American Journal of Respiratory and Critical Care Medicine

The human β -adrenoceptor is a member of the seven-transmembrane family of receptors, encoded by a gene on chromosome 5. β -Adrenoceptors have been classified into β1, β2, and β3 subgroups, with β2-receptors being widely distributed in the respiratory tract, particularly in airway smooth muscle. Intracellular signaling following β2-adrenoceptor activation is largely affected through a trimeric Gs protein coupled to adenylate cyclase. Cyclic AMP (cAMP) induces airway relaxation through phosphorylation of muscle regulatory proteins and attenuation of cellular Ca2 + concentrations. Alternative cAMP-independent pathways involving activation of membrane maxi-K+ channels and coupling through Gi to the MAP kinase system have also been described. Site-directed mutagenesis has identified Asp 113 and Ser 204/207 within the third and fourth membrane domains as the active site of the β2-receptor, critical for β2-agonist binding and activity. β2-Agonists have been characterized as those that directly activate the receptor (albuterol), those that are taken up into a membrane depot (formoterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol). These differences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxation and bronchodilation in patients with asthma. β -Adrenoceptor desensitization associated with β2-agonist activation is a consequence of phosphorylation by β -ARK and uncoupling of the receptor from Gs following β -arrestin binding, of internalization and recycling of the receptor through processes of sequestration and resensitization and downregulation, modulated by an effect on receptor gene expression. The degree of receptor desensitization appears to differ, depending on the cell or tissue type, and is reflected in the different profiles of clinical tolerance to chronic β2-agonist therapy. A number of polymorphisms of the β2-receptor have been described that appear to alter the behavior of the receptor following agonist exposure. These include Arg-Gly 16, Glu-Gln 27, and Thr-lle 164. The Gly 16 receptor downregulates to a greater extent and is associated with increased airway hyperreactivity, nocturnal symptoms, and more severe asthma. The Glu 27 form appears to protect against downregulation and is associated with less reactive airways. An individual can be homozygous or heterozygous for given polymorphisms, and large populations will have to be studied to determine their importance to the asthma phenotype. Johnson M. The β -adrenoceptor.

β -Adrenoceptor Structure

The human β-adrenoceptor gene is situated on the long arm of chromosome 5 and codes for an intronless gene product of approximately 1,200 base pairs (1). The β-adrenoceptor is a member of the seven-transmembrane family of receptors (Figure 1) related to bacteriorhodopsin, which was used for the early structural work (2). It is composed of 413 amino acid residues of approximately 46,500 daltons (Da) (2). β-Adrenoceptors have been subdivided into at least three distinct groups: β1, β2, and β3, classically identified in cardiac, airway smooth muscle, and adipose tissue, respectively (3). There is a 65–70% homology between β13- and β2-receptors. This discussion will focus primarily on β2-receptors in the respiratory tract.

β -Receptor Density

Autoradiographic studies of human lung have suggested that β2-adrenoceptors are widely distributed, occurring not only in airway smooth muscle but also on other cells in the lung, such as epithelial and endothelial cells, type II cells, and mast cells (4). Until recently, quantification of these pulmonary receptors has only been possible in vitro. Radioligand binding studies on lobectomy specimens have shown β2-receptor density to increase with increasing airway generation, with high levels in the alveolar region (5). Computed tomography (CT) scanning has confirmed that β2-receptor distribution is greater for small than for large airways (6).

Alternatively, the density of β2-receptors on peripheral blood lymphocytes has been used as an index of β-receptors in the airways (7), but numbers (700–750 receptors per cell) are substantially less than in smooth muscle (30,000–40,000 per cell). Position emission tomography (PET) has now made possible the noninvasive quantification of β-receptors in vivo using the radioligand (IIC)CGP12177 (8). Serial measurements have shown pulmonary β2-receptor density to be 10.9 ± 1.0 picomole (pmol)/g tissue, compared with 8.8 ± 2.3 pmol/g for cardiac tissue (8). There was no difference between normal subjects and patients with asthma (9), but an inverse relationship was reported between FEV1 (% predicted) and lung β2-receptor density (9).

β -Receptor Kinetics

The temporal aspects of β2-receptor trafficking have not been well defined. Using an epitope-tagged human β-receptor, recycling, as measured by radioligand binding using the hydrophilic ligand, (3H)-CGP12177, proceeded with an apparent rate constant of 0.09, which reflects a one-phase exponential kinetic model with a recycling half-life (t1/2) of 7.5 min (10).

It has been the accepted dogma since the 1960s that β-adrenoceptor activation increases intracellular cyclic adenosine monophosphate (cAMP) levels. The coupling of the β-adrenoceptor to adenylate cyclase is affected through a trimeric Gs protein, which consists of α, β, and γ subunits (11).

There is now good evidence that β-adrenoceptors exist in two forms, activated and inactivated, and that under resting conditions these two forms are in equilibrium but with the inactivated state being predominant (12). The β2-receptor is in the activated form when it is associated with the α subunit of the G protein, together with a molecule of guanosine triphosphate (GTP), and it is through this α subunit that the receptor is coupled to adenylate cyclase. The replacement of the GTP by guanosine diphosphate (GDP) both catalyzes the conversion of ATP to cAMP by the enzyme and dramatically reduces the affinity of the α subunit for the receptor, causing dissociation and the receptor to return to its low-energy, inactivated form. It is probable that β2-agonists have their effects, not through inducing a conformational change in the receptor, but rather by binding to and temporarily stabilizing receptors in their activated state, i.e., bound to Gs-GTP, and therefore shifting the equilibrium (12). Implicit in this is the possibility that the spontaneous, albeit low, frequency of interconversion of inactivated to activated β-adrenoceptor that occurs in the absence of β-agonist results in a basal level of activity (12). Thus, the role of the β-agonist molecule is to amplify this low inherent receptor activity. In the case of the β2-adrenoceptor, this would be manifested as a basal level of cAMP turnover. Indeed, there is some evidence for this mechanism since single amino acid mutations made to β-adrenoceptors, which result in a shift in the resting equilibrium toward the activated state, are coupled to sustained increases in intracellular second messengers in the absence of agonist (12).

The corollary is that β-antagonists bind with high affinity to the low-energy inactivated form of the β-adrenoceptor, and thus shift the equilibrium away from the activated form. This is supported by the observation that addition of GDP inhibits the ability of β-agonists to bind to the receptor and enhances the binding of β-antagonists (13). If this is the case, β-antagonists should not be considered as competing directly for the same receptor, but instead as binding to a different form of the β-adrenoceptor protein and moving the equilibrium in opposite directions. While this would result in a competitive interaction, it is not competitive in the sense that β-agonist and β-antagonist molecules simultaneously compete for the same region of the β-adrenoceptor protein.

The mechanism by which cAMP induces airway smooth muscle cell relaxation is not fully understood, but it is believed that it catalyzes the activation of protein kinase A (PKA), which in turn phosphorylates key regulatory proteins involved in the control of muscle tone (Figure 2). cAMP also results in inhibition of calcium ion (Ca2+) release from intracellular stores, reduction of membrane Ca2+ entry, and sequestration of intracellular Ca2+, leading to relaxation of the airway smooth muscle (14). However, it has been suggested recently that some of the relaxant response to β2-agonists may be mediated through cAMP-independent mechanisms, involving direct interaction of Gsα with potassium channels, which are present in the airway smooth muscle cell membrane (15).

This has followed the observation that some effects of β2-adrenoceptor agonist stimulation may be inhibited by charybdotoxin and iberiotoxin, inhibitors of high-conductance, Ca2+-activated potassium ion (K+) channels (maxi-K channels) (16). Although these agents can markedly inhibit β-adrenoceptor–mediated effects on airway smooth muscle, they have no such effects on mast cells, suggesting a degree of tissue specificity in transduction processes. In support of the involvement of K+ fluxes in β-adrenoceptor agonist activity is the observation that in bovine tracheal smooth muscle cells, isoproterenol and albuterol both depolarize the cell membrane and cause the opening of K+ channels, as indicated by an increase in rubidium efflux (16). It is interesting, however, that although this effect is clearly β-adrenoceptor–mediated, only isoproterenol and albuterol appear to cause depolarization and induce rubidium efflux, salmeterol being without effect, although all three β2-agonists relax the preparation (16). It is difficult to reconcile these data, but it suggests that K+ channel activation may be a function of ligand efficacy and is not obligatory in airway smooth muscle relaxation.

Although most of the actions of the β2-receptor appear to be mediated through Gs proteins and the cAMP-dependent PKA system, β-receptors can also couple to Gi proteins. Stimulation of mitogen-activated protein (MAP) kinase by the β2-receptor has recently been demonstrated (17) and reported to be mediated by the βγ subunits of pertussis toxin–sensitive G proteins through a pathway involving the nonreceptor tyrosine kinase cSrc and the G protein RaS. Activation of this pathway by the β2-receptor requires that the receptor be phosphorylated by PKA, since inhibitors of PKA block the response and a mutant lacking the normal phosphorylation sites can activate adenylate cyclase, but not MAP kinase. This mechanism may serve not only to mediate uncoupling of the β2-receptor from Gs and thus heterologous desensitization, but may also switch the coupling of the receptor from Gs to Gi and represent direct feedback inhibition as a means of terminating the β2-agonist/receptor signal and response.

Site-directed mutagenesis has been able to identify regions of the β2-adrenoceptor protein important for β2-agonist binding to G protein coupling (18). The active site of the receptor, with which β2-agonists must interact in order to exert their biological effects, is located approximately one-third of the way (15 Ångström units [Å]) into the receptor core (Figure 1). It is generally agreed that there are residues of critical importance with respect to agonist binding to the active site, namely aspartate (Asp)-residue 113 (counted from the extracellular or N-terminus end) of the third domain, two serine (Ser) residues, 204 and 207, which are both on the fifth domain, and two phenylalanines (Phe), 259 and 290, on the sixth domain (19). Thus, a model has emerged for the agonist binding site of the β2-adrenoceptor, in which the ligand is bound within the hydrophobic core of the protein, intercalated among the transmembrane helices, and anchored by specific molecular interactions between amino acid residues in the receptor and functional groups on the ligand (19).

Asp binds to the nitrogen of the β-adrenoceptor agonist molecule, while the two Ser residues interact with the hydroxyl groups on the phenyl ring. Other residues may also be important, e.g., there is evidence that Asp residue 79 on the second domain and threonine (Thr)-164 are involved in agonist recognition (19). It is becoming increasingly clear that antagonists do not interact with the same amino acids as agonists in binding to the β-adrenoceptor. Thus, it appears that although antagonists probably bind to Asp-113, they do not interact with the two Ser residues on the fifth domain but rather with an asparagine (Asn) residue, 312 in the seventh domain (20).

All β-adrenoceptor agonists have an asymmetric center due to the presence of the β-OH group on the ethanolamine function (21). The presence of an asymmetric center results in the molecule existing as a pair of optical isomers (or mirror images), referred to as the R and S [or (−) and (+)] enantiomers, in a racemic mixture. In fact, some agonists—for example, fenoterol, formoterol, and procaterol—have two asymmetric centers, and there are four enantiomers—RR, SS, RS, and SR—present. It is a feature of most biological systems that they are stereospecific, and this is true of ligand/β-receptor interactions. Where the individual enantiomers of β2-adrenoceptor agonists have been resolved and tested, it is clear that the activity lies predominantly in the R-enantiomer, probably as a result of an optimal interaction between the “down” orientation of the β-OH group and Ser 165. For albuterol, for example, the R-enantiomer is at least 100-fold more potent as a β2-agonist than the S-enantiomer (21), whereas this difference is greater than 1,000-fold for the RR and SS forms of formoterol (22).

In the case of salmeterol, where enantiomerically pure samples have been prepared, there is still significant β2-agonist activity in the S-enantiomer, which is only 40-fold less potent than the R-form and 15-fold weaker than the racemic mixture. Interestingly, both the R- and S-enantiomers of salmeterol are long-acting (23). There is no evidence of the S-isomer of salmeterol antagonizing the effects of the corresponding R-form, or of the S-enantiomer having pharmacologic effects different from those of the racemic mixture (23).

β -Agonist Affinity and Efficacy

The affinity of a ligand is a measure of the avidity of its binding to its receptor. Few β2-agonists have been shown to have much higher affinity than isoproterenol and, indeed, albuterol has a relatively low affinity for β2-adrenoceptors. In contrast, salmeterol and formoterol have high affinities for the β2-adrenoceptor with a KI of 53 nM and 74 nM, respectively, compared with 200 and 2,500 nM for isoproterenol and albuterol (24).

β-Agonist potency, however, is a function not only of receptor affinity, but also of efficacy. A full agonist will have a high efficacy while a pure antagonist will have low or zero efficacy. The majority of β2-adrenoceptor agonists have an intermediate efficacy, and if tissue factors permit, they will behave as full agonists; however, if receptor density is too low or coupling is inadequate, the β-agonist may behave in a partial manner, i.e., it will be incapable of achieving the same maximum effect as an agonist of higher efficacy, and it may even behave as an antagonist. Examples of compounds of high efficacy (approximately equivalent to isoproterenol) are procaterol, fenoterol, and formoterol, whereas most saligenins and resorcinols, albuterol and terbutaline, for example, tend to be of moderate efficacy (65–85%), and the efficacy of the dichloroaniline, clenbuterol, is low (40%). Salmeterol has an efficacy at β2-adrenoceptors in airway smooth muscle of approximately 65% (25). Low efficacy in a β2-adrenoceptor agonist does not, however, compromise its clinical effectiveness as a bronchodilator drug.

Kinetics of β2-Agonist–Induced Airway Smooth Muscle Relaxation

The molecular size and structure of a β2-agonist determines the manner in which it interacts with the β2-adrenoceptor in airway smooth muscle. The albuterol molecule, which is 11 Å in length and hydrophilic in nature, accesses the active site of the β2-adrenoceptor directly from the extracellular compartment (26). There is therefore a rapid onset of airway tissue relaxation and of bronchodilation in patients. However, the drug rapidly re-equilibrates, its residency time at the active site is limited, and the resulting duration of action short (4–6 h).

Formoterol is moderately lipophilic in nature (27). It is taken up into the cell membrane in the form of a depot, from where it progressively leaches out to interact with the active site of the β2-receptor (27). The size of the depot is determined by the concentration or dose of formoterol applied. In airway preparations, the onset of action of formoterol is somewhat delayed compared with albuterol, and the duration of relaxant activity, although longer, is concentration-dependent (28). This profile has been confirmed clinically in patients with asthma, where bronchodilation was observed for 8, 10, and 12 h following doses of 6, 12, and 24 μg, respectively (29).

The salmeterol molecule is 25 Å in length and it is greater than 10,000 times more lipophilic than albuterol (30). The use of low-angle neutron diffraction techniques to study the interaction of salmeterol with the cell membrane has indicated that it partitions rapidly (< 1 min) into the outer phospholipid monolayer by a factor approaching 30,000:1 (31). Molecular modeling suggests that the orientation is such that the saligenin moiety is the same plane as the polar head groups, with the side chain in close association with the hydrophobic tails of the phospholipids. It is of interest that 17 Å side chain of salmeterol, which was found to be optimal for duration of action, is the same as the depth of the phospholipid monolayer (30). There is no evidence that salmeterol “flip-flops” from the outer to the inner monolayers of the surface phospholipids, but instead the molecule diffuses laterally to approach the active site of the β-adrenoceptor through the membrane (31). This translocation process appears to be slow (> 30 min).

The experimental data indicates that the receptor binding of salmeterol is only slowly reversible and noncompetitive, whereas functional responses to the molecule are both fully reversible and competitive (32). In order to rationalize these findings, the “exo-site” hypothesis was proposed (33). The original concept was that the long side chain of the molecule interacted with a nonpolar region in the cell membrane, the exo-site, in the vicinity of the β2-receptor. High-affinity binding of the side chain to the exo-site then allowed the saligenin head to repeatedly activate the receptor, enabling salmeterol to be long-acting. From the molecular modeling studies, it has been predicted that there is a preferred “down” conformation of the molecule in the receptor protein (34), whereby the saligenin head binds to the active site in an analogous position to that of albuterol, and the long, flexible side chain is located deep into a hydrophobic core domain of the receptor, suggesting that the specific exo-site for salmeterol may be an integral part of the β2-adrenoceptor protein itself.

Site-directed mutagenesis studies (35) showed that it was possible to replace a discrete length of the fourth transmembrane domain of the β2-adrenoceptor (specifically, residues 149–158), believed to be associated with exo-site binding from molecular modeling (34), with the corresponding section of the β1-adrenoceptor, while maintaining the affinity of salmeterol for the resulting hybrid receptor. However, significantly, this modification resulted in a decreased persistence of agonist activity after washout. Even more significantly, when the corresponding β1-adrenoceptor hybrid was constructed with the same key amino acids (methionine, leucine, isoleucine, isoleucine, valine) from the β2-adrenoceptor, this resulted in markedly enhanced persistence of salmeterol activity (35).

The mechanism of action of salmeterol therefore involves the interaction of the side chain with an auxiliary binding site (exo-site), a domain of highly hydrophobic amino acids within the fourth domain of the β2-adrenoceptor. When the side chain is in association with the exo-site, the molecule is prevented from dissociating from the β2-adrenoceptor, but the saligenin head can freely engage and disengage the active site by the Charniére (hinge) principle, flexion being about the oxygen atom in the side chain (Figure 3). The position of this oxygen atom was shown in structure–activity studies to be critical for duration of action (34).

The onset of action of salmeterol on airway smooth muscle is therefore slower than that of other β2-agonists, such as albuterol and formoterol. However, whereas the duration of action of the latter can be increased by increasing the concentration applied to the tissue, salmeterol appears to be inherently long-acting, in that its effects are independent of dose, as a result of exo-site binding. The duration of action of β2-agonists against spasmogen-induced, neuronally mediated, and inherent tone in the human bronchus is in the order: salmeterol >> formoterol ⩾ albuterol ⩾ terbutaline > fenoterol (36).

In terms of intracellular mediators, McCrea and Hill (37) have shown that the increment in cAMP in cultured smooth muscle cells is rapid with isoproterenol and albuterol, whereas salmeterol increases intracellular cAMP more slowly, consistent with the membrane access of the molecule to the β2-adrenoceptor. In addition, the maximum elevation of cAMP to salmeterol achieves only 45% of that to isoproterenol, confirming the partial agonist nature of the response. However, whereas cAMP responses to isoproterenol and albuterol are transient, and rapidly reversed toward basal levels by washing the cells, salmeterol induces a sustained (> 120 min) elevation of intracellular cAMP. The changes in intracellular cAMP with β2-agonists such as albuterol and salmeterol are therefore consistent with the kinetics of their effects on airway relaxation.

Associated with β-adrenoceptor activation is the auto-regulatory process of receptor desensitization. This process operates as a safety device to prevent overstimulation of receptors in the face of excessive β-agonist exposure. Desensitization occurs in response to the association of receptor with the agonist molecule, and is prevented by the interaction of the receptor with an antagonist. The mechanisms by which desensitization can occur consist of three main processes: (1) uncoupling of the receptors from adenylate cyclase; (2) internalization of uncoupled receptors; and (3) phosphorylation of internalized receptors (38). The extent of desensitization depends on the degree and duration of the β-adrenoceptor/β-agonist response.

The principal mechanism of homologous short-term, β2- agonist–promoted desensitization of the β2-adrenoceptor is phosphorylation of the receptor by the cAMP-independent kinase (βARK) or other closely related G protein–coupled receptor kinases (GRKs). Mutation studies on the β-adrenoceptor protein have shown that the third intracellular loop and the intracellular C-terminus are the major sites of phosphorylation (38). Such phosphorylation ultimately results in binding of β-arrestin and partial uncoupling of the agonist-occupied form of the receptor from the stimulatory guanine nucleotide– binding protein Gs, thereby limiting receptor function. Simple uncoupling is a transient process and may be reversed within minutes of removal of the agonist.

After more prolonged agonist exposure, an internalization of receptors occurs, which results in a loss of some proportion of cell surface receptors. This process, termed sequestration, has also been considered to be another mechanism of desensitization, but recent studies have suggested that its major role in short-term regulation of the receptor may be in resensitization (Figure 4), since it appears that the sequestered pool is the site of dephosphorylation of the receptor. Internalization takes longer to reverse than uncoupling, but full reversal normally occurs within hours.

After hours of agonist exposure, a net loss of cellular receptors occurs (denoted downregulation) via several mechanisms that are independent of receptor phosphorylation. β2-Receptor trafficking as part of the overall process of receptor desensitization has now been investigated in the form of kinetic analysis of internalization and recycling of the human β2-receptor. Cellular trafficking was measured by flow cytometry, quantifying the cell surface levels of a monoclonal Ab (12CA5) against the hemagglutinin epitope of the receptor ectodomain (39). In the presence of a β-agonist (isoproterenol, 5 μM), steady-state rate constants of 0.38 and 0.25 for internalization and recycling, respectively, were determined, with a total transit time for the receptor cycling between the cell surface and the endocytic compartment of 6.6 min (39).

The process of desensitization may differ markedly from tissue to tissue. It is clear, for example, that human lymphocytes desensitize very rapidly on exposure to β2-adrenoceptor agonists, whereas human bronchial smooth muscle is singularly resistant. The level of βARK mRNA in airway smooth muscle cells was only about 20% of that in bronchial epithelial cells and approximately 11% of that in mast cells (40). At the protein level, βARK expression in airway smooth muscle cells was nearly undetectable, being about 10-fold less than that expressed in mast cells. A marked discrepancy in GRK activities was also observed with mast cells (90.7 ± 0.5 relative units) as compared with airway smooth muscle cells (9.3 ± 0.6 relative units, p < 0.001). In contrast, the activities of cAMP-dependent PKA were not different (40). This predicts that airway smooth muscle β2-receptors would undergo minimal short-term (5 min) agonist-promoted desensitization as compared with the β2-receptor expressed on mast cells. In response to isoproterenol (1 μM), mast cell cAMP reached maximum levels after 90 s and did not further increase over time, indicative of receptor desensitization in this cell. In contrast, cAMP levels of airway smooth muscle cells did not plateau, increasing at a rate of 103 ± 9% per min, consistent with little desensitization over the study period (40). This may explain the clinical observation that repetitive administration of β2-agonists to subjects with asthma appears to result in desensitization of bronchoprotective responses (41) thought to be mediated by the pulmonary mast cell β2-receptor, but not the bronchodilatory response of β2-receptor expressed on bronchial smooth muscle (42). This type of difference may also be manifested in the well documented decline in the side effects associated with β2-adrenoceptor agonist therapy (e.g., tachycardia and physiologic tremor) in patients with asthma, but the maintenance of bronchodilatation despite regular treatment for prolonged periods (42).

As desensitization results from agonist occupancy and can be inhibited by antagonists, it follows that a partial agonist would be less prone to induce receptor desensitization than a full agonist. Indeed, this has been demonstrated to be the case with β2-agonists clinically, where a degree of bronchodilator tolerance was observed with the high-efficacy agonist formoterol on chronic exposure and despite the presence of the corticosteroid budesonide (43), but not with the partial agonist salmeterol (44).

It is now well appreciated that in addition to desensitization processes that negatively regulate the function of the β2-receptor protein itself, β-agonists, acting through the cAMP pathway, also dramatically modulate β2-receptor gene expression. Isoproterenol resulted in a significant decline (50%) in β2-receptor transcripts at 4 and 8 h, respectively (45). In comparison to isoproterenol, cells treated with salmeterol had no such downregulating effect on β2-receptor gene expression (45). These data are consistent with the hypothesis that the long-acting characteristics of salmeterol may be due, at least in part, to the ability of this agonist to maintain a population of functional β2-receptors through persistent elevation of gene transcription, despite a prolonged, low-level exposure to the agonist.

Two weeks of albuterol treatment (4 mg orally twice daily and 200 μg four times daily) resulted in a decrease in β-receptor density, assessed by PET scanning, of 22% in the lung (46). This was associated with a reduction in bronchodilator response to albuterol (46). Corticosteroids have facilitatory effects on the β2-adrenoceptor, increasing β2-receptor gene transcription, through binding and activation of cAMP response element binding protein (CREB), regulating both the numbers of the receptors and the coupling of the receptor to adenylate cyclase (47). Systemic corticosteroids have been shown to reverse β2-adrenoceptor downregulation in normal subjects and subjects with asthma who have been exposed to β2-agonists (48). It is of interest, however, that an inhaled corticosteroid does not apparently prevent tolerance to the bronchoprotective effects of a long-acting β2-agonist such as formoterol (49) or salmeterol (50).

A number of common variants (polymorphisms) of β2-receptor have recently been described (51) that alter the behavior of the receptor following agonist exposure. The main clinical interest in these polymorphisms lies in the possibility that they may determine the extent to which the receptor downregulates in the airways and as such may modify bronchodilator responses through changes in the expression and coupling of β2-receptors in airway cells. There are two genes for the β2-adrenoceptor, and therefore an individual can be homozygous or heterozygous for a given polymorphism.

Studies on the β2-adrenoceptor identified a total of nine different polymorphisms (51). All of these differed from the accepted wild-type sequence by a single base change at different positions in the coding sequence of the gene. Because of redundancy in the amino acid code, a number of these polymorphisms are clinically silent. However, four polymorphisms resulting from single base changes were identified that altered the amino acid sequence of the receptor protein (51). Three of these polymorphisms have now been studied in some detail, and all three appear to alter the functional properties of the receptor, such that the airways of individuals with these forms of the receptor might be expected to behave differently when exposed to circulating catecholamines or exogenously applied β2-agonists.

The initial studies focused (51) on amino acid 16 (Figure 1), which can be either arginine (Arg) or glycine (Gly), depending on whether base 46 is A or G. The data suggest that the ability of a receptor to desensitize is markedly influenced by the presence of Gly 16. The Gly 16 receptor downregulates following exposure to an agonist to a much greater extent than the Arg 16 form in both transfected cell systems and in primary cultured human airway smooth muscle cells (51). Two recent clinical studies have supported the possibility that the Gly 16 form of the receptor is associated with markers of more severe asthma. Preliminary data from Dutch families with asthma suggest that Gly 16 may be associated with airway hyperreactivity (52). In addition, patients with significant nocturnal worsening of their asthma were more likely to have the Gly 16 form of the receptor than patients with asthma without nocturnal falls in peak flow rate (53). The allelic frequencies for Arg 16 and Gly 16 are 35% and 65%, respectively (54).

The second polymorphism is at codon 27 (Figure 1), which exists as either glutamine (Gln) or as glutamate (Glu), depending on whether base 76 is C or G. The allelic frequency for Gln 27 and Glu 27 is 55% and 45%, respectively (54). In contrast to Gly 16, the Glu 27 form of the receptor appears to protect against downregulation (55). Using primary cultured human airway smooth muscle cells, following prolonged exposure to β2-agonists, the Glu 27 form downregulated to a much lesser extent than the Gln 27 receptor, as assessed by changes in receptor number (56). In addition, a similar relative resistance to downregulation was observed using β2-agonist–mediated cAMP formation as an end point for receptor coupling (56). In a group of 65 patients with mild to moderate asthma, individuals with the Glu 27 form of the receptor had four times less reactive airways than those with Gln 27 when assessed using methacholine challenge. Heterozygotes had an intermediate mean PD20 value (57). Where homozygous Glu 27, which is predicted to protect against receptor desensitization, is combined with homozygous Gly 16, the effects of Gly 16 are dominant (58).

The third polymorphism is at amino acid 164, which can either be Thr or isoleucine (Ile) (Figure 1). This polymorphism is much rarer than that at amino acid 16 or 27, with an allelic frequency of about 1% (59), but it is potentially interesting in that amino acid 164 is situated in the fourth transmembrane spanning domain of the receptor and is adjacent to Ser 165, which has been predicted to interact with the β-OH group of adrenergic ligands. This polymorphism has been studied in a transfected cell system and has been shown to alter the agonist-binding properties of the receptor. Cells expressing lle 164 were found to have approximately four times less ligand affinity (59). This alteration in binding affinity was reflected in a reduced capacity for the receptor to activate adenylate cyclase, relative to the wild-type (Thr 164) form of the receptor (59).

Given that most individuals will be heterozygous and that Arg-Gly 16 and Gln-Glu 27 polymorphisms may be in linkage disequilibrium, large populations will have to be studied to determine the importance of β2-adrenoceptor polymorphisms to the asthma phenotype. However, the relationship between polymorphisms of the β2-adrenoceptor and pulmonary and systemic exposure to chronic dosing with a β2-agonist has been investigated (58, 60). In 10 of 14 subjects with nonresistant genotypes (Gly/Gly 16; Gly/Arg 16), there was a significant reduction (mean, 24%) in pulmonary β-adrenoceptors, as assessed by PET scanning after 2 wk dosing with albuterol. Four subjects who were heterozygous for the Glu 27 polymorphism were resistant to downregulation of pulmonary β2-receptors (60). Similarly, homozygous Gly 16 was significantly more prone to bronchodilator tolerance (46%) than Arg 16 (8%) following administration of formoterol (24 μg bd) for 4 wk (58).

1. Kobilka B. K., Dixon R. A., Frielle H. G., Dohlman M. A., Bolanowski I., Sigal I. S.cDNA for the human β2-adrenergic receptor: a protein with multiple spanning domains and encoded by a gene whose chromosomal location is shared with that of a receptor for platelet growth factor. Proc. Natl. Acad. Sci. U.S.A.8419874650
2. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H.Model for the structure of bacteriorhodopsin based on high-resolution electron cyro-microscopy. J. Mol. Biol.2131990899929
3. Frielle T., Daniel K. W., Caron M. G., Lefkowitz R. J.Structural basis of β-adrenergic receptor subtype specificity studies with chimeric β22-adrenergic receptors. Proc. Natl. Acad. Sci. U.S.A.85198894949498
4. Johnson, M. 1992. Mechanisms of action of β-adrenoceptor agonists. In J. F. Costello and R. D. Mann, editors. Beta-Agonists in the Treatment of Asthma. Parthenon, Carnforth. 27–42.
5. Spina D., Rigby R. J., Paterson J. W., Goldie R. G.Autoradiographic localization of β-adrenoceptors in asthmatic human lung. Am. Rev. Respir. Dis.140198914101415
6. Hoffman E. A., Chiplunkar R., Casale T. B.CT scanning confirms beta receptor distribution is greater for small versus large airways (abstract). Am. J. Respir. Crit. Care Med.1551997A855
7. Martinsson A., Larsson K., Hjemdahl P.Studies in vivo and in vitro of terbutaline-induced beta-adrenoceptor desensitization in healthy subjects. Clin. Sci.7219874754
8. Ueki J., Rhodes C. G., Hughes J. M. B., DeSilva R., Lefroy D., Ind P. W., Qing F., Brady F., Luthra S. K., Steel C., Waters S. L., Lammertsma A. A., Camici P. G., Jones T.In vivo quantification of pulmonary β-adrenoceptor density in humans with (S)-11C-CGP12177 and PET. J. Appl. Physiol.751993559565
9. Qing F., Rahman S. U., Rhodes C. G., Hayes M., Ind P. W., Hughes J. M.β-Adrenergic receptors in vivo and lung function in drug-free asthmatic subjects (abstract). Am. J. Respir. Crit. Care Med.1551997A855
10. Moore R. H., Morrison K. J., Carsrud N. D. V., Trial J., Millman E., Dickey B. F., Knoll B. J.Kinetic analysis of internalization and recycling of the human β2-adrenoceptor (abstract). Am. J. Respir. Crit. Care Med.1531997A240
11. Robison G. A., Butcher R. W., Sutherland E. W.Adenyl cyclase as an adrenergic receptor. Ann. N.Y. Acad. Sci.3191967703723
12. Onaran H. O., Costa T., Rodbard D.Subunits of guanine nucleotide-binding proteins and regulation of spontaneous receptor activity: thermodynamic model for the interaction between receptors and guanine nucleotide-binding protein subunits. Mol. Pharmacol.431993245256
13. Costa T., Ogino Y., Munson P. J., Onaran H. O., Rodbard D.Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol. Pharmacol.411992549560
14. Johnson, M., and R. A. Coleman. 1995. Mechanisms of action of β2-adrenoceptor agonists. In W. W. Busse and S. T. Holgate, editors. Asthma and Rhinitis. Blackwell, Cambridge. 1278–1295.
15. Cook S. J., Small R. C., Berry J. L., Chiu P., Downing S. J., Foster R. W.β-Adrenoceptor subtypes and plasmalemmal K+-channels in trachealis muscle. Br. J. Pharmacol.109199311401148
16. Chiu P., Cook S. J., Small R. C.β-Adrenoceptor subtypes and the opening of plasmalemmal K+-channels in bovine tracheal muscle: studies of mechanical activity and ion fluxes. Br. J. Pharmacol.109199311491156
17. Daaka Y., Luttrell L. M., Lefkowitz R. J.Switching of the coupling of the β2-adrenergic receptor to different G-proteins by protein kinase A. Nature39019978891
18. Tota M. R., Candelore M. R., Dixon R. A. F., Strader C. D.Biophysical and genetic analysis of the ligand binding site of the beta-adrenoceptor. Trends Pharmacol. Sci.12199146
19. Strader C. D., Candelore M. R., Hill W. S., Dixon R. A. F., Sigal I. S.Identification of two serine residues involved in agonist activation of the β-adrenergic receptor. J. Biol. Chem.26419891357213580
20. Strader C. D., Sigal I. S., Candelore M. R., Hill W. S., Dixon R. A. F.Conserved aspartic residues 9 and 113 of the β-adrenergic receptor have different roles in receptor function. J. Biol. Chem.26319881026710271
21. Buckner C. K., Abel P.Studies on the effects of the enantiomers of soterenol, trimetoquinol and salbutamol on β-adrenergic receptors of isolated guinea pig atria and trachea. J. Pharm. Exp. Ther.1891974616625
22. Johnson M.Salmeterol. Med. Res. Rev.151995225257
23. Johnson M., Butchers P. R., Coleman R. A., Nials A. T., Strong P., Summer M. J., Vardey C. J., Whelan C. J.The pharmacology of salmeterol. Life Sci.52199321312147
24. Brittain R. T., Jack D., Sumer M. J.Further studies on the long duration of action of salmeterol, a new selective β2-stimulant bronchodilator. J. Pharm. Pharmacol.40198893P
25. Jack D.A way of looking at agonism and antagonism: lessons from salbutamol, salmeterol and other β-adrenoceptor agonists. Br. J. Clin. Pharmacol.311991501514
26. Johnson, M. 1993. β2-Adrenoceptor agonists: optimal pharmacological profile. In The Role of β2-Agonists in Asthma Management. The Medicine Group, Oxford. 6–8.
27. Anderson G. P.Formoterol: pharmacology, molecular basis of agonism and mechanism of long duration of a highly potent and selective β2-adrenoceptor agonist bronchodilator. Life Sci.52199321452160
28. Coleman R. A., Johnson M., Nials A. T., Sumer M. J.Salmeterol but not formoterol persists at β2-adrenoceptors. Br. J. Pharmacol.991990121P
29. Ringdahl N., Derom E., Pauwels R.Onset and duration of action of single doses of formoterol inhaled via Turbuhaler in mild to moderate asthma. Eur. Respir. J.8199568S
30. Johnson M.Salmeterol. Drug News and Perspectives61993316324
31. Rhodes D. G., Newton R., Butler R., Herbette L.Binding and structural studies of the interactions of salmeterol with membrane bi-layers. FASEB J.61992A374
32. Johnson M.The pharmacology of salmeterol. Lung1681990115119
33. Brittain R. T.Approaches to a long-acting selective β2-adrenoceptor stimulant. Lung1681990111114
34. Lewell X. Q.A model of the adrenergic beta-2 receptor and binding sites for agonist and antagonist. Drug Des. Discov.919922948
35. Green S. A., Spasoff A. P., Coleman R. A., Johnson M., Liggett S. B.Sustained activation of a G-protein–coupled receptor via anchored agonist binding. J. Biol. Chem.27119962402924035
36. Coleman R. A., Nials A. T., Vardey C. J.Effects of salmeterol, albuterol and formoterol on human bronchial smooth muscle (abstract). Am. Rev. Respir. Dis.1451992A391
37. McCrea K. E., Hill S. J.Salmeterol, a long-acting β2-adrenoceptor agonist mediating cyclic AMP accumulation in a neuronal cell line. Br. J. Pharmacol.1101993619626
38. Freedman N. I., Lefkowitz R. J.Desensitization of G protein-coupled receptors. Rec. Progr. Horm. Res.511996319353
39. Moore R. H., Morrison K. J., Carsrud N. D. V., Trial J., Millman E., Dickey B. F., Knoll B. J.Kinetic analysis of internalization and recycling of the human β2-adrenoceptor (abstract). Am. J. Respir. Crit. Care Med.1531997A240
40. McGraw D. W., Liggett S. B.Heterogeneity of β-adrenergic receptor kinase expression in the lung accounts for cell-specific desensitization of the β2-adrenergic receptor. J. Biol. Chem.272199773387344
41. O'Connor B. J., Aikman S., Barnes P. J.Tolerance to the non-bronchodilator effects of inhaled β2-agonists. N. Engl. J. Med.327199212041208
42. Dahl R., Earnshaw J. S., Palmer J. B. D.Salmeterol: a four week study of a long-acting beta-adrenoceptor agonist for the treatment of reversible airways disease. Eur. Respir. Dis.4199111781184
43. Pauwels R. A., Lofdahl C. G., Postma D. S., Tattersfield A. E., O'Byrne P., Barnes P. J., Ullman A.Effect of inhaled formoterol and budesonide on exacerbations of asthma. N. Engl. J. Med.337199714051411
44. Ullman A., Hedner J., Svedmyr N.Inhaled salmeterol in asthmatic patients: an evaluation of asthma symptoms and the possible development of tachyphlyaxis. Am. Rev. Respir. Dis.1421990571575
45. Wang S., Collins S.Regulation of the β2-adrenergic receptor by the long-acting agonist salmeterol in human bronchial epithelial cells (abstract). Am. J. Respir. Crit. Care Med.1531996A730
46. Hayes M. J., Qing F., Rhodes C. G., Rahman S. U., Ind P. W., Sriskandan S., Jones T., Hughes J. M. B.In vivo quantification of human pulmonary β-adrenoceptors: effect of β-agonist therapy. Am. J. Respir. Crit. Care Med.154199612771283
47. Hui K. K. P., Connolly M. E., Taskin D. P.Reversal of human lymphocyte β-adrenoceptor desensitization by glucocorticoids. Clin. Pharmacol. Ther.321982566571
48. Brodde O. E., Howe U., Egerzegi S., Konietzko N., Michel M. C.Effects of prednisolone on β2-adrenoceptors in asthmatic patients receiving β2-bronchodilators. Eur. J. Clin. Pharmacol.341988145150
49. Yates D. H., Sussman H., Shaw M. J., Barnes P. J., Chung K. F.Regular formoterol treatment in mild asthma: effect on bronchial responsiveness during and after treatment. Am. J. Respir. Crit. Care Med.152199511701174
50. Yates D. H., Kharitonov S. A., Barnes P. J.An inhaled glucocorticoid does not prevent tolerance to the bronchoprotective effect of a long-acting inhaled β2-agonist. Am. J. Respir. Crit. Care Med.154199616031607
51. Reishaus E., Innis M., MacIntyre N., Liggett S. B.Mutations in the gene encoding for the β2-adrenergic receptor in normal and asthmatic subjects. Am. J. Respir. Cell Mol. Biol.81993334339
52. Holroyd K. J., Levitt R. C., Dragwa C., Amelung P. J., Panhuysen C. M., Meyers D. A.Evidence for β2-adrenergic receptor polymorphism at amino acid 16 as a risk factor for bronchial hyper-responsiveness (abstract). Am. J. Respir. Crit. Care Med.1511995A673
53. Turki J., Pak J., Green S., Martin R., Liggett S. B.Genetic polymorphisms of the β2-adrenergic receptor in nocturnal and non-nocturnal asthma: evidence that Gly 16 correlates with the nocturnal phenotype. J. Clin. Invest.95199516351641
54. Hall I. P.β2-Adrenoceptor polymorphisms: are they clinically important? Thorax511996351353
55. Green S. A., Turki J., Innis M., Liggett S. B.Amino terminal polymorphisms of the human β2-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry33199494149419
56. Green S. A., Turki J., Bejarano P., Hall I. P., Liggett S. B.Influence of β2-adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol.1319952533
57. Hall I. P., Wheatley A., Wilding P., Liggett S. B.Association of the Glu 27 β2-adrenoceptor polymorphism with lower airway reactivity in asthmatic subjects. Lancet345199512131214
58. Tan S., Hall I. P., Dewar J., Dow E., Lipworth B.Association between β2-adrenoceptor polymorphism and susceptibility to bronchodilator desensitization in moderately severe stable asthmatics. Lancet3501997995999
59. Green S. A., Cole G., Jacinto M., Innis M., Liggett S. B.A polymorphism of the human β2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J. Biol. Chem.26419931357213578
60. Rahman S. U., Qing F., Rhodes C. G., Hall I. P., Ind P. W., Jones T., Hughes J. M. B.Regulation of pulmonary β2-adrenergic receptor expression: concordance between receptor density, function and genotypes (abstract). Am. J. Respir. Crit. Care Med.1551997A855
Correspondence and requests for reprints should be addressed to Malcolm Johnson, Respiratory Therapeutic Development, Glaxo Wellcome Research and Development, Uxbridge, Middlesex UB 11 1BT, UK.


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