The Ile164 β2-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to Gs

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Abstract

β2-adrenoceptors (β2AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (Kis were 1.2–3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase (∼40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31±4.8% vs. 49±4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a ∼50% decrease in the duration of action of salmeterol at Ile164 (t1/2=21.0±3.6 vs. 46.8±4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

Introduction

The β2-adrenoceptor (β2AR) is expressed in many cell-types in the body subserving a wide range of physiologic functions in the sympathetic nervous system Green and Liggett, 1996a, Liggett, 1997. β2AR expressed on bronchial smooth muscle act to relax the muscle (Liggett, 1997), affording bronchodilation and increased airflow. β2AR agonists, directed at these airway receptors, are the most commonly utilized therapeutic agents for the treatment of bronchospasm in asthma and chronic obstructive lung disease (COPD) (Tattersfield, 1997). Of particular concern within these populations are those who overuse β-adrenoceptor agonists or have significantly depressed responsiveness, since these individuals may be at risk of increased morbidity and mortality Spitzer et al., 1992, Suissa et al., 1994, Sears et al., 1990, Beasley et al., 1999.

In the human population, the β2AR displays genetic heterogeneity (Reihsaus et al., 1993), with nonsynonymous single nucleotide polymorphisms occurring at four loci within the coding region. The common polymorphisms occur at amino acid positions 16 (Arg or Gly) and 27 (Gln or Glu). At amino acid position 164, either Thr or Ile can be found, with the latter occurring with a heterozygous frequency of ∼5%. This polymorphism is localized within the fourth transmembrane spanning domain (TMD) of the receptor, and influences the binding “pocket” for conventional agonists which is formed by the TMDs (Green et al., 1993). Using site-directed mutagenesis and recombinant expression in Chinese hamster fibroblasts, we have shown that the affinity of the Ile164 receptor for epinephrine, norepinephrine and isoproterenol is decreased. Furthermore, the coupling of Ile164 to Gs as assessed in adenylyl cyclase assays, in response to the agonist epinephrine, is depressed compared to that of the wild-type Thr164 receptor (Green et al., 1993). This functional phenotype appears to be due to altered physical coupling of the receptor to Gαs and thus decreased formation of the high-affinity agonist-receptor-Gαs complex (Green et al., 1993). The consequences of the Ile164 receptor have also been explored in transgenic mice (Turki et al., 1996). In these studies, the Thr164 and the Ile164 receptors were overexpressed in the heart (where β2AR exert positive inotropic and chronotropic effects) and in vitro adenylyl cyclase signaling and in vivo cardiac function assessed. In response to the β-adrenoceptor agonist isoproterenol, adenylyl cyclase activities were reduced as were the inotropic and chronotropic responses.

All the above functional studies have been carried out only with the aforementioned prototypic β-adrenoceptor agonists. However, the β-adrenoceptor agonists currently available for the treatment of bronchospasm have a wide range of structures and pharmacologic properties. Structurally, these vary from minimal substitutions at the catechol ring and the amine head groups (albuterol) to complex substitutions such as the aralkyloxalkyl substitution at the amine group found with salmeterol (Derom and Pauwels, 1997). These drugs differ in their subtype specificities, potencies and efficacies for activation of β2AR, which has led to the notion that these synthetic agents can interact with the receptor in agonist-specific ways (Derom and Pauwels, 1997). Indeed, the long duration of action of salmeterol (∼12 h) has been shown to be due to binding of the aforementioned side chain to TMDs (including TMD4), which anchors the agonist to the receptor providing for repetitive binding events Green et al., 1996b, Rong et al., 1999. Such binding has been termed exosite binding (Liggett and Green, 1996), since it lies outside the critical contact points for conventional agonists Strader et al., 1989, Strader et al., 1995, Wieland et al., 1996, which are in TMDs 3, 5 and 6. In contrast, a similar long duration of action found with formoterol is thought to be due to a lipid depot effect in the cell membrane (Nials et al., 1993). Given this agonist pleiotropy, we have considered that the phenotype of the Ile164 receptor may be dependent on agonist structure. We have thus pursued the pharmacogenomic properties of this receptor using the common β-adrenoceptor agonists used for the treatment of asthma and COPD.

Section snippets

Mutagenesis and cell transfections

Site-directed mutagenesis was carried out as described previously (Green et al., 1993) such that a cytosine or a thymine is present at nucleotide 491 of the human β2AR open reading frame encoding Thr or Ile, respectively, at amino acid 164. The constructs consisted of these coding blocks cloned into the mammalian expression vector pcDNA/Neo1. Both receptors had Arg at position 16 and Gln at position 27. Chinese hamster fibroblasts (CHW 1102 cells) were permanently transfected using a calcium

Results

Initial studies focused on conventional ligand binding affinities and stimulation of adenylyl cyclase for the six agonists. The results of competition binding studies are shown in Table 1. As previously described (Green et al., 1993), isoproterenol had a lower affinity for the Ile164 receptor, as indicated by a ∼3-fold higher Ki as compared to that for the Thr164 receptor. The other agonists had Kis that varied between 1.2- and 2.4-fold greater with the Ile164 receptor compared to the wild-type

Discussion

Variation in the β2AR gene in the human population has led to the hypothesis that the known interindividual variability in the clinical response to therapeutic β-adrenoceptor agonists may be, in part, due to such genetic variation. Our approach to studying polymorphisms of the β2AR Green et al., 1993, Green et al., 1994b, Drysdale et al., 2000 and other adrenoceptors Mason et al., 1999, Small et al., 2000 has been to carry out in vitro studies in recombinant cells or transgenic mice before

Acknowledgements

The authors thank Nicole Tepe for the technical assistance and Esther Getz for the manuscript preparation. Supported by NIH grants GM61376, HL45967, and HD07463.

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