Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor

Journal of Pharmacological Sciences

J Pharmacol Sci 102, 167 – 172 (2006)

©2006 The Japanese Pharmacological Society

Full Paper

Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor Maruf Ahmed1, Habib Abul Muntasir2, Murad Hossain2, Masaji Ishiguro3, Tadazumi Komiyama4, Ikunobu Muramatsu5, Hitoshi Kurose6, and Takafumi Nagatomo2,* 1

Pharmacy Department, University of Rajshahi, Rajshahi-6205, Bangladesh Departments of 2Pharmacology and 4Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan 3 Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimahon-cho, Mishima-gun, Osaka 618-8503, Japan 5 Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Matsuoka, Fukui 910-1193, Japan 6 Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received June 19, 2006; Accepted August 2, 2006

Abstract. We obtained a new mutant of the β1-adrenergic receptor (β1-AR) by point mutations that can constitutively activate β1-AR. Aspartate104 of the β1-AR in the 2nd transmembrane was replaced with alanine. The β1-AR mutant expressed in human embryonic kidney (HEK)-293 cells displayed high level of constitutive activity with respect to wild-type (P<0.05), which could be partially inhibited by some beta-blockers. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the β1-AR as well as the mechanism of action of beta-blockers. Keywords: β1-adrenoceptor, constitutive activity, overexpression, inverse agonism

humans (for review, see ref. 2). As these mutants are able to activate G proteins in the absence of a ligand, they are useful tools in the study of conformational changes leading to receptor activation and in the drug discovery process. Initial discovery that point mutations in a GPCR could trigger receptor activation was done on the α1b-AR (3). When Ala293 in the C-terminal portion of the third intracellular loop (3i loop) of the α1b-AR was substituted by leucine, the mutant became constitutively active. Later, the same Ala293 was systematically mutated by other amino acids which induced variable levels of constitutive activity (4). Mutations of a single residue Thr348 in α2A-AR (5) and four residues Leu272, His269, Lys267, and Leu266 in β2-AR (6) C-terminal portions of 3i loop resulted in constitutively active mutants of these receptors. An activating mutation of the receptor for luteinizing hormone (LH) was identified as the most common cause of familial male precocious puberty (FMPP).

Introduction The adrenergic receptors (AR) mediate the functional effects of epinephrine and norepinephrine by coupling to several of the major signaling pathways modulated by G proteins. The AR family includes nine different gene products: three β (β1, β2, β3), three α2 (α2A, α2B, α2C), and three α1 (α1a, α1b, α1d) receptor subtypes. Like all G protein-coupled receptors (GPCR), the ARs display seven transmembrane α-helices that contribute to form the ligand binding pocket, whereas amino acid sequences of the intracellular (i) loop mediate the interaction of the receptor with G protein as well as with other signaling and regulatory factors (1). Constitutively active mutants (CAMs) of GPCRs are found naturally in disease states and have stimulated research for a naturally occurring GPCR mutant in *Corresponding author. [email protected] Published online in J-STAGE: October 7, 2006 doi: 10.1254/jphs.FP0060640

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The substitution of an aspartate with glycine at position 578 of helix 6 has been observed in a number of individuals suffering from FMPP (7). Other activating mutations of the LH receptor have been identified, clustering on helix 6 (8). In another study with A3 adenosine receptor, the mutation of Ala229Glu in transmembrane helical domain 6 has been found to constitutively activate the receptor (9). Altogether these findings identified the boundary between helix 6 and the 3i loop of GPCRs as a domain crucially involved in the process of receptor activation. However, it became soon apparent that the location of activating mutations was not limited to this region of GPCRs. Mutation of the highly conserved DRY motif, which is located at the bottom of transmembrane 3 of the rhodopsin-like GPCR family, has also been shown to result in constitutive activity (10 – 12). Other mutations, Cys128Phe (13) and Ala204Val (14) located in the 3rd and 5th transmembrane of the α1b-AR, have led to constitutive activation of the receptor. Later, a mutation (Leu322Lys in C-terminus of 3i loop) has been found in vitro that maintained the β1-AR in a constitutively active form (15). Another mutation in Arg156Ala of β1-AR was also found to be a CAM but the extent of its constitutive activity was lower than that of Leu322Lys as assessed by immunoprecipitation of mutant receptor-Gsα fusion protein (16). In the present study, we showed that when one of the two residues in β1-AR (Asp104 in the 2nd transmembrane domain), which were shown to be involved in determining the binding pockets for the β1AR agonist, SWR-0342SA (17) by molecular dynamics simulation (18), was mutated to alanine, the resulting mutant was found to be constitutively active in the second messenger assay. Our study provides the fact that β1-AR can be activated by point mutations and that the position of the mutant is in the 2nd transmembrane domain. It has been proposed that disorders resulting from activating mutations may be treated by a new class of therapeutic agents, termed inverse agonists, which can attenuate ligand-independent signaling (19, 20). We also report that some beta-blockers showed inverse agonism to the mutant receptor unlike to the wild-type receptor where they showed neutral antagonism. Materials and Methods Mutagenesis and cell transfection The cDNA encoding human β1-AR in pCMV5 was a kind gift from Dr. Lefkowitz at Duke University Medical Center, Durham, NC, USA. The mutant human β1-AR, Asp104Ala, was created by a QuikChange® sitedirected mutagenesis kit (Stratagene, La Jolla, CA, USA)

using the forward primer 5'-CTGGCCAGCGCCGCC CTGGTCATGGGG CTG-3' and reverse primer 5'-CAG CCCCATGACCAGGGCGGCGCTGGCCAG-3', according to the manufacturer’s protocol. The mutant receptor was confirmed by DNA sequencing analysis (Beckman Coulter CEQ2000XL; Beckman / Coulter, Fullerton, CA, USA). Expression construct of either β1-AR wild-type or mutant gene were introduced into human embryonic kidney (HEK)-293 cells together with selection vector pcDNA3.1(+) (Invitrogen, San Diego, CA, USA) using LipofectamineTM2000 (Invitrogen, Carlsbad, CA, USA), a lipofection transfection kit, according to the manufacturer’s protocol. The transfected cells were grown as monolayers in 35-mm dishes containing Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 µg/ ml of streptomycin under an atmosphere of 95% air and 5% CO2 at 37°C. Ligand binding Twelve hours after transfection, the confluent cells were subcultured into two 100 mm dishes. In order to select stable transformants, at 24 h after the transfection, medium of the transfected cells was changed into selection medium containing Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 500 µg / ml G418 under an atmosphere of 95% air and 5% CO2 at 37°C. The media were changed every 3/ 4 days for 21 days. Colonies of stable cells were passaged into a 35-mm dish and kept in the maintenance medium, Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 200 µg / ml G418 under an atmosphere of 95% air and 5% CO2 at 37°C. Stable cells were screened by radioligand binding assay. Crude membranes were prepared from cells expressing the β1-AR and its mutant. Radioligand binding studies were carried out in assay buffer containing 75 mM Tris-HCl (pH 7.4), 12.5 mM MgCl2, and 2 mM EDTA (21) at 37°C for 60 min using 5 – 10 µg of membrane protein. The total reaction volume was 250 µl. For saturation isotherms, membranes of WT or mutant receptor were incubated with varying concentrations (10 – 2000 pM) of [3H]-CGP12177 in the absence (total binding) or presence (nonspecific binding) of 1 µM (−)-propranolol. Competition binding studies were carried out using 100 pM [3H]-CGP12177 for β1-AR and 300 pM for Asp104Ala mutation of β1-AR. The reactions were stopped by dilution with 4 ml cold wash buffer containing 25 mM Tris (pH 7.5) and 1 mM MgCl2 and rapid filtration using Brandel cell harvester over Whatman GF / C glass fiber filters that had been treated with 0.1% polyethylenimine. The filters were washed

Beta-Blockers in β1-AR Mutant

with an additional ice-cold wash buffer (4 ml). The radioactivity remaining on the filter was counted by liquid scintillation counting. cAMP accumulation assay Adherent cells in 12-well plates (8 to 8.3 × 105 cells / well) were incubated for 30 min. at 37°C in 500 µl / well of Hank’s balance salt solution (HBSS) buffered with 20 mM HEPES (pH 7.4), 1 mM ascorbic acid, and 1 mM isobutyl-methylxanthine, in agonist or other drugs. The reaction was stopped with aspiration and washing with 1 ml ice cold (−) phosphate buffered saline. After 20 min. at 37°C in 1 N sodium hydroxide the samples were neutralized and centrifuged at 3000 × g for 10 min at 4°C. The amount of adenosine-3',5' cyclic monophosphate (cAMP) produced was determined on 50 µl of supernatant using an Amersham [3H]-cAMP assay kit according to the manufacturer’s protocol. Protein assay Protein contents of the membrane preparation were measured by the method of Lowry et al. using bovine serum albumin as the standard (22). Drugs [3H]-CGP12177 (1221.0 GBq/ mmol) was purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). Isoproterenol and (−)-epinephrine were purchased from Sigma-Aldrich (Irvine, UK). The following drugs were donated: atenolol hydrochloride and (−)propranolol (ICI Japan Ltd., Tokyo) and carvedilol (Daiichi Pharmaceuticals Co., Ltd., Tokyo). All other chemicals used were of the highest purity available. Data analyses Binding data were evaluated by a non-linear regression analysis using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). The results of experiments are expressed as means ± S.E.M. The inhibition concentration (IC50) in displacement analysis were determined as the concentrations of ligands that inhibited [3H]-CGP12177 binding by 50%; the values of inhibition constants (Ki) were calculated by the equation of Cheng and Prusoff (23) and expressed as pKi (−log Ki). In the cAMP accumulation assay, the cAMP contents in wells were calculated in pmol / well according to the manufacturer’s protocol. Student’s unpaired t-test was performed to assess the significance of the difference. A P value of less than 0.05 was taken as significant.

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Results Active mutation of the β1-AR Aspartate 104 in the 2nd transmembrane domain of the β1-AR was mutated into alanine. The wild-type β1AR and its mutant (Asp104Ala) were expressed in HEK293. Clear differences were observed in the basal levels of cAMP in cells expressing the wild-type and mutant receptor (Fig. 1). In cells expressing the wild-type β1-AR and Asp104Ala mutation of β1-AR, the basal levels of cAMP were 101% and 287% higher, respectively, than in control cells expressing the vectors alone (1.623 ± 0.14 pmol / well). Thus both the wild-type and the mutant display a very small, but significant spontaneous activity. These results demonstrate that mutations of Asp104 can constitutively activate β1-AR, which could be a novel finding that the 2nd transmembrane domain could also be an important switch regulating the activation of many GPCRs. Effect of different ligands on receptor-mediated cAMP accumulation Stimulation of the wild type β1-AR with isoproterenol or (−)-epinephrine resulted in a large increase of cAMP accumulation. Stimulations of cells with agonists could also increase the cAMP response mediated by the β1-AR mutant. The maximal cAMP levels induced by isoproterenol for the wild type β1-AR was 6.81 pmol / well

Fig. 1. Basal cAMP response in cells expressing the wild type (WT) and Asp104Ala mutation of β1-AR. Receptor expression was 2.6 pmol/mg protein for wild type and 10.2 pmol/mg protein for mutant β1-AR. Results are the mean ± S.E.M. of four independent experiments. Statistical significance was analyzed by unpaired Student’s t-test, *P<0.05. Basal level of the mutant was compared to that of the wild type receptor.

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and the maximal level induced by (−)-epinephrine for the Asp104Ala mutation of β1-AR was 71.25 pmol / well. However, accumulation by isoproterenol was lower than that by (−)-epinephrine in the mutant receptor. At the present time, we have no explanation for the effects, and further studies should be done to settle this. cAMP increase expressed as fold of basal was greater for Asp104Ala as compared to the wild type receptors because of its increased constitutive activity. Beta-blockers did not display any change in agonist or inverse agonist activity in the wild type but showed significant (P<0.05) inverse agonist activity in its constitutively active mutant (Fig. 2). Inverse agonism

at the wild type receptor was difficult to assess because of their low spontaneous activity. Ligand binding affinity for the wild type β1-AR and mutated receptors The affinities of the agonists, isoproterenol and (−)epinephrine, as well as of most beta-blockers were measured in membranes derived from HEK-293 cells expressing the wild type β1-AR or its mutant (Table 1). In the saturation experiment, the Kd and Bmax values of wild-type and mutant β1-AR calculated from Scatchard plots (n = 6) were 89.99 ± 18.52 pM and 2644 ± 124.5 fmol / mg protein, respectively, for the β1-AR and 228.4 ± 42.49 pM and 10242 ± 513.4 fmol / mg protein, respectively, for the Asp104Ala mutation of β1-AR. The dissociation constant and the receptor density in the mutant have been significantly (P<0.05 and P<0.0001, respectively) increased, suggesting a lower affinity of the radioligand [3H]-CGP12177 and increased receptor expression. The affinity of isoproterenol and (−)-epinephrine was decreased at the receptor mutants in a manner that, to a large extent, related to the decreased affinity of the radioligand with the mutant receptor. In fact, the Ki values of β1-non selective antagonists, (−)-propranolol and carvedilol, and a β1-selective antagonist, atenolol, were also decreased in the mutant with respect to the wild type. However, none of these declines in affinity to the mutant was significant statistically. These results are in agreement with the previous study that the ligand binding and signaling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked (24). Our results demonstrate that a mutation in the second transmembrane domain is able to increase the basal signaling activity of the human β1-AR. Table 1. Ligand binding affinity for the wild type (WT) β1-AR and its mutant Asp104Ala β1-AR

Ligand

Fig. 2. Effects of agonists and antagonists on the basal cAMP response mediated by β1-AR and constitutively active Asp104Ala mutation of β1-AR. Ligands are grouped accordingly: agonists (light stippling), non-selective antagonists (horizontal stripes), and β1selective antagonist (vertical stripes). Results are the mean ± S.E.M. of three to four experiments. Statistical significance was analyzed by the unpaired Student’s t-test on the inhibition of basal value, *P<0.05 (ligand vs basal).

WT

Asp104Ala 5.46 ± 0.2

Isoproterenol

6.22 ± 0.52

(−)-Epinephrine

5.15 ± 0.2

4.63 ± 0.1

(−)-Propranolol

8.87 ± 0.7

7.88 ± 1.07

Carvedilol

8.83 ± 0.5

7.42 ± 1.3

Atenolol

6.52 ± 0.3

5.37 ± 0.72

The affinities expressed as pKi were determined in competition binding experiments using [3H]-CGP12177 in cell membranes as described in the Materials and Methods section. The best fit of the competition was monophasic and the Hill coefficient was near to unity. The results for the wild type and the mutants are the mean of three or four experiments ± S.E.M.

Beta-Blockers in β1-AR Mutant

Discussion The main finding of our study is that point mutation in the 2nd transmembrane domain can constitutively activate the β1-AR. In a previous study, a point mutation at leucine 322 in the C-terminal portion of the 3i loop of the β1-AR constitutively activates the receptor (15). From the binding data, it is evident that the overexpression of the Asp104Ala mutation of β1-AR mutant receptor is responsible for the elevated basal cAMP, which was partially inhibited by the beta-blockers used in this study. The allosteric ternary complex model of receptor activation (6) predicts that constitutively active receptor mutants display increased affinity for agonists and decreased affinity for inverse agonists as compared to wild type GPCRs. In agreement with this concept, the beta-blockers displayed decreased affinity to the mutant. However, in disagreement with the predictions of the ternary complex model, the affinity of isoproterenol and (−)-epinephrine was decreased at the receptor mutants. In a study with constitutively active cholecystokinin type 2 receptor (CCK-2R) mutants, not all mutants showed the hallmark features of constitutively active receptor variant such as increased affinity of agonists. Most ligand affinities to mutants Met134Ala, Ser219His and Ser379His of CCK-2R were lower compared with the corresponding wild type values (25). These different pharmacological characteristics of the constitutively active mutant may be attributed to the location of the respective amino acid substitutions within the wild type receptor protein. A decrease in agonist activity could be a result of mutation-induced disruption of direct receptor-ligand interactions. Furthermore, there is precedent in the literature in which mutation-induced constitutive activation of GPCRs led to affinity decreases of certain agonists compared with wild type values (26, 27), rather than increases, as might have been predicted with receptor variants in a more active conformation. As with our findings, these investigations reported that amino acid substitutions were introduced within transmembrane domains. Another investigation confirmed that replacement of eight amino acids of transmembrane domain 2 of the β1-AR had shown that this transmembrane domain is a major determinant of the highaffinity binding of the β1-AR selective agonists (28). However, an amino acid in the same domain was not considered in the present study. However, it is likely that such an amino acid may also contribute to agonist binding to β1-AR; and for this reason, the agonist affinity is decreased in the mutant. Our findings on the effects of beta-blockers on the constitutive activity of the Asp104Ala mutation of β1-

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AR showed that non-selective and selective antagonists act as inverse agonists of the mutant. Unlike the results in a previous study by Lattion and coworkers, carvedilol, (−)-propranolol, and atenolol showed inverse agonism at this CAM of β1-AR. These beta-blockers showed neutral antagonism to the wild type receptor. In conclusion, the constitutively active β1-AR mutant described in this work might represent a new useful tool to further investigate the activation process of β1-AR and the mechanism of action of beta-blockers. Acknowledgments This research was supported by a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan. References 1 Weiss J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 1997;5:346–354. 2 Seifert R, Wenzel-Seifert K. Constitutive activity of G-proteincoupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol. 2002;366:381–416. 3 Cotecchia S, Exum S, Caron MG, Lefkowitz RJ. Regions of α1adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc Natl Acad Sci U S A. 1990;87:2896–2900. 4 Kjelsberg MA, Cotecchia S, Ostrowski J, Caron MG, Lefkowitz RJ. Constitutive activation of the α1B-adrenergic receptor by all amino acid substitution at a single site. J Biol Chem. 1992;67: 1430–1433. 5 Ren Q, Kurose H, Lefkowitz RJ, Cotecchia S. Constitutively active mutants of the α2A-adrenergic receptor. J Biol Chem. 1993;68:16483–16487. 6 Samama P, Cotecchia S, Costa T, Lefkowitz RJ. A mutationinduced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J Biol Chem. 1993;68:4625–4636. 7 Shenker A, Laue L, Kosugi S, Merenddino JJ Jr, Menegichi T, Cutler JB Jr. A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature. 1993;365:649–651. 8 Ascoli M, Fanelli F, Segaloff DL. The lutropin /choriogonadotropin receptor, a 2002 perspective. Endocr Rev. 2002;23:141– 174. 9 Chen A, Gao Z-G, Barak D, Liang BT, Jacobson KA. Constitutive activation of A3 adenosine receptors by site-directed mutagenesis. Biochem Biophy Res Comm. 2001;284:596–601. 10 Scheer A, Fanelli F, Costa T, De Benedetti PG, Cotecchia S. The activation process of the alpha1B-adrenergic receptor: Potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc Natl Acad Sci U S A. 1997;4:808–813. 11 Alewijnse AE, Timmerman H, Jacobs EH, Smit MJ, Roovers E, Cotecchia S, et al. The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine

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