. Dissociation Constant, Kd, and Number of Binding Sites [P] Determined by Frontal Affinity Chromatography on Immobilized β2-ARa ligand

[P] (pmol)

Kd (nM)

lit. Kd (nM)

(S)-propranolol (()-CGP 12177A

65.6 114.7

84.3 552.6

0.728 80-20029

a Data presented are from a single β -AR column. Each value is the 2 average of two chromatographic runs, n ) 2.

ligands produced no displacement of the radiolabeled markers, indicating that the observed retention was nonspecific in nature. Thus, the data indicate that the IAM support did contribute to the total retention of CGP but should not interfere with the specific displacement studies and with the calculated Kd values. Zonal Displacement Studies. 1. (R)- versus (S)-Propranolol. Zonal displacement studies were conducted using [3H]CGP as the marker ligand and (R)- and (S)-propranolol as the displacers. The results indicate that (S)-propranolol had a greater effect than (R)-propranolol on the retention of the marker, Figure 3A. This result is consistent with previous studies that have established that (S)-propranolol is a more effective β-AR inhibitor than (R)-propranolol.31-33

Enantiomers have the same physicochemical properties, and the observed enantioselectivity must be a function of specific interactions with the stationary phase. Since the stationary phase is based upon immobilized membranes, the possibility exists that the observed enantioselectively may be due to the binding of propranolol to constituents of the immobilized membrane other than the β2-AR. However, in an immobilized membrane column, this possibility can be controlled by utilizing a marker that is specific for the target receptor. This has been previously demonstrated with a column that contained the neuronal nicotinic acetylcholine receptor, the NMDA receptor, and the GABA receptor, in which each of the receptors could be studied independently by using a specific marker for each receptor.34 CGP is a specific marker for the β2-AR, and therefore, the observed displacements were a function of the propranolol enantiomers binding to the β2-AR. (31) Stoschitzky, K.; Lindner, W. Wiener Medizinische Wochenschrift 1990, 140, 156-162. (32) Stoschitzky, K.; Lindner, W.; Kiowski, W.J. Cardiovasc. Pharm. 1995, 25, 268-272. (33) Caron, M. G.; Lefkowitz, R. J. J. Biol. Chem. 1976, 251, 2374-2384. (34) Moaddel, R.; Cloix, J. F.; Ertem, G.; Wainer, I. W. Pharm. Res. 2002, 19, 104-107.

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Figure 4. Zonal chromatographic elution profile of 1.25 nM [3H]fenoterol on an immobilized β2-AR column: (A) before exposure to fenoterol; (B) after exposure to fenoterol and 15-h washing.

Figure 3. Zonal chromatographic profile of [3H]-CGP on the immobilized β2-AR column (A) displaced by antagonists, in panel A, 200 nM (R)-propranolol (B) and 200 nM (S)-propranolol (C), and in panel B, 70 nM butoxamine (B), 70 nM (S)-propranolol (C), and 70 nM nadolol (D). Table 2. Retention Volume of 70 nM Antagonists Displacing the 2 nM [3H]-CGP Chromatographic Peak (R1 ) 2.8 mL) to the Right (-) or Left (+) upon Zonal Chromatographya

a

ligand

R2 (mL)

∆R ) R1- R2 (µL)

butoxamine (S)-propranolol nadolol

2.95 2.54 2.54

-150 +260 +260

Values are duplicates obtained from two separate columns.

2. β2-AR Antagonists. Zonal displacement studies were also conducted using the β2-AR antagonists (R,S)-propranolol and (R,S)nadolol, Figure 3B. Both compounds significantly reduced the retention of the marker indicating a competitive binding interaction, Table 2. (R,S)-butoxamine has also been identified as an antagonist of the β2-AR. However, instead of decreasing the observed retention of the marker, the addition of (R,S)-butoxamine to the running buffer produced an increase in the retention of CGP, Table 2. Computational studies of the binding of agonists and antagonists at the active site of the β2-AR indicate that propranolol and butoxamine bind to the same pocket on the receptor and interact in essentially the same manner with amino acid moieties within this pocket.35 However, the binding simulations suggested that butoxamine makes an additional strong hydrogen bond with the 7192 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

side chain of an asparagine moiety (N293) at the sixth transmembrane helix (TM6). This interaction improves the binding energy by ∼2.5 kcal/mol but requires an 180° rotation of the N293 side chain. The results from our study suggest that the binding of (R,S)butoxamine to the active site of the immobilized β2-AR may have produced the predicted rotation of the N293 side chain and that dissociation of the (R,S)-butoxamine-β2-AR complex did not result in an immediate reversal of this conformational change. Thus, the marker bound to an altered protein, which, in this case, resulted in an enhanced stability of the CGP-β2-AR complex. The data indicate that the immobilized β2-AR retained its ability to undergo induced conformational changes, but either the solubilization or immobilization or affected the ability of the receptor to rapidly reverse these changes. However, the conformational change was reversible, and after the column was washed for 6 h, the retention volume of CGP returned to its initial level. Fenoterol-Induced Conformational Change in the β2-AR. A chromatogram from a zonal chromatographic study of [3H]fenoterol on a β2-AR column that had not been previously exposed to an agonist contained two distinct peaks, Figure 4. After exposure of the receptor to 100 nM of fenoterol and washing for 15 h, the same [3H]-fenoterol sample was reinjected onto the column and was eluted as a single peak, Figure 4. This result was reproducible and the effect was partially reversed upon 10 h of rinsing. The β2-AR is a dynamic molecule, and its conformational plasticity in the presence and absence of ligands has been demonstrated using fluorescence lifetime analysis.11 The agonistinduced activation of the β2-AR, as well as most GPCRs, is thought to be the result of conformational changes that transform the receptor from a resting to a higher energy active state.8-10 Recent data have extended the existing two-state (resting and active) model of GPCR activation into a multistep process.8-10 The data from the chromatographic studies with fenoterol on the β2-AR column are consistent with agonist-induced conformational changes. The results also suggest that the immobilized cellular membranes contained two populations of the β2-AR, one (35) Freddolino, P. L.; Kalani, M. Y. S.; Vaidehi, N.; Floriano, W. B.; Hall, S. E.; Trabanino, R. J.; Wai Tak Kam, V.; Goddard, W. A., III. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2736-2741.

reflecting the resting state of the receptor and the other the active state, and that the exposure of the β2-AR to an agonist transformed the majority of the receptors into the active state. However, as illustrated by the studies with butoxamine, the conformational changes and the recovery from these changes are slow relative to the rates observed in intact cells. A contributing factor is that the studies were conducted at room temperature. For the β2-AR, the rate of conformational change has been shown to be temperature dependent; i.e., at 37 °C the rate is ∼3-fold higher than at 25 °C.7 A key component in the GPCR activation mechanism is the phosphorylation/dephosphorylation of the G-protein, which also drives the cycling of the receptor between the resting and active states. In this study, the immobilized membranes did not possess the ability to phosphorylate or dephosphorylate G-proteins, indicating that required proteins and cofactors were removed by the solubilization process. This may contribute to the slow reversal of the ligand-induced conformational change. This is supported by the observation that the addition of 5 µM GTP to the running buffer produced a decrease in the retention volume of [3H]-CGP (data not shown). The data suggest that the immobilized β2-AR retained its ability to bind G-proteins and that the binding reduced the affinity of the receptor for CGP. This result is consistent with previous studies with a partially solubilized R2-AR that maintained its ligand binding activity and where the addition of GTP induced a rapid decrease in the affinity for a marker ligand.36,37 CONCLUSIONS The results of these studies demonstrate that membranes obtained from cells expressing the β2-AR have been immobilized on an IAM liquid chromatographic stationary phase. The immobilized β2-AR retained their ability to bind ligands and could be used to qualitatively rank antagonists by their relative Kd values. The immobilized receptors also retained their ability to undergo antagonist- and agonist-induced conformational changes. The later property suggests that the β2-AR columns can be used to selectively study the resting and active states of the receptor and perhaps the intermediate states as well. This possibility is being explored and will be reported elsewhere. (36) Nanoff, C.; Stiles, G. L. J. Recept. Res. 1993, 13, 961-973. (37) Sladeczek, F.; Bockaert, J.; Rouot, B. Biochem. Biophyus. Res. Commun. 1984, 119, 1116-1121.

The retention of the conformational mobility of the immobilized β2-AR is consistent with previous results of studies with the β2AR immobilized on gold and glass surfaces.12 In these experiments, the β2-AR was purified, labeled with a reporter fluorophore at a conformationally sensitive site and immobilized on a surface layered with biotinylated bovine serum albumin and biotinylated M1 antibody. The conformational changes were initiated by the presence of the agonist (-)-isoproterenol and monitored using fluorescence microscopy. The method described in this paper is an alternative approach to the investigation of ligand-β2-AR interactions. It does not utilize a purified receptor or a reporter fluorophore, but it does require a specific marker. While the current studies employed a radiolabeled marker, previous studies have demonstrated that this is not necessary as membranes-based columns can be used in LCMS systems.30 Thus, the use of immobilized membranes obtained from cell lines that express a target receptor in liquid affinity chromatography may be a rapid and general method to screen interactions between potential ligands and the receptor. The data obtained using the parent HEK293 cell line suggests that it may be possible to use endogenously expressed β2-AR rather than overexpressed receptors. In addition, this also raises the possibility that tissues obtained from target organs, such as cardiac tissue, could be used to produce columns for the study of target receptors, such as the β2-AR. The latter possibilities are currently under investigation and the results of these studies will be reported elsewhere. Abbreviations: β-adrenergic receptor (β-AR); G-proteincoupled receptor (GPCR); immobilized artificial membrane (IAM); phosphatidylcholine (PC); phosphatidylserine, (PS); 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS); n-dodecyl-β-D-maltoside (DDM); (-)-[3H]-CGP-12177 ([3H]-CGP); (R,S)-[4-3H]-propranolol HCl (R,S-propranolol); (()[3H]-fenoterol (fenoterol); (()-CGP-12177A (CGP). ACKNOWLEDGMENT We thank Dr. Tienian Zhu for assistance in producing the β2AR expressing cells. Received for review July 26, 2004. Accepted September 14, 2004. AC048910C

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