Study of interaction between agonists and Asn293 in helix VI of human β2-adrenergic receptor

Article abstract of DOI:10.1124/mol.56.5.909

Previously, we demonstrated the involvement of Asn293 in helix VI of the human β₂-adrenergic receptor in stereoselective agonist recognition and activation. In the present study, we have further explored the role of this residue by synthesizing

Full text of DOI:10.1124/mol.56.5.909

0026-895X/99/050909-08$3.00/0
Copyright © The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY, 56:909–916 (1999).
Study of Interaction between Agonists and Asn293 in Helix VI
of Human ␤2-Adrenergic Receptor
¨
HELENE M. ZUURMOND, JUTTA HESSLING, KLAUS BLUML, MARTIN LOHSE, and ADRIAAN P. IJZERMAN
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Leiden, the Netherlands (H.M.Z., A.P.IJ.); and University of
Wu¨ rzburg, Institute of Pharmacology and Toxicology, Wu¨ rzburg, Germany (J.H., K.B., M.L.)
Received February 23, 1999; accepted July 22, 1999
This paper is available online at http://www.molpharm.org
ABSTRACT
Previously, we demonstrated the involvement of Asn293 in compounds studied. It appeared that one derivative of isoproter-
helix VI of the human ␤2-adrenergic receptor in stereoselective enol, but not of clenbuterol, showed a gain in affinity from the wild
agonist recognition and activation. In the present study, we type to the mutant receptor. This derivative had a methyl substitu-
have further explored the role of this residue by synthesizing ent instead of the usual ␤-OH group in the aliphatic side chain of
derivatives of isoproterenol and clenbuterol, two ␤-adrenergic isoproterenol, compatible with the more lipophilic nature of the
receptor agonists. We analyzed their efficacy and affinity on the leucine side chain. Such a “gain of function” approach through a
wild-type and a mutant receptor (Asn293Leu). Each compound combination of synthetic chemistry with molecular biology, may
had similar efficacy (␶ values) on both the wild-type and mutant be useful to enhance our insight into the precise atomic events
receptor, although ␶ values varied considerably among the eight that govern ligand-receptor interactions.
␤2-Adrenergic receptors serve as a prototypic member of
We thought it worthwhile to further characterize the
the large superfamily of G protein-coupled receptors. Apart atomic details of this stereoselective interaction. Therefore,
from the visual pigment rhodopsin, they were the first to be we synthesized “mutated” derivatives of ␤-adrenergic recep-
cloned (Dixon et al., 1986) and subjected to site-directed tor agonists in the present study and analyzed their effects
mutagenesis (Strader et al., 1988, 1989). The latter two stud- on the wild-type and aforementioned mutant receptor in
ies identified Asp113 in helix III and two serines in helix V which Asn293 was changed for a leucine residue. We rea-
(Ser204 and 207) of the human ␤2-adrenergic receptor as soned that such an approach (mutant ligands for mutant
anchor points for the protonated amine function and the receptors) might lead to a “gain of function” in terms of
catechol group of the endogenous agonist epinephrine, re- affinity. This would provide further physical evidence for a
spectively. In the previous two decades, thorough medicinal direct interaction between a specific receptor residue and a
chemistry had already led to the introduction of an important functional group in the ligand, indeed allowing an interpre-
class of antiasthmatic drugs, all ␤2-receptor agonists, exem- tation at almost the atomic rather than the molecular level of
plified by salbutamol and clenbuterol. The nonselective cat- the ligand-receptor interaction.
echolamine isoproterenol has been and still is the reference
compound when the pharmacology and molecular biology of
␤-adrenergic receptors are the subject of investigation. In a
recent study, we identified Asn293 in helix VI as a residue
that determines the stereoselectivity of catecholamines by
virtue of its interaction with the ␤-OH group in the aliphatic
side chain of this class of compounds (Wieland et al., 1996).
The hypothesis for this interaction was based on the molec-
ular modeling of the ␤2-adrenergic receptor for which we
used the atomic coordinates of bacteriorhodopsin as a tem-
plate. Figure 1 is a visualization of this receptor model,
defining the positions of the four above-mentioned amino
acid residues and the levorotamer of isoproterenol.
Experimental Procedures
Synthesis of Isoproterenol and Clenbuterol Analogs
General Methods and Materials. Schleicher and Schu¨ll DC
Fertigfolien F 1500 LS 254 were used for thin layer chromatography
analysis. Compounds were detected under UV light. For structures
of compounds, see Fig. 2. Column chromatography was performed on
silica gel 60, 230 to 400 mesh (Merck, Darmstadt, Germany). 1H
NMR spectra (300 MHz) were recorded at 25°C with a Bruker WM
300 spectrometer. 13C NMR spectra (50 MHz) were recorded with a
Jeol JNM-FX 200 spectrometer. 1H chemical shifts (␦) are given in
parts per million relative to that of Me4Si [CDCl3, MeOD, or di-
methyl sulfoxide (DMSO)]. 13C chemical shifts are not included but
are available on request from the corresponding author. All mass
experiments were performed on a Finnigan MAT900, equipped with
This study was supported by grants from the European Community (pro-
grams EUROCEPTOR and InverseA).
ABBREVIATIONS: CDCl3, deuterated chloroform; MeOD, deuterated methanol; DMSO, dimethylsulfoxide; CYP, cyanopindolol.
909

910
Interaction of Agonists and Asn293 of ␤2-Adrenergic Receptor
a programmable direct insertion probe. Temperature was raised MeOH (15 ml) was added. The cooling bath was removed and the
from 30 to 300°C in 5 min. Mass spectra were recorded in electron
impact mode with 70 eV. Melting points were uncorrected. All com-
pounds appeared pure on thin layer chromatography, and in 1H
NMR, 13C NMR, and mass spectrometry.
amine (25 mmol) added. Stirring was continued for 2 h during while
the temperature was allowed to rise to room temperature. The mix-
ture was stirred for another 2 h, and 1 N HCl (50 ml) was added. The
organic layer was extracted with an additional 1 N HCl (30 ml). The
aqueous layer was made alkaline with 5 N NaOH and then extracted
with CH2Cl2 (3 ϫ 25 ml). The combined organic layers were dried
(K2CO3) and evaporated. The residue was purified on silica gel (1:0 to
9:1 CH2Cl2/MeOH).
2-(4-Amino-3,5-dichlorophenyl)-1-(tert-butylamino)ethane
(2). Prepared as described above, starting from cyanide 9 and with
tert-BuNH2 as the amine in a yield of 45%. 1H NMR (DMSO) ␦ 1.27
(s, 9H, 3ϫ CH3-tert-Bu), 2.78 (m, 2H, CH2NH), 3.02 (m, 2H,
CH2CH2NH), 7.21 (s, 2H, Harom).
4-Amino-3,5-dichlorobenzylcyanide (9). To a stirred solution
of commercially available 4-aminobenzylcyanide (0.5 g, 3.8 mmol) in
acetic acid (10 ml) was added dropwise N-chlorosuccinimide (1.1 g,
8.4 mmol) in acetic acid (5 ml). After 17 h at room temperature, the
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in CH2Cl2 (25 ml) and extracted with water (10
ml), dried (MgSO4), and concentrated. The residue was purified by
column chromatography on silica gel (CH2Cl2) to give pure 9 (0.46 g,
60%). 1H NMR (MeOD) ␦ 3.65 (s, 2H, CH2CN), 7.15 (s, 2H, Harom).
MS: m/z 200 (Mϩ).
The hydrochloride salt of compound 2 was prepared from the free
base. The solid was recrystallized (MeOH-CH2Cl2): mp 224–226°C.
MS: m/z 260 (Mϩ), and used as such in the test systems.
2-(4-Amino-3,5-dichlorophenyl)-1-(tert-butylamino)propane
(3). Prepared as described above, starting from cyanide 10 and with
tert-BuNH2 as the amine in a yield of 51%. 1H NMR (CDCl3) ␦ 1.07 (s,
4-Amino-3,5-dichloro-␣-methylbenzylcyanide (10). Com-
pound 9 (0.4 g, 2.0 mmol) was dissolved in tert.-BuOH (10 ml). To this
solution was added potassium tert-butanolate (0.2 g, 2 mmol) dis-
solved in tert-BuOH (5 ml), and subsequently MeI (0.3 g, 2.2 mmol).
After 4 h at room temperature, the reaction mixture was diluted with
CH2Cl2 (20 ml) and the organic layer extracted with water (10 ml),
dried (MgSO4), and concentrated. Purification of the remaining oil on 9H, 3 ϫ CH3-tBu), 1.21 (d, 3H, CH3, J ϭ 6.4 Hz), 2.66 (m, 2H, CH2NH),
silica gel [1:0 to 0:1 light petroleum (bp 40–60°C)/ether] afforded 10
(0.3 g, 67%). 1H NMR (CDCl3) ␦ 1.54 (d, 3H, CH3, J ϭ 7.2 Hz), 3.60
(q, 1H, CH), 7.18 (s, 2H, Harom).
2.69 (m, 1H, CHCH2NH), 7.05 (s, 2H, Harom).
The hydrochloride salt of 3 was prepared from the free base. The
solid was recrystallized (MeOH-diisopropylether): mp 190–193°C.
MS: m/z 274 (Mϩ), and used as such in the test systems.
2-(3,4-Dimethoxyphenyl)-1-(iso-propylamino)ethane (12). Pre-
pared as described above, starting from dimethoxybenzylcyanide (Brus-
see et al., 1978) and with iPrNH2 as the amine in a yield of 89%. 1H
NMR (CDCl3) ␦ 1.05 (d, 6H, 2ϫ CH3-iPr, J ϭ 6.2 Hz), 2.71–2.89 (m, 5H,
CH2CH2NH, CH2NH, CH-iPr), 3.86, 3.87 (2ϫ s, 6H, 2ϫ OCH3), 6.75
(dd, 1H, Harom, Jo 8.2 Hz, Jm 1.9 Hz), 6.80–6.85 (m, 2H, Harom).
2-(3,4-Dimethoxyphenyl)-1-(iso-propylamino)propane (13).
Prepared as described above, starting from cyanide 11 and with
iPrNH2 as the amine in a yield of 91%. 1H NMR (CDCl3) ␦ 1.02 (d,
3H, CH3-iPr, J ϭ 6.2 Hz), 1.05 (d, 3H, CH3-iPr, J ϭ 6.3 Hz), 1.25 (d,
3H, CH3, J ϭ 7.2 Hz), 2.42–2.95 (m, 4H, CHCH2NH, CH2NH, CH-
iPr), 3.85, 3.87 (2 ϫ s, 6H, 2 ϫ OCH3), 6.78 (dd, 1H, Harom, Jo 8.1 Hz,
Jm 2.1 Hz), 6.70–6.75 (m, 2H, Harom).
3,4-Dimethoxy-␣-methylbenzylcyanide (11). A solution of po-
tassium tert-butanolate (8.3 g, 73 mmol) in tert-BuOH (100 ml) was
added at once to a solution of commercially available 3,4-dimethoxy-
benzylcyanide (10 g, 57 mmol) and MeI (13.3 g, 57 mmol) in tert-
BuOH (100 ml). The temperature was maintained at 35–38°C for 60
min. Then the reaction mixture was concentrated and the residue
diluted with CH2Cl2 (250 ml). The organic layer was washed with
water (125 ml), dried (MgSO4), and concentrated to give 11 (13 g,
85%). 1H NMR (CDCl3) ␦ 1.06 (t, 3H, CH3, J ϭ 7.2 Hz), 3.67 (t, 1H,
CH, J ϭ 7.2 Hz), 3.86 (s, 6H, 2ϫ OCH3), 6.83 (dd, 1H, Harom, Jo 8.1
Hz, Jm 2.0 Hz), 7.25 (d, 1H, Harom, Jm 2.0 Hz), 7.28 (d, 1H, Jo 2.0 Hz).
General Procedure for One-Pot Reduction-Transimina-
tion-Reduction. To a cooled (Ϫ70°C) solution of cyanide (5 mmol) in
dry ether (40 ml) was added 1 M diisobutylaluminum hydride in
cyclohexane (10 ml, 10 mmol). After stirring at Ϫ70°C for 3 h, dry
Fig. 1. Model visualizing the binding of
(Ϫ)-isoproterenol to the human ␤2-adren-
ergic receptor. The receptor ␣-helical
backbone (helices I-VII) is viewed from
the extracellular side. (Ϫ)-Isoproterenol
and the receptor residues thought to be
involved in ligand binding are shown in
red and yellow (Asp113, Ser204, Ser207,
and Asn293, respectively).

Zuurmond et al.
911
General Procedure for Demethylation. The dimethoxy deriv- position 293 from asparagine to leucine) (Wieland et al., 1996) were
ative (2 mmol) was dissolved in 48% HBr (10 ml) and the solution grown in monolayers on Dulbeccos’s modified Eagle’s medium
was refluxed for 17 h. Then the solvent was evaporated and the last (DMEM F-12) supplemented with 10% fetal calf serum, 2 mM glu-
traces of the solvent were removed with the aid of repeated addition and
evaporation of toluene (3 ϫ 25 ml). The residue was purified by column
chromatography on silica gel (EtAc/MeOH/NH4OH, 85/15/1, v/v).
tamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin (all purchased
at Pan Systems, Berlin, Germany) and 0.5 mg/ml of G418 (Gibco
Laboratories, Eggenstein, Germany) in a 7.5% CO2 incubator at
2-(3,4-Dihydroxyphenyl)-1-(iso-propylamino)ethane (6). Pre- 37°C. Clones with comparable densities (ϳ0.2 pmol/mg membrane
pared as described above, starting from dimethoxy derivative 12 in a protein) were selected for the experiments.
yield of 52%. 1H NMR (DMSO) ␦ 1.21 (d, 6H, 2 ϫ CH3-iPr, J ϭ 6.6 Hz),
Preparation of Crude Cell Membranes
2.72 (m, 2H, CH2NH), 3.02 (m, 2H, CH2CH2NH), 3.30 (q, 1H, CH3-iPr,
J ϭ 6.2 Hz), 6.49 (dd, 1H, Harom, Jo 8.0 Hz, Jm 2.1 Hz), 6.63 (d, 1H,
Harom, Jm 2.1 Hz), 6.67 (d, 1H, Harom, Jo 8.0 Hz). MS: m/z 195 (Mϩ).
The fumaric acid salt of 6 was prepared from the free base. The
solid thus obtained was Ͼ97% pure and was used as such in the test
systems.
2-(3,4-Dihydroxyphenyl)-1-(iso-propylamino)propane (7). Pre-
pared as described above, starting from dimethoxy derivative (com-
pound 13) in a yield of 63%. 1H NMR (MeOD) ␦ 1.28 (d, 3H, CH3, J ϭ
6.1 Hz), 1.30 (d, 6H, 2ϫ CH3-iPr, J 6.2 Hz), 2.95 (m, 1H, CHCH2NH),
3.12 (m, 2H, CH2NH), 3.32 (q, 1H, CH-iPr, J ϭ 6.2 Hz), 6.62 (dd, 1H,
Harom, Jo 8.1 Hz, Jm 2.2 Hz), 6.72 (d, 1H, Jm 2.2 Hz), 6.76 (d, 1H, Jo 8.1
Hz). MS: m/z 209 (Mϩ).
Cells were washed three times with ice-cold phosphate-buffered
saline, scraped in 50 ml of ice-cold lysis buffer (5 mM Tris-HCl, 2 mM
EDTA, pH 7.4), homogenized with an Ultra Turrax for 30 s at full
speed and centrifuged at 1000g for 10 min (4°C). The supernatants
were then centrifuged at 50,000g for 15 min (4°C), and the resulting
pellets were resuspended in an appropriate volume of incubation
buffer (50 mM Tris-HCl, pH 7.4) to give a final concentration of 2
mg/ml. Protein content was determined by the method of Bradford
(1976) with bovine serum albumin (Sigma Chemical Co., Deisen-
hofen, Germany) as the standard.
Radioligand Binding Assay
The fumaric acid salt of 7 was prepared from the free base. The
solid thus obtained was Ͼ97% pure and was used as such in the test
systems.
For inhibition assays, 0.1 ml of fresh membranes was incubated in
triplicates with 50 pM 125I-cyanopindolol (125I-CYP) and various
concentrations of isoproterenol/clenbuterol (Sigma Chemical Co.)
and their derivatives (100 nM–1 mM) in the presence of 100 ␮M
Gpp(NH)p (Sigma Chemical Co.) for 1 h at 37°C. The latter addition
was used to uncouple the ␤2-adrenergic receptors from Gs and
thereby generate monophasic competition curves for agonists. Non-
specific binding was determined in the presence of 10 ␮M (Ϫ)-pro-
pranolol (Sigma Chemical Co.). The incubations were terminated by
filtration through Whatman GF/C filters and washing with ice-cold
50 mM Tris-HCl, pH 7.4.
2-(4-Amino-3,5-dichlorophenyl)-2-[(methyl)oxy]-1-(tert-bu-
tylamino)ethane (4). Clenbuterol (0.5 g, 1.8 mmol) was suspended in
MeOH (10 ml) and hydrogen chloride was passed in with stirring at
10°C until a clear solution resulted. The reaction mixture was concen-
trated and the hydrochloride salt thus obtained (87% yield) was recrys-
tallized (MeOH-diisopropylether): mp 212–214°C. 1H NMR (MeOD) ␦
1.34 (s, 9H, 3 ϫ CH3-tBu), 3.07 (m, 2H, CH2NH), 3.28 (s, 3H, OCH3),
4.28 (t, 1H, CHCH2NH, J ϭ 5.8 Hz), 7.23 (s, 2H, Harom). MS: m/z 290
(Mϩ).
2-(3,4-Dihydroxyphenyl)-2-[(methyl)oxy]-1-(isopropylamin-
o)ethane (8). Prepared as described for compound 4 starting from
(Ϯ)-isoproterenol in a yield of 85%. The hydrochloride salt thus
obtained was Ͼ97% pure and was used as such in the test systems.
1H NMR (MeOD) ␦ 1.33 (d, 3H, CH3-iPr), J ϭ 6.5 Hz), 1.35 (d, 3H,
CH3-iPr, J ϭ 6.1 Hz), 3.05 (dd, 1H, CHCHHNH, J ϭ 12.9 Hz, J ϭ 3.5
Hz), 3.13 (dd, 1H, CHCHHNH, J ϭ 12.9 Hz, J ϭ 10.3 Hz), 3.25 (s, 3H,
OCH3), 3.31 (q, 1H, CH-iPr), 4.35 (dd, 1H, CHCH2NH, J ϭ 3.5 Hz,
J ϭ 10.3 Hz), 6.70 (dd, 1H, Harom, Jo 8.1 Hz, Jm 1.9 Hz), 6.80 (d, 1H,
Harom, Jm 1.9 Hz), 6.81 (d, 1H, Harom, Jo 8.1 Hz). MS: m/z 225 (Mϩ).
Adenylyl Cyclase Assays
Adenylyl cyclase activity was determined as described in Wieland
et al. (1996). Crude cell membranes were prepared freshly immedi-
ately before the assay as described above. Incubations contained 40
to 50 ␮g of protein; 50 mM Tris-HCl, pH 7.4; 500 ␮M RO 20–1724;
100 ␮M cAMP; 2 mg/ml BSA; 1 mM MgCl2; 5 mM creatine phos-
phate; 0.4 mg/ml creatine kinase, 1 ␮M GTP; 100 ␮M [␣-32P]ATP (0.2
␮Ci/tube; Amersham Corp., Braunschweig, Germany); and the de-
sired concentrations of the test compounds in a final volume of 100
␮l. Incubations were done for 30 min at 30°C. Adenylyl cyclase
activities measured in the presence of 100 ␮M (Ϫ)-isoproterenol were
set to 100%. Basal activities were not subtracted.
Expression of Wild-Type and Mutant ␤2-Adrenoceptors in
Stable CHO Cells
UV-Spectrometry
CHO cell lines stably expressing either wild-type or mutated ␤2-
adrenoceptor cDNA (encoding a single point mutation at amino acid
Isoproterenol and clenbuterol (100 ␮M in 0.1 M KCl) were ana-
lyzed at 25°C, under N2 atmosphere and in the dark, essentially to
prevent oxidation of isoproterenol during measurements. Solutions
were adjusted to either pH 5 or pH 11 by addition of minute amounts
of HCl or NaOH, respectively. UV spectra were recorded on an
Aminco DW-2A UV/VIS spectrophotometer.
Molecular Modeling
The receptor-ligand model presented in Fig. 1 was developed ex-
actly as described in Wieland et al. (1996). The figure was generated
with programs MOLSCRIPT and RASTER3D.
Data Analysis
Determination of ligand-binding parameters was performed by
nonlinear curve fitting with SCTFIT and plotted with KALEIDA-
GRAPH, respectively. KD values of 125I-CYP were 7.1 and 7.3 pM for
the wild-type and mutant receptor, respectively (Wieland et al.,
1996).
Fig. 2. Structural formulae of clenbuterol and isoproterenol and their
derivatives used in this study.
Concentration-response curves of adenylyl cyclase experiments

912
Interaction of Agonists and Asn293 of ␤2-Adrenergic Receptor
were fitted to the operational model developed by Black and Leff suggesting an improved fit of this ligand on the mutant over
(1983) as described in Lohse (1990) to obtain an estimate of the
transducer ratio ␶:
the wild-type receptor.
Next, activation of adenylyl cyclase in the cell membranes
was studied. Concentration-response curves of the various
ligands were obtained on membranes containing either the
wild-type or the N293L mutant receptors. In both types of
membranes, (Ϫ)-isoproterenol stimulated adenylyl cyclase
activity by 10- to 15-fold, and in agreement with earlier data
(Wieland et al., 1996), there was no difference in the extent of
stimulation between wild-type and mutant receptors. How-
ever, the position of the isoproterenol stimulation curve was
shifted to the right by more than one order of magnitude in
the mutant receptors (Fig. 5). Smaller rightward shifts (2–3-
fold) were seen with isoproterenol derivatives 6 and 8. Again,
E ϭ Em ϫ ␶A/͓͑KA ϩ A͒ ϩ ␶A͔
with E denoting the effect, Em the maximum possible effect, A the
agonist concentration, and KA the dissociation constant of the ago-
nist-receptor complex. The transducer ratio ␶ is a parameter describ-
ing the signal transduction efficacy of the system and is estimated
from the fit of the data.
Results
Chemistry. The clenbuterol derivatives 2–4 were pre- the exception was the isoproterenol methyl derivative 7, dis-
pared as follows. First, commercially available 4-aminoben- playing an ϳ3-fold leftward shift. Both clenbuterol (1) and its
zylcyanide was chlorinated with N-chlorosuccinimide to give methoxy analog 4 showed a small leftward shift as well; it
its 3,5-dichloro derivative 9, which was converted into the proved impossible to estimate EC50 values for the other low-
desired end product 2 by a one-pot reduction/transimination/ efficacy clenbuterol derivatives 2 and 3.
hydride reduction sequence in a yield of 45% (Zandbergen et
al., 1992). Methylation of derivative 9 with methyl iodide in
the presence of potassium tert-butoxide gave methyl deriva-
tive 10 in a yield of 67%. Conversion of the latter into clen-
buterol analog 3 was achieved by the one-pot reduction/transimi-
nation/hydride reduction sequence. The protected isoproterenol
analogs 12 and 13 were likewise obtained. Demethylation of
analogs 12 and 13 in aqueous HBr gave the unprotected
isoproterenol analogs 6 and 7. The methoxy derivatives 4 and
8 were synthesized by HCl/MeOH treatment of clenbuterol
and isoproterenol, respectively.
Biology. The parent compounds clenbuterol and isoproter-
enol and their derivatives modified at the ␤-OH position (Fig.
2) were tested in radioligand binding studies and adenylate
cyclase assays. For that purpose the wild-type ␤2-adrenergic
receptor was expressed in CHO cells and membranes were
prepared, as was done for a mutant ␤2-adrenergic receptor in
which an asparagine residue in the sixth transmembrane
domain was changed into a leucine (N293L) (Wieland et al.,
1996). All clenbuterol and isoproterenol derivatives were ca-
pable of displacing 125I-CYP, a radiolabelled antagonist, from
both wild-type and mutant receptors (Fig. 3 and Table 1). The
Ki values listed in Table 1 show that the methoxy analogs 4
and 8 had highest affinity on the wild-type receptor among
the derivatives. All derivatives had lower affinities for the
wild-type receptor than their parent compounds isoprotere-
nol and clenbuterol. This latter observation held also on the
mutant receptor. Here, the methyl derivative 7 was most
potent among the isoproterenol derivatives, whereas both the
unsubstituted (2) and methoxy derivative 4 were the most
potent clenbuterol analogs.
More interesting, however, were the differential affinities
at the two receptors, as quantified by the ratios given in Fig.
4. Apparently, clenbuterol and each of its analogs had essen-
tially equal affinity for wild-type as well as mutant receptor
because all ratios were equal or close to unity. However,
results were different for isoproterenol and its analogs. In
our previous study (Wieland et al., 1996; data also in Table 1)
Fig. 3. Stereospecific binding of isoproterenol derivatives to CHO cell
it had been observed that (Ϫ)-isoproterenol, but not clen-
buterol, lost its stereoselective recognition of the receptor
upon the N293L mutation, due to the decrease in affinity on
the mutant receptor of this stereoisomer. A 6-fold gain in
membranes containing wild-type and mutant ␤2-adrenergic receptors.
Data are means Ϯ S.E. (n ϭ 3). Top, competition for 125I-CYP binding to
the wild-type ␤2-adrenergic receptor by various isoproterenol derivatives
(6, E; 7, F; 8, f). Bottom, competition for 125I-CYP binding to the mutant
N293L ␤2-adrenergic receptor by various isoproterenol derivatives (6, E;
affinity, however, was observed for the methyl derivative 7, 7, F; 8, f).

Zuurmond et al.
913
Intrinsic activities were estimated from these data by fit- buterol are virtually identical, whereas at pH 5 the UV
spectra of both do not match.
ting them to the operational model as described in Experi-
mental Procedures (the fitted curves shown in Fig. 5 were
obtained with this model) to obtain the transducer ratios ␶.
These values are depicted in Fig. 6. There were no obvious
differences in results obtained for wild-type and mutant re-
ceptors. Compared with (Ϫ)-isoproterenol, all compounds
were partial agonists, and the extent of agonism was very
similar for wild-type and mutant receptors. Clenbuterol (1)
had a slightly higher ␶ value in the mutant receptors,
whereas those of 6 and 7 were slightly lower. These data
indicate that there was no correlation between the alter-
ations in affinities as seen in the binding experiments and
the intrinsic activities.
UV-Spectroscopy. The UV spectra of isoproterenol and
clenbuterol were recorded at two pH values. At pH 5, the
aromatic ring systems of both compounds are neutral,
whereas at pH 11 the catechol ring of isoproterenol is nega-
tively charged, and no changes occur in clenbuterol’s ring
system (IJzerman et al., 1984). As a result, the UV spectra of
clenbuterol at both pH values are almost coinciding (Fig. 7A),
whereas isoproterenol’s spectra are different (Fig. 7B). Inter-
estingly, at pH 11 the spectra of isoproterenol and clen-
Discussion
In Fig. 1, a possible binding mode of (Ϫ)-isoproterenol, the
prototypic ␤-adrenergic receptor agonist, to the human ␤2-
adrenergic receptor is visualized. This representation is en-
tirely based on the receptor model developed by Wieland et
al. (1996), but viewed from a different angle. In that previous
study, we demonstrated the involvement of Asn293 in the
stereospecific agonist recognition and activation of the hu-
man ␤2-adrenergic receptor, through its interaction with the
␤-OH group in the ligand side chain (see also Fig. 2). In
particular, catecholamines such as isoproterenol proved
highly sensitive to a mutation from asparagine to leucine on
this position. The mutant receptor displayed strongly dimin-
ished affinity for the active (Ϫ)-stereoisomer of isoproterenol,
whereas the (ϩ)-isomer was less affected. However, clen-
buterol, a structurally dissimilar agonist (Fig. 2), did not
appear to suffer from the mutation.
In the present study, we focused on this apparent discrep-
TABLE 1
Affinities of clenbuterol/isoproterenol and their derivatives (all
racemates, except (Ϫ)-isoproterenol) for wild-type and N293L mutant
␤2-adrenergic receptors
The affinities were determined in competition experiments with 50 pM 125I-CYP.
The data were analyzed by nonlinear curve fitting to obtain estimates for the affinity.
Data are means Ϯ S.E., n ϭ 3.
Affinity
Compound
Wild-type
N293L
Ki, ␮M
1 ((Ϯ)-clenbuterol)
0.024 Ϯ 0.008a
3.3 Ϯ 1.1
3.1 Ϯ 0.9
1.6 Ϯ 0.5
0.28 Ϯ 0.07a
28 Ϯ 9.1
0.025 Ϯ 0.007a
2.3 Ϯ 0.4
2
3
4
7.9 Ϯ 1.8
2.3 Ϯ 0.2
5 ((Ϫ)-isoproterenol)
2.5 Ϯ 0.4a
29 Ϯ 1.2
6
7
8
39 Ϯ 14
6.4 Ϯ 1.1
24.5 Ϯ 1.6
12 Ϯ 0.1
a Data were taken from Wieland et al. (1996).
Fig. 5. Concentration-response curves (adenylyl cyclase activation) for all
compounds on both wild-type and N293L mutant ␤-adrenergic receptors.
Maximal stimulation by (Ϫ)-isoproterenol was set to 100%. A typical
experiment is shown, which was repeated at least three more times in
duplicate with similar results.
Fig. 4. Affinity ratios (Ki, wild-type/Ki, N293L) for all eight compounds, cal-
culated from the data in Table 1. Data are means Ϯ S.E. (n ϭ 3).

914
Interaction of Agonists and Asn293 of ␤2-Adrenergic Receptor
ancy at the agonist ligand binding site in more detail. By chain (Fig. 2). Radioligand binding studies and adenylate
“mutating” isoproterenol and clenbuterol, we sought to com- cyclase assays on both wild-type and mutant receptors were
plement our analysis of the receptor-agonist interaction. performed with the two parent compounds and their deriva-
Hence, we synthesized a number of isoproterenol and clen-
buterol analogs, all varied at the ␤-OH position in the side
tives. We reasoned that the mutation of an asparagine resi-
due into a leucine would cause a change in the characteristics
of part of the ligand binding site from essentially hydrophilic
with hydrogen-bonding capacity to far more lipophilic. As a
consequence, certain analogs of isoproterenol should be able
to recognize the mutant receptor better than the wild-type
receptor, and we hypothesized that replacement of the ␤-OH
group in the aliphatic side chain of the ligand by a more
lipophilic group might do in this respect. Such a gain of
function also would complement and corroborate the findings
in our previous study (Wieland et al., 1996), and, moreover,
help us in appreciating atomic rather than molecular mech-
anisms in receptor recognition.
Indeed, we succeeded in making an isoproterenol deriva-
tive (7) that displayed higher affinity for the mutant receptor
(Table 1; Fig. 4). Apparently, a methyl substituent (7) instead
of a hydrogen atom (6) or a methoxy group (8) is an appro-
priate point of interaction for the leucine side chain. The
affinity of 7 for the N293L mutant receptor was comparable
to isoproterenol’s affinity, given the fact that 7 is a racemate
and isoproterenol was tested as the active enantiomer only.
Thus, the ϳ100-fold affinity difference on the wild-type re-
ceptor between isoproterenol and its methyl derivative 7 was
fully canceled on the mutant receptor. Interestingly, 7 did not
prove superior to isoproterenol on the mutant receptor, sug-
gesting that the receptor environment around the ␤-position
in the aliphatic side chain of the ligands remains hydrophilic
in nature, despite the presence of the leucine side chain.
It might be argued that such a gain in affinity as observed
for 7 could be due to more indirect effects, e.g., changes in
overall receptor conformation. However, in this case a direct
effect is more probable due to the other observations we
made. First, the two ligands clenbuterol and isoproterenol
behaved differently. The ratios shown in Fig. 4 demonstrate
that hardly any gain or loss of function was found for clen-
buterol and its derivatives, suggesting that Asn293 is not
relevant for the recognition of this ␤-adrenergic receptor
agonist. If the effect observed for isoproterenol were due to
Fig. 6. Transducer ratios ␶ (see Experimental Procedures) for all eight
compounds on both wild-type and N293L mutant receptors. Data are
means Ϯ S.E. (n ϭ 4–5).
Fig. 7. UV spectra of clenbuterol
(A) and isoproterenol (B) at pH 5
and pH 11 (⑀mol: molar extinc-
tion; ␭: wavelength).

Zuurmond et al.
915
general changes in receptor conformation, a similar change without the need to undergo some sort of proton abstraction.
would have been expected for clenbuterol and analogs. Sec- This would make a direct interaction with the two serine
ond, the data from the adenylate cyclase experiments show residues unnecessary, and allow a different orientation of
that the intrinsic activities on the two receptors are almost clenbuterol in the receptor.
similar for each derivative (Fig. 5). Also, the transducer ra-
Interestingly, ␤-receptor antagonists having an oxypro-
tios ␶ (Fig. 6) hardly varied between the wild-type and the panolamine side chain as in propranolol and in the radioli-
mutant receptors. Apparently, the transduction mechanism gand used in the present study (125I-CYP), also show a ste-
for the wild-type and the mutant receptor had not drastically reoselective interaction through their side chain OH group
changed. Such a change would have been more probable if with an asparagine residue, but now in helix VII. This has
the mutant receptor had acquired a different overall confor- been convincingly demonstrated, in particular on several
mation.
subtypes of the 5-hydroxytryptamine receptor, because com-
The mutated ligands were all less effective than their par- pounds such as propranolol and pindolol display nanomolar
ent compounds because maximal cyclase activation was not affinity for these receptors, too (Guan et al., 1992). In a recent
observed (Fig. 5) and their ␶ values were lower (Fig. 6). study, we applied a similar strategy as described above to a
Apparently, the ␤-OH group is preferred over the other sub- series of propranolol analogs (Kuipers et al., 1997). Asn386
stituents for full receptor activation. This finding corrobo- (helix VII) of the human 5-hydroxytryptamine1A receptor
rates the observations in our previous study (Wieland et al., was mutated into a valine rather than a leucine residue. It
1996) that this position is not only important for stereoselec- was found that a propranolol derivative with methoxy in-
tive receptor recognition but also for receptor activation. The stead of hydroxy in the oxypropanolamine side chain showed
adenylyl cyclase experiments confirmed the preference of 7 a 25-fold affinity gain on the mutant receptor. Valine has a
for the mutant receptor. Its EC50 value on the mutant recep- branched side chain with one -CH2 group less than leucine,
tor was 20 Ϯ 9 ␮M compared with 58 Ϯ 5 ␮M on the wild-type which might explain this receptor’s preference for the bigger
receptor.
methoxy group compared with the methyl substituent in
The present study also provides evidence that there is not isoproterenol.
one single agonist binding site on the ␤2-adrenergic receptor,
In summary, our study demonstrates that by simultaneous
based upon the above-mentioned observations that the rec- mutations in both ligands and receptors, gain of function can
ognition of clenbuterol and analogs is different from isopro- be established. To our knowledge, this is the first study of its
terenol and derivatives. Hence, the critical involvement of kind focusing on agonist-receptor interactions. This approach
Ser204 and Ser207 on helix V in agonist binding (Fig. 1), as is helpful to rule out mutations that have indirect effects on
elegantly shown by Strader et al. (1989), may be limited to this interaction; it is a first step in the unraveling of ligand-
catecholamines. These two amino acids are supposed to in- receptor interactions at atomic detail. It is expected that
teract with the two catechol-OH groups via hydrogen bonding future elucidation of the three-dimensional architecture of G
and, as a consequence, the ligand is directed to position its protein-coupled receptors combined with the approach out-
␤-OH group in the vicinity of Asn293. In binding clenbuterol, lined in the present study, i.e., a combination of synthetic
Asn293 does not seem to play a significant role, and, thus, chemistry with molecular biology, will enhance our insight in
Ser204 and Ser207 are hypothesized to be of less importance, the atomic events that govern the ligand-receptor interac-
too. Preliminary experiments in which we analyzed the bind- tion.
ing of a series of agonists to a “double mutant” ␤2-adrenergic
receptor (Ser204Ala, Ser207Ala) strongly corroborated this
notion (data not shown). Isoproterenol was Ͼ100-fold more
Acknowledgments
potent on the wild-type receptor, whereas clenbuterol’s affin-
ity was only slightly diminished on the double mutant recep-
tor (ϳ3-fold).
We thank Dr. T. Costa (ISS, Rome) for helpful discussions.
References
Another line of evidence for this hypothesis came from the
UV experiments (Fig. 7). In hydrogen bonding to the two
serine residues, the two catechol-OH groups in isoproterenol
may undergo a weakening of their O—H bond. This gradual
transition from a fully intact OH group to a situation that
resembles a deprotonated form is mimicked in the UV record-
ings at pH 5 and pH 11, respectively. Under slightly acidic
conditions (pH 5) the catechol structure is intact, whereas
basic conditions (pH 11) promote the dissociation of a proton
from the catechol OH groups (IJzerman et al., 1984). Clen-
buterol’s UV spectrum, unlike that of isoproterenol, is not pH
dependent. The electronic characteristics of the aromatic ring
systems in clenbuterol and isoproterenol (as exemplified by
their UV spectra) are almost coinciding at pH 11. It has been
shown that the electronic features of the aromatic ring sys-
tem in ␤-adrenergic receptor agonists are strongly correlated
to their intrinsic activity (IJzerman et al., 1986). Apparently,
clenbuterol’s dichloro-anilino ring system may have the ap-
propriate characteristics for productive receptor interaction
Black JW and Leff P (1983) Operational models of pharmacological agonism. Proc R
Soc Lond B Biol Sci 220:141–162.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
72:248–254.
Brussee H, Zonneveld EA, Jansen ACA and Gerritsma KW (1978) Syntheses of some
derivatives of dopamine. Arch Pharm (Weinbeim) 311:796–798.
Dixon RAF, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG, Frielle T, Bol-
anowski MA, Bennett CD, Rands E, Diehl RE, Mumford RA, Slater EE, Sigal IS,
Caron MG, Lefkowitz RJ and Strader CD (1986) Cloning of the gene and cDNA for
mammalian ␤-adrenergic receptor and homology with rhodopsin. Nature (Lond)
321:75–79.
Guan X, Peroutka SJ and Kobilka BK (1992) Identification of a single amino acid
residue responsible for the binding of a class of ␤-adrenergic receptor antagonists
to 5-hydroxytryptamine1a receptors. Mol Pharmacol 41:695–698.
IJzerman AP, Bultsma T, Timmerman H and Zaagsma H (1984) The ionization of
␤-adrenoceptor agonists: A method for unravelling ionization schemes. J Pharm
Pharmacol 36:11–15.
IJzerman AP, Bultsma T and Timmerman H (1986) Quantitative evaluation of the
␤2-adrenoceptor intrinsic activity of N-tert-butylphenylethanolamines.
Chem 29:549–554.
J Med
Kuipers W, Link R, Standaar PJ, Stoit AR, Van Wijngaarden I, Leurs R and
IJzerman AP (1997) Study of the interaction between aryloxypropanolamines and
Asn386 in helix VII of the human 5-hydroxytryptamine1A receptor. Mol Pharmacol
51:889–896.
Lohse MJ (1990) Quantitation of receptor desensitization by an operational model of
agonism. J Biol Chem 265:3210–3211.

916
Interaction of Agonists and Asn293 of ␤2-Adrenergic Receptor
Strader CD, Candelore MR, Hill WS, Sigal IS and Dixon RAF (1989) Identification of
two serine residues involved in agonist activation of the ␤-adrenergic receptor.
J Biol Chem 264:13572–13578.
Zandbergen P, Van den Nieuwendijk AMCH, Brussee J and Van der Gen A (1992) A
one-pot reduction-transimination-reduction synthesis of N-substituted ␤-ethano-
lamines from cyanohydrins. Tetrahedron 48:3977–3982.
Strader CD, Sigal IS, Candelore MR, Rands E, Hill WS and Dixon RAF (1988)
Conserved aspartic acid residues 79 and 113 of the ␤-adrenergic receptor have
different roles in receptor function. J Biol Chem 263:10267–10271.
Wieland K, Zuurmond HM, Krasel C, IJzerman AP and Lohse MJ (1996) Involve-
ment of Asn-293 in stereospecific agonist recognition and in activation of the
␤2-adrenergic receptor. Proc Natl Acad Sci USA 93:9276–9281
Send reprint requests to: Adriann P. IJzerman, Ph.D., Receptor Medical
Chemistry, Leiden/Amsterdam Center for Drug Research, P.O. Box 9502,
2300RA Leiden, the Netherlands. E-mail: ijzerman@lacdr.leidenuniv.nl