THE -ADRENOCEPTOR

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-Adrenoceptor Structure

The human -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 -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). -Adrenoceptors have been subdivided into at least three distinct groups: ₁, ₂, and ₃, classically identified in cardiac, airway smooth muscle, and adipose tissue, respectively (3). There is a 65-70% homology between ₁/₃- and ₂-receptors. This discussion will focus primarily on ₂-receptors in the respiratory tract.

View larger version (38K): [in this window] [in a new window]   Figure 1.   Structure of the human 2-adrenoceptor.

-Receptor Density

Autoradiographic studies of human lung have suggested that ₂-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 ₂-receptor density to increase with increasing airway generation, with high levels in the alveolar region (5). Computed tomography (CT) scanning has confirmed that ₂-receptor distribution is greater for small than for large airways (6).

Alternatively, the density of ₂-receptors on peripheral blood lymphocytes has been used as an index of -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 -receptors in vivo using the radioligand (^IIC)CGP12177 (8). Serial measurements have shown pulmonary ₂-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 ₂-receptor density (9).

-Receptor Kinetics

The temporal aspects of ₂-receptor trafficking have not been well defined. Using an epitope-tagged human -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 (t_1/2) of 7.5 min (10).

	-RECEPTOR COUPLING: AIRWAY INTRACELLULAR SIGNALING PATHWAYS

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

There is now good evidence that -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 ₂-receptor is in the activated form when it is associated with the  subunit of the G protein, together with a molecule of guanosine triphosphate (GTP), and it is through this  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  subunit for the receptor, causing dissociation and the receptor to return to its low-energy, inactivated form. It is probable that ₂-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 -adrenoceptor that occurs in the absence of -agonist results in a basal level of activity (12). Thus, the role of the -agonist molecule is to amplify this low inherent receptor activity. In the case of the ₂-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 -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 -antagonists bind with high affinity to the low-energy inactivated form of the -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 -agonists to bind to the receptor and enhances the binding of -antagonists (13). If this is the case, -antagonists should not be considered as competing directly for the same receptor, but instead as binding to a different form of the -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 -agonist and -antagonist molecules simultaneously compete for the same region of the -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 ₂-agonists may be mediated through cAMP-independent mechanisms, involving direct interaction of Gs with potassium channels, which are present in the airway smooth muscle cell membrane (15).

View larger version (25K): [in this window] [in a new window]   Figure 2.   2-Adrenoceptor signal transduction pathways in airway smooth muscle.

This has followed the observation that some effects of ₂-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 -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 -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 -adrenoceptor-mediated, only isoproterenol and albuterol appear to cause depolarization and induce rubidium efflux, salmeterol being without effect, although all three ₂-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 ₂-receptor appear to be mediated through Gs proteins and the cAMP-dependent PKA system, -receptors can also couple to Gi proteins. Stimulation of mitogen-activated protein (MAP) kinase by the ₂-receptor has recently been demonstrated (17) and reported to be mediated by the 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 ₂-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 ₂-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 ₂-agonist/receptor signal and response.

	22-RECEPTOR AGONISTS

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Site-directed mutagenesis has been able to identify regions of the ₂-adrenoceptor protein important for ₂-agonist binding to G protein coupling (18). The active site of the receptor, with which ₂-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 ₂-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 -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 -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 -adrenoceptor agonists have an asymmetric center due to the presence of the -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 agonistsfor example, fenoterol, formoterol, and procaterolhave two asymmetric centers, and there are four enantiomersRR, SS, RS, and SRpresent. It is a feature of most biological systems that they are stereospecific, and this is true of ligand/-receptor interactions. Where the individual enantiomers of ₂-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 -OH group and Ser 165. For albuterol, for example, the R-enantiomer is at least 100-fold more potent as a ₂-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 ₂-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).

-Agonist Affinity and Efficacy

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

-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 ₂-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 -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 ₂-adrenoceptors in airway smooth muscle of approximately 65% (25). Low efficacy in a ₂-adrenoceptor agonist does not, however, compromise its clinical effectiveness as a bronchodilator drug.

Kinetics of ₂-Agonist-Induced Airway Smooth Muscle Relaxation

The molecular size and structure of a ₂-agonist determines the manner in which it interacts with the ₂-adrenoceptor in airway smooth muscle. The albuterol molecule, which is 11 Å in length and hydrophilic in nature, accesses the active site of the ₂-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 ₂-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 -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 ₂-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 ₂-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 ₂-adrenoceptor (specifically, residues 149-158), believed to be associated with exo-site binding from molecular modeling (34), with the corresponding section of the ₁-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 ₁-adrenoceptor hybrid was constructed with the same key amino acids (methionine, leucine, isoleucine, isoleucine, valine) from the ₂-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 ₂-adrenoceptor. When the side chain is in association with the exo-site, the molecule is prevented from dissociating from the ₂-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).

View larger version (28K): [in this window] [in a new window]   Figure 3.   The 2-receptor exo-site mechanism of action of salmeterol.

The onset of action of salmeterol on airway smooth muscle is therefore slower than that of other ₂-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 ₂-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 ₂-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 ₂-agonists such as albuterol and salmeterol are therefore consistent with the kinetics of their effects on airway relaxation.

	-RECEPTOR DESENSITIZATION

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Associated with -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 -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 -adrenoceptor/-agonist response.

The principal mechanism of homologous short-term, ₂- agonist-promoted desensitization of the ₂-adrenoceptor is phosphorylation of the receptor by the cAMP-independent kinase (ARK) or other closely related G protein-coupled receptor kinases (GRKs). Mutation studies on the -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 -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.

View larger version (24K): [in this window] [in a new window]   Figure 4.   -Adrenoceptor trafficking.

After hours of agonist exposure, a net loss of cellular receptors occurs (denoted downregulation) via several mechanisms that are independent of receptor phosphorylation. ₂-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 ₂-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 -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 ₂-adrenoceptor agonists, whereas human bronchial smooth muscle is singularly resistant. The level of ARK 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, ARK 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 ₂-receptors would undergo minimal short-term (5 min) agonist-promoted desensitization as compared with the ₂-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 ₂-agonists to subjects with asthma appears to result in desensitization of bronchoprotective responses (41) thought to be mediated by the pulmonary mast cell ₂-receptor, but not the bronchodilatory response of ₂-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 ₂-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 ₂-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 ₂-receptor protein itself, -agonists, acting through the cAMP pathway, also dramatically modulate ₂-receptor gene expression. Isoproterenol resulted in a significant decline (50%) in ₂-receptor transcripts at 4 and 8 h, respectively (45). In comparison to isoproterenol, cells treated with salmeterol had no such downregulating effect on ₂-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 ₂-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 -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 ₂-adrenoceptor, increasing ₂-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 ₂-adrenoceptor downregulation in normal subjects and subjects with asthma who have been exposed to ₂-agonists (48). It is of interest, however, that an inhaled corticosteroid does not apparently prevent tolerance to the bronchoprotective effects of a long-acting ₂-agonist such as formoterol (49) or salmeterol (50).

	POLYMORPHISM OF THE -ADRENOCEPTOR

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A number of common variants (polymorphisms) of ₂-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 ₂-receptors in airway cells. There are two genes for the ₂-adrenoceptor, and therefore an individual can be homozygous or heterozygous for a given polymorphism.

Studies on the ₂-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 ₂-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 ₂-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 ₂-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 -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 ₂-adrenoceptor polymorphisms to the asthma phenotype. However, the relationship between polymorphisms of the ₂-adrenoceptor and pulmonary and systemic exposure to chronic dosing with a ₂-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 -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 ₂-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).