Am J Respir Crit Care Med Vol 158. pp S146–S153, 1998Internet address: www.atsjournals.org

The

b

-Adrenoceptor

MALCOLM JOHNSON

Respiratory Therapeutic Development, Glaxo Wellcome Research and Development, Uxbridge, Middlesex, United Kingdom

The human

b

-adrenoceptor is a member of the seven-transmembrane family of receptors, encoded

by a gene on chromosome 5.

b

-Adrenoceptors have been classified into

b

1

,

b

2

, and

b

3

subgroups,with

b

2

-receptors being widely distributed in the respiratory tract, particularly in airway smooth mus-cle. Intracellular signaling following

b

2

-adrenoceptor activation is largely affected through a trimericGs protein coupled to adenylate cyclase. Cyclic AMP (cAMP) induces airway relaxation through phos-

phorylation of muscle regulatory proteins and attenuation of cellular Ca

2

1

concentrations. Alterna-tive cAMP-independent pathways involving activation of membrane maxi-K

1

channels and couplingthrough Gi to the MAP kinase system have also been described. Site-directed mutagenesis has identi-fied Asp 113 and Ser 204/207 within the third and fourth membrane domains as the active site of the

b

2

-receptor, critical for

b

2

-agonist binding and activity.

b

2

-Agonists have been characterized as thosethat directly activate the receptor (albuterol), those that are taken up into a membrane depot (for-moterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol). Thesedifferences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxationand bronchodilation in patients with asthma.

b

-Adrenoceptor desensitization associated with

b

2

-ago-nist activation is a consequence of phosphorylation by

b

-ARK and uncoupling of the receptor from Gsfollowing

b

-arrestin binding, of internalization and recycling of the receptor through processes of se-questration and resensitization and downregulation, modulated by an effect on receptor gene ex-pression. The degree of receptor desensitization appears to differ, depending on the cell or tissuetype, and is reflected in the different profiles of clinical tolerance to chronic

b

2

-agonist therapy. Anumber of polymorphisms of the

b

2

-receptor have been described that appear to alter the behaviorof 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 hyper-reactivity, nocturnal symptoms, and more severe asthma. The Glu 27 form appears to protect againstdownregulation and is associated with less reactive airways. An individual can be homozygous or het-erozygous for given polymorphisms, and large populations will have to be studied to determine their

importance to the asthma phenotype.

Johnson M. The

b

-adrenoceptor.

AM J RESPIR CRIT CARE MED 1998;158:S146–S153.

THE

b

-ADRENOCEPTOR

b

-Adrenoceptor Structure

The human

b

-adrenoceptor gene is situated on the long armof chromosome 5 and codes for an intronless gene product ofapproximately 1,200 base pairs (1). The

b

-adrenoceptor is amember of the seven-transmembrane family of receptors (Fig-ure 1) related to bacteriorhodopsin, which was used for theearly structural work (2). It is composed of 413 amino acid res-idues of approximately 46,500 daltons (Da) (2).

b

-Adrenocep-tors have been subdivided into at least three distinct groups:

b

1

,

b

2

, and

b

3

, classically identified in cardiac, airway smoothmuscle, and adipose tissue, respectively (3). There is a 65–70%homology between

b

1

/

b

3

- and

b

2

-receptors. This discussionwill focus primarily on

b

2

-receptors in the respiratory tract.

b

-Receptor Density

Autoradiographic studies of human lung have suggested that

b

2

-adrenoceptors are widely distributed, occurring not only inairway smooth muscle but also on other cells in the lung, suchas epithelial and endothelial cells, type II cells, and mast cells(4). Until recently, quantification of these pulmonary recep-

tors has only been possible

in vitro

. Radioligand binding stud-ies on lobectomy specimens have shown

b

2

-receptor density toincrease with increasing airway generation, with high levels inthe alveolar region (5). Computed tomography (CT) scanninghas confirmed that

b

2

-receptor distribution is greater for smallthan for large airways (6).

Alternatively, the density of

b

2

-receptors on peripheralblood lymphocytes has been used as an index of

b

-receptors inthe airways (7), but numbers (700–750 receptors per cell) aresubstantially less than in smooth muscle (30,000–40,000 percell). Position emission tomography (PET) has now made pos-sible the noninvasive quantification of

b

-receptors

in vivo

us-ing the radioligand (

II

C)CGP12177 (8). Serial measurementshave shown pulmonary

b

2

-receptor density to be 10.9

6

1.0picomole (pmol)/g tissue, compared with 8.8

6

2.3 pmol/g forcardiac tissue (8). There was no difference between normal

Correspondence and requests for reprints should be addressed to Malcolm John-son, Respiratory Therapeutic Development, Glaxo Wellcome Research and Devel-opment, Uxbridge, Middlesex UB 11 1BT, UK.

Johnson: The

b

-Adrenoceptor

S147

subjects and patients with asthma (9), but an inverse relation-ship was reported between FEV

1

(% predicted) and lung

b

2

-receptor density (9).

b

-Receptor Kinetics

The temporal aspects of

b

2

-receptor trafficking have not beenwell defined. Using an epitope-tagged human

b

-receptor, re-cycling, as measured by radioligand binding using the hydro-philic ligand, (

3

H)-CGP12177, proceeded with an apparentrate constant of 0.09, which reflects a one-phase exponentialkinetic model with a recycling half-life (t

1/2

) of 7.5 min (10).

b

-RECEPTOR COUPLING: AIRWAY INTRACELLULAR SIGNALING PATHWAYS

It has been the accepted dogma since the 1960s that

b

-adreno-ceptor activation increases intracellular cyclic adenosine mono-phosphate (cAMP) levels. The coupling of the

b

-adrenoceptorto adenylate cyclase is affected through a trimeric Gs protein,which consists of

a

,

b

, and

g

subunits (11).There is now good evidence that

b

-adrenoceptors exist intwo forms, activated and inactivated, and that under restingconditions these two forms are in equilibrium but with the in-activated state being predominant (12). The

b

2

-receptor is inthe activated form when it is associated with the

a

subunit ofthe G protein, together with a molecule of guanosine triphos-phate (GTP), and it is through this

a

subunit that the receptoris coupled to adenylate cyclase. The replacement of the GTP

by guanosine diphosphate (GDP) both catalyzes the conver-sion of ATP to cAMP by the enzyme and dramatically reducesthe affinity of the

a

subunit for the receptor, causing dissocia-tion and the receptor to return to its low-energy, inactivatedform. It is probable that

b

2

-agonists have their effects, notthrough inducing a conformational change in the receptor, butrather by binding to and temporarily stabilizing receptors intheir activated state, i.e., bound to Gs-GTP, and thereforeshifting the equilibrium (12). Implicit in this is the possibilitythat the spontaneous, albeit low, frequency of interconversionof inactivated to activated

b

-adrenoceptor that occurs in theabsence of

b

-agonist results in a basal level of activity (12).Thus, the role of the

b

-agonist molecule is to amplify this lowinherent receptor activity. In the case of the

b

2

-adrenoceptor,this would be manifested as a basal level of cAMP turnover.Indeed, there is some evidence for this mechanism since singleamino acid mutations made to

b

-adrenoceptors, which resultin a shift in the resting equilibrium toward the activated state,are coupled to sustained increases in intracellular second mes-sengers in the absence of agonist (12).

The corollary is that

b

-antagonists bind with high affinity tothe low-energy inactivated form of the

b

-adrenoceptor, andthus shift the equilibrium away from the activated form. Thisis supported by the observation that addition of GDP inhibitsthe ability of

b

-agonists to bind to the receptor and enhancesthe binding of

b

-antagonists (13). If this is the case,

b

-antago-nists should not be considered as competing directly for thesame receptor, but instead as binding to a different form of the

Figure 1. Structure of the human b2-adrenoceptor.

S148

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 158 1998

b

-adrenoceptor protein and moving the equilibrium in oppo-site directions. While this would result in a competitive inter-action, it is not competitive in the sense that

b

-agonist and

b

-antagonist molecules simultaneously compete for the sameregion of the

b

-adrenoceptor protein.The mechanism by which cAMP induces airway smooth

muscle cell relaxation is not fully understood, but it is believedthat it catalyzes the activation of protein kinase A (PKA),which in turn phosphorylates key regulatory proteins involvedin the control of muscle tone (Figure 2). cAMP also results in

inhibition of calcium ion (Ca

2

1

) release from intracellularstores, reduction of membrane Ca

2

1

entry, and sequestration ofintracellular Ca

2

1

, leading to relaxation of the airway smoothmuscle (14). However, it has been suggested recently thatsome of the relaxant response to

b

2

-agonists may be mediatedthrough cAMP-independent mechanisms, involving direct in-teraction of Gs

a

with potassium channels, which are presentin the airway smooth muscle cell membrane (15).

This has followed the observation that some effects of

b

2

-adrenoceptor agonist stimulation may be inhibited by charyb-dotoxin and iberiotoxin, inhibitors of high-conductance, Ca

2

1

-activated potassium ion (K

1

) channels (maxi-K channels)(16). Although these agents can markedly inhibit

b

-adreno-ceptor–mediated effects on airway smooth muscle, they haveno such effects on mast cells, suggesting a degree of tissuespecificity in transduction processes. In support of the involve-ment of K

1

fluxes in

b

-adrenoceptor agonist activity is the ob-servation that in bovine tracheal smooth muscle cells, isopro-terenol and albuterol both depolarize the cell membrane andcause the opening of K

1

channels, as indicated by an increasein rubidium efflux (16). It is interesting, however, that al-though this effect is clearly

b

-adrenoceptor–mediated, onlyisoproterenol and albuterol appear to cause depolarization

and induce rubidium efflux, salmeterol being without effect,although all three

b

2

-agonists relax the preparation (16). It isdifficult to reconcile these data, but it suggests that K

1

chan-nel activation may be a function of ligand efficacy and is notobligatory in airway smooth muscle relaxation.

Although most of the actions of the

b

2

-receptor appear tobe mediated through Gs proteins and the cAMP-dependentPKA system,

b

-receptors can also couple to Gi proteins. Stim-ulation of mitogen-activated protein (MAP) kinase by the

b

2

-receptor has recently been demonstrated (17) and reported tobe mediated by the

bg

subunits of pertussis toxin–sensitive Gproteins through a pathway involving the nonreceptor tyro-sine kinase cSrc and the G protein RaS. Activation of thispathway by the

b

2

-receptor requires that the receptor be phos-phorylated by PKA, since inhibitors of PKA block the re-sponse and a mutant lacking the normal phosphorylation sitescan activate adenylate cyclase, but not MAP kinase. Thismechanism may serve not only to mediate uncoupling of the

b

2

-receptor from Gs and thus heterologous desensitization,but may also switch the coupling of the receptor from Gs to Giand represent direct feedback inhibition as a means of termi-nating the

b

2

-agonist/receptor signal and response.

b

2

-RECEPTOR AGONISTS

Site-directed mutagenesis has been able to identify regions ofthe b2-adrenoceptor protein important for b2-agonist bindingto G protein coupling (18). The active site of the receptor,with which b2-agonists must interact in order to exert their bi-ological effects, is located approximately one-third of the way(15 Ångström units [Å]) into the receptor core (Figure 1). It isgenerally agreed that there are residues of critical importancewith respect to agonist binding to the active site, namely as-

Figure 2. b2-Adrenoceptor signal transduction pathways in airway smooth muscle.

Johnson: The b-Adrenoceptor S149

partate (Asp)-residue 113 (counted from the extracellular orN-terminus end) of the third domain, two serine (Ser) resi-dues, 204 and 207, which are both on the fifth domain, and twophenylalanines (Phe), 259 and 290, on the sixth domain (19).Thus, a model has emerged for the agonist binding site of theb2-adrenoceptor, in which the ligand is bound within the hy-drophobic core of the protein, intercalated among the trans-membrane helices, and anchored by specific molecular inter-actions between amino acid residues in the receptor andfunctional groups on the ligand (19).

Asp binds to the nitrogen of the b-adrenoceptor agonistmolecule, while the two Ser residues interact with the hy-droxyl groups on the phenyl ring. Other residues may also beimportant, e.g., there is evidence that Asp residue 79 on thesecond domain and threonine (Thr)-164 are involved in ago-nist recognition (19). It is becoming increasingly clear that an-tagonists do not interact with the same amino acids as agonistsin binding to the b-adrenoceptor. Thus, it appears that al-though antagonists probably bind to Asp-113, they do not in-teract with the two Ser residues on the fifth domain but ratherwith an asparagine (Asn) residue, 312 in the seventh domain(20).

All b-adrenoceptor agonists have an asymmetric centerdue to the presence of the b-OH group on the ethanolaminefunction (21). The presence of an asymmetric center results inthe molecule existing as a pair of optical isomers (or mirrorimages), referred to as the R and S [or (2) and (1)] enanti-omers, in a racemic mixture. In fact, some agonists—for exam-ple, fenoterol, formoterol, and procaterol—have two asym-metric centers, and there are four enantiomers—RR, SS, RS,and SR—present. It is a feature of most biological systems thatthey are stereospecific, and this is true of ligand/b-receptor in-teractions. Where the individual enantiomers of b2-adreno-ceptor agonists have been resolved and tested, it is clear thatthe activity lies predominantly in the R-enantiomer, probablyas a result of an optimal interaction between the “down” ori-entation of the b-OH group and Ser 165. For albuterol, for ex-ample, the R-enantiomer is at least 100-fold more potent as ab2-agonist than the S-enantiomer (21), whereas this differenceis greater than 1,000-fold for the RR and SS forms of formot-erol (22).

In the case of salmeterol, where enantiomerically puresamples have been prepared, there is still significant b2-ago-nist activity in the S-enantiomer, which is only 40-fold less po-tent than the R-form and 15-fold weaker than the racemicmixture. Interestingly, both the R- and S-enantiomers of sal-meterol are long-acting (23). There is no evidence of the S-iso-mer of salmeterol antagonizing the effects of the correspond-ing R-form, or of the S-enantiomer having pharmacologiceffects different from those of the racemic mixture (23).

b-Agonist Affinity and Efficacy

The affinity of a ligand is a measure of the avidity of its bind-ing to its receptor. Few b2-agonists have been shown to havemuch higher affinity than isoproterenol and, indeed, albuterolhas a relatively low affinity for b2-adrenoceptors. In contrast,salmeterol and formoterol have high affinities for the b2-adrenoceptor with a KI of 53 nM and 74 nM, respectively,compared with 200 and 2,500 nM for isoproterenol and al-buterol (24).

b-Agonist potency, however, is a function not only of re-ceptor affinity, but also of efficacy. A full agonist will have ahigh efficacy while a pure antagonist will have low or zero effi-cacy. The majority of b2-adrenoceptor agonists have an inter-mediate efficacy, and if tissue factors permit, they will behaveas full agonists; however, if receptor density is too low or cou-

pling is inadequate, the b-agonist may behave in a partial man-ner, i.e., it will be incapable of achieving the same maximumeffect as an agonist of higher efficacy, and it may even behaveas an antagonist. Examples of compounds of high efficacy (ap-proximately equivalent to isoproterenol) are procaterol, fe-noterol, and formoterol, whereas most saligenins and resor-cinols, albuterol and terbutaline, for example, tend to be ofmoderate efficacy (65–85%), and the efficacy of the dichloro-aniline, clenbuterol, is low (40%). Salmeterol has an efficacyat b2-adrenoceptors in airway smooth muscle of approxi-mately 65% (25). Low efficacy in a b2-adrenoceptor agonistdoes not, however, compromise its clinical effectiveness as abronchodilator drug.

Kinetics of b2-Agonist–Induced AirwaySmooth Muscle Relaxation

The molecular size and structure of a b2-agonist determinesthe manner in which it interacts with the b2-adrenoceptor inairway smooth muscle. The albuterol molecule, which is 11 Åin length and hydrophilic in nature, accesses the active site ofthe b2-adrenoceptor directly from the extracellular compart-ment (26). There is therefore a rapid onset of airway tissue re-laxation and of bronchodilation in patients. However, the drugrapidly re-equilibrates, its residency time at the active site islimited, and the resulting duration of action short (4–6 h).

Formoterol is moderately lipophilic in nature (27). It istaken up into the cell membrane in the form of a depot, fromwhere it progressively leaches out to interact with the activesite of the b2-receptor (27). The size of the depot is deter-mined by the concentration or dose of formoterol applied. Inairway preparations, the onset of action of formoterol is some-what delayed compared with albuterol, and the duration ofrelaxant activity, although longer, is concentration-dependent(28). This profile has been confirmed clinically in patients withasthma, where bronchodilation was observed for 8, 10, and12 h following doses of 6, 12, and 24 mg, respectively (29).

The salmeterol molecule is 25 Å in length and it is greaterthan 10,000 times more lipophilic than albuterol (30). The useof low-angle neutron diffraction techniques to study the inter-action of salmeterol with the cell membrane has indicated thatit partitions rapidly (, 1 min) into the outer phospholipidmonolayer by a factor approaching 30,000:1 (31). Molecularmodeling suggests that the orientation is such that the salige-nin moiety is the same plane as the polar head groups, withthe side chain in close association with the hydrophobic tailsof the phospholipids. It is of interest that 17 Å side chain ofsalmeterol, which was found to be optimal for duration of ac-tion, is the same as the depth of the phospholipid monolayer(30). There is no evidence that salmeterol “flip-flops” fromthe outer to the inner monolayers of the surface phospholip-ids, but instead the molecule diffuses laterally to approach theactive site of the b-adrenoceptor through the membrane (31).This translocation process appears to be slow (. 30 min).

The experimental data indicates that the receptor bindingof salmeterol is only slowly reversible and noncompetitive,whereas functional responses to the molecule are both fullyreversible and competitive (32). In order to rationalize thesefindings, the “exo-site” hypothesis was proposed (33). Theoriginal concept was that the long side chain of the moleculeinteracted with a nonpolar region in the cell membrane, theexo-site, in the vicinity of the b2-receptor. High-affinity bind-ing of the side chain to the exo-site then allowed the saligeninhead to repeatedly activate the receptor, enabling salmeterolto be long-acting. From the molecular modeling studies, it hasbeen predicted that there is a preferred “down” conformationof the molecule in the receptor protein (34), whereby the sali-

S150 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 158 1998

genin head binds to the active site in an analogous position tothat of albuterol, and the long, flexible side chain is locateddeep into a hydrophobic core domain of the receptor, suggest-ing that the specific exo-site for salmeterol may be an integralpart of the b2-adrenoceptor protein itself.

Site-directed mutagenesis studies (35) showed that it waspossible to replace a discrete length of the fourth transmem-brane domain of the b2-adrenoceptor (specifically, residues149–158), believed to be associated with exo-site binding frommolecular modeling (34), with the corresponding section ofthe b1-adrenoceptor, while maintaining the affinity of salme-terol for the resulting hybrid receptor. However, significantly,this modification resulted in a decreased persistence of agonistactivity after washout. Even more significantly, when the cor-responding b1-adrenoceptor hybrid was constructed with thesame key amino acids (methionine, leucine, isoleucine, isoleu-cine, valine) from the b2-adrenoceptor, this resulted in mark-edly enhanced persistence of salmeterol activity (35).

The mechanism of action of salmeterol therefore involvesthe interaction of the side chain with an auxiliary binding site(exo-site), a domain of highly hydrophobic amino acids withinthe fourth domain of the b2-adrenoceptor. When the sidechain is in association with the exo-site, the molecule is pre-vented from dissociating from the b2-adrenoceptor, but thesaligenin head can freely engage and disengage the active siteby the Charniére (hinge) principle, flexion being about the ox-ygen atom in the side chain (Figure 3). The position of thisoxygen atom was shown in structure–activity studies to be crit-ical for duration of action (34).

The onset of action of salmeterol on airway smooth muscleis therefore slower than that of other b2-agonists, such as al-buterol and formoterol. However, whereas the duration of ac-tion of the latter can be increased by increasing the concentra-tion applied to the tissue, salmeterol appears to be inherentlylong-acting, in that its effects are independent of dose, as a re-sult of exo-site binding. The duration of action of b2-agonistsagainst spasmogen-induced, neuronally mediated, and inher-ent 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 smoothmuscle cells is rapid with isoproterenol and albuterol, whereas

salmeterol increases intracellular cAMP more slowly, consis-tent with the membrane access of the molecule to the b2-adrenoceptor. In addition, the maximum elevation of cAMPto salmeterol achieves only 45% of that to isoproterenol, con-firming the partial agonist nature of the response. However,whereas cAMP responses to isoproterenol and albuterol aretransient, and rapidly reversed toward basal levels by wash-ing the cells, salmeterol induces a sustained (. 120 min) eleva-tion of intracellular cAMP. The changes in intracellular cAMPwith b2-agonists such as albuterol and salmeterol are thereforeconsistent with the kinetics of their effects on airway relax-ation.

b-RECEPTOR DESENSITIZATION

Associated with b-adrenoceptor activation is the auto-regula-tory process of receptor desensitization. This process operatesas a safety device to prevent overstimulation of receptors inthe face of excessive b-agonist exposure. Desensitization oc-curs in response to the association of receptor with the agonistmolecule, and is prevented by the interaction of the receptorwith an antagonist. The mechanisms by which desensitizationcan occur consist of three main processes: (1) uncoupling ofthe receptors from adenylate cyclase; (2) internalization of un-coupled receptors; and (3) phosphorylation of internalized re-ceptors (38). The extent of desensitization depends on the de-gree and duration of the b-adrenoceptor/b-agonist response.

The principal mechanism of homologous short-term, b2-agonist–promoted desensitization of the b2-adrenoceptor isphosphorylation of the receptor by the cAMP-independent ki-nase (bARK) or other closely related G protein–coupled re-ceptor kinases (GRKs). Mutation studies on the b-adrenocep-tor protein have shown that the third intracellular loop andthe intracellular C-terminus are the major sites of phosphory-lation (38). Such phosphorylation ultimately results in bindingof b-arrestin and partial uncoupling of the agonist-occupiedform of the receptor from the stimulatory guanine nucleotide–binding protein Gs, thereby limiting receptor function. Simpleuncoupling is a transient process and may be reversed withinminutes of removal of the agonist.

After more prolonged agonist exposure, an internalizationof receptors occurs, which results in a loss of some proportionof cell surface receptors. This process, termed sequestration,has also been considered to be another mechanism of desensi-tization, but recent studies have suggested that its major rolein short-term regulation of the receptor may be in resensitiza-tion (Figure 4), since it appears that the sequestered pool isthe site of dephosphorylation of the receptor. Internalizationtakes longer to reverse than uncoupling, but full reversal nor-mally occurs within hours.

After hours of agonist exposure, a net loss of cellular re-ceptors occurs (denoted downregulation) via several mecha-nisms that are independent of receptor phosphorylation. b2-Receptor trafficking as part of the overall process of receptordesensitization has now been investigated in the form of ki-netic analysis of internalization and recycling of the human b2-receptor. Cellular trafficking was measured by flow cytome-try, quantifying the cell surface levels of a monoclonal Ab(12CA5) against the hemagglutinin epitope of the receptorectodomain (39). In the presence of a b-agonist (isoprotere-nol, 5 mM), steady-state rate constants of 0.38 and 0.25 forinternalization and recycling, respectively, were determined,with a total transit time for the receptor cycling between thecell surface and the endocytic compartment of 6.6 min (39).

The process of desensitization may differ markedly fromtissue to tissue. It is clear, for example, that human lympho-

Figure 3. The b2-receptor exo-site mechanism of action of salme-terol.

Johnson: The b-Adrenoceptor S151

cytes desensitize very rapidly on exposure to b2-adrenoceptoragonists, whereas human bronchial smooth muscle is singu-larly resistant. The level of bARK mRNA in airway smoothmuscle cells was only about 20% of that in bronchial epithelialcells and approximately 11% of that in mast cells (40). At theprotein level, bARK expression in airway smooth muscle cellswas nearly undetectable, being about 10-fold less than that ex-pressed in mast cells. A marked discrepancy in GRK activitieswas also observed with mast cells (90.7 6 0.5 relative units) ascompared with airway smooth muscle cells (9.3 6 0.6 relativeunits, p , 0.001). In contrast, the activities of cAMP-depen-dent PKA were not different (40). This predicts that airwaysmooth muscle b2-receptors would undergo minimal short-term (5 min) agonist-promoted desensitization as comparedwith the b2-receptor expressed on mast cells. In response toisoproterenol (1 mM), mast cell cAMP reached maximum lev-els after 90 s and did not further increase over time, indicativeof receptor desensitization in this cell. In contrast, cAMP lev-els of airway smooth muscle cells did not plateau, increasing ata rate of 103 6 9% per min, consistent with little desensitiza-tion over the study period (40). This may explain the clinicalobservation that repetitive administration of b2-agonists tosubjects with asthma appears to result in desensitization ofbronchoprotective responses (41) thought to be mediated bythe pulmonary mast cell b2-receptor, but not the bronchodila-tory response of b2-receptor expressed on bronchial smoothmuscle (42). This type of difference may also be manifested inthe well documented decline in the side effects associated withb2-adrenoceptor agonist therapy (e.g., tachycardia and physio-logic tremor) in patients with asthma, but the maintenance ofbronchodilatation despite regular treatment for prolonged pe-riods (42).

As desensitization results from agonist occupancy and canbe inhibited by antagonists, it follows that a partial agonistwould be less prone to induce receptor desensitization than afull agonist. Indeed, this has been demonstrated to be the casewith b2-agonists clinically, where a degree of bronchodilatortolerance was observed with the high-efficacy agonist formot-erol on chronic exposure and despite the presence of the corti-costeroid budesonide (43), but not with the partial agonist sal-meterol (44).

It is now well appreciated that in addition to desensitiza-tion processes that negatively regulate the function of the b2-receptor protein itself, b-agonists, acting through the cAMPpathway, also dramatically modulate b2-receptor gene expres-sion. Isoproterenol resulted in a significant decline (50%) in

b2-receptor transcripts at 4 and 8 h, respectively (45). In com-parison to isoproterenol, cells treated with salmeterol had nosuch downregulating effect on b2-receptor gene expression(45). These data are consistent with the hypothesis that thelong-acting characteristics of salmeterol may be due, at least inpart, to the ability of this agonist to maintain a population offunctional b2-receptors through persistent elevation of genetranscription, despite a prolonged, low-level exposure to theagonist.

Two weeks of albuterol treatment (4 mg orally twice dailyand 200 mg four times daily) resulted in a decrease in b-recep-tor density, assessed by PET scanning, of 22% in the lung (46).This was associated with a reduction in bronchodilator re-sponse to albuterol (46). Corticosteroids have facilitatory ef-fects on the b2-adrenoceptor, increasing b2-receptor gene tran-scription, through binding and activation of cAMP responseelement binding protein (CREB), regulating both the num-bers of the receptors and the coupling of the receptor to ade-nylate cyclase (47). Systemic corticosteroids have been shownto reverse b2-adrenoceptor downregulation in normal subjectsand subjects with asthma who have been exposed to b2-ago-nists (48). It is of interest, however, that an inhaled cortico-steroid does not apparently prevent tolerance to the broncho-protective effects of a long-acting b2-agonist such as formoterol(49) or salmeterol (50).

POLYMORPHISM OF THE b-ADRENOCEPTOR

A number of common variants (polymorphisms) of b2-recep-tor have recently been described (51) that alter the behaviorof the receptor following agonist exposure. The main clinicalinterest in these polymorphisms lies in the possibility that theymay determine the extent to which the receptor downregu-lates in the airways and as such may modify bronchodilatorresponses through changes in the expression and coupling ofb2-receptors in airway cells. There are two genes for the b2-adrenoceptor, and therefore an individual can be homozygousor heterozygous for a given polymorphism.

Studies on the b2-adrenoceptor identified a total of ninedifferent polymorphisms (51). All of these differed from theaccepted wild-type sequence by a single base change at differ-ent positions in the coding sequence of the gene. Because ofredundancy in the amino acid code, a number of these poly-morphisms are clinically silent. However, four polymorphismsresulting from single base changes were identified that alteredthe amino acid sequence of the receptor protein (51). Three ofthese polymorphisms have now been studied in some detail,and all three appear to alter the functional properties of thereceptor, such that the airways of individuals with these formsof the receptor might be expected to behave differently whenexposed to circulating catecholamines or exogenously appliedb2-agonists.

The initial studies focused (51) on amino acid 16 (Figure 1),which can be either arginine (Arg) or glycine (Gly), dependingon whether base 46 is A or G. The data suggest that the abilityof a receptor to desensitize is markedly influenced by the pres-ence of Gly 16. The Gly 16 receptor downregulates followingexposure to an agonist to a much greater extent than the Arg16 form in both transfected cell systems and in primary cul-tured human airway smooth muscle cells (51). Two recentclinical studies have supported the possibility that the Gly 16form of the receptor is associated with markers of more severeasthma. Preliminary data from Dutch families with asthmasuggest that Gly 16 may be associated with airway hyperreac-tivity (52). In addition, patients with significant nocturnalworsening of their asthma were more likely to have the Gly 16

Figure 4. b-Adrenoceptor trafficking.

S152 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 158 1998

form of the receptor than patients with asthma without noc-turnal falls in peak flow rate (53). The allelic frequencies forArg 16 and Gly 16 are 35% and 65%, respectively (54).

The second polymorphism is at codon 27 (Figure 1), whichexists as either glutamine (Gln) or as glutamate (Glu), de-pending on whether base 76 is C or G. The allelic frequencyfor Gln 27 and Glu 27 is 55% and 45%, respectively (54). Incontrast to Gly 16, the Glu 27 form of the receptor appears toprotect against downregulation (55). Using primary culturedhuman airway smooth muscle cells, following prolonged expo-sure to b2-agonists, the Glu 27 form downregulated to a muchlesser extent than the Gln 27 receptor, as assessed by changesin receptor number (56). In addition, a similar relative resis-tance to downregulation was observed using b2-agonist–medi-ated 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 fourtimes less reactive airways than those with Gln 27 when as-sessed using methacholine challenge. Heterozygotes had anintermediate mean PD20 value (57). Where homozygous Glu27, which is predicted to protect against receptor desensitiza-tion, is combined with homozygous Gly 16, the effects of Gly16 are dominant (58).

The third polymorphism is at amino acid 164, which can ei-ther be Thr or isoleucine (Ile) (Figure 1). This polymorphismis much rarer than that at amino acid 16 or 27, with an allelicfrequency of about 1% (59), but it is potentially interesting inthat amino acid 164 is situated in the fourth transmembranespanning domain of the receptor and is adjacent to Ser 165,which has been predicted to interact with the b-OH group ofadrenergic ligands. This polymorphism has been studied in atransfected cell system and has been shown to alter the ago-nist-binding properties of the receptor. Cells expressing lle 164were found to have approximately four times less ligand affinity(59). This alteration in binding affinity was reflected in a re-duced 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 thatArg-Gly 16 and Gln-Glu 27 polymorphisms may be in linkagedisequilibrium, large populations will have to be studied to de-termine the importance of b2-adrenoceptor polymorphisms tothe asthma phenotype. However, the relationship betweenpolymorphisms of the b2-adrenoceptor and pulmonary andsystemic exposure to chronic dosing with a b2-agonist hasbeen investigated (58, 60). In 10 of 14 subjects with nonresis-tant genotypes (Gly/Gly 16; Gly/Arg 16), there was a signifi-cant reduction (mean, 24%) in pulmonary b-adrenoceptors,as assessed by PET scanning after 2 wk dosing with albuterol.Four subjects who were heterozygous for the Glu 27 polymor-phism were resistant to downregulation of pulmonary b2-recep-tors (60). Similarly, homozygous Gly 16 was significantlymore prone to bronchodilator tolerance (46%) than Arg 16(8%) following administration of formoterol (24 mg bd) for4 wk (58).

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