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REVIEWS

The most readily observed drug effect is tissue response,and those molecules that produce responses are calledagonists. Such molecules are thought to have ‘efficacy’,a molecular property that has been discussed innumerous reviews on classical drug receptor theory1–4.This review will be confined to molecules that induceeffects through interaction with receptors, specificallyG-protein-coupled receptors (GPCRs), but not neces-sarily only through G-protein activation. Physiologicalresponses due to interactions with other cellular com-ponents, such as phosphorylating kinases and proteinsthat modify receptor–G-protein coupling, will not beconsidered.

On the assumption that all drugs acting at receptorsrequire affinity for the receptor to induce an effect,agonists seem to be a subset of all the drugs in pharma-cological space — they have both affinity for theirreceptors and efficacy to produce an effect. The basicpremise of this review is that efficacy should not beconfined to G-protein activation, but should also beexpanded to the complete range of behaviours ofGPCRs, such as receptor internalization, desensitization,oligomerization, phosphorylation and association withother membrane proteins. There are many types ofefficacy, and ligands might selectively influence somereceptor behaviours and not others. Given this, abroader definition would be that efficacy is the propertyof a ligand that causes the receptor to change itsbehaviour towards the cell. This review will also present

a probabilistic model of receptor behaviour5,6, whichsuggests that a single thermodynamic mechanismmight account for this apparently diverse range of ligandeffects. This model predicts that affinity and efficacy areinextricably linked, and that molecules with affinitymight well also have some form of efficacy. The discoverythat efficacy exists in a wider range of molecules thanperhaps previously thought could have practical rami-fications, as many molecules might have unrealizedtherapeutic value.

Efficacy: the historical perspectiveMisconceptions about efficacy might have resulted fromthe limited vantage point that pharmacologists have hadto observe receptor behaviour. In the 1950s, the firstmethod that was used to express and quantify the abilityof agonists to evoke a response was the comparison ofagonist maximal responses (intrinsic activity)7. A short-coming of this approach is that the extent of theresponse is tightly controlled by the efficiency of theorgan system being examined. So, depending on thereceptor density and the efficiency of receptor couplingin different organs, ligands with low levels of efficacycould be defined experimentally as being full agonists,partial agonists or antagonists; that is, the environmentof the receptor dictates the apparent property of thedrug8. Shortly after these initial experiments, Stephensonfirst referred to the term efficacy to denote the power ofan agonist to produce a response9.A method to compare

EFFICACY AT G-PROTEIN-COUPLEDRECEPTORSTerry Kenakin

At present, the drug-discovery process centres on ligands that either block or producephysiological responses. However, there are therapeutic uses for ligands that do neither of thesethings, but which still affect receptors in other ways. This review discusses the intimaterelationship between the affinity of a ligand for its receptor, and the probability that the binding ofthe ligand will produce some change in the receptor, resulting in efficacy. This, in turn, argues thatligands that have affinity should be tested more broadly, for a wider range of efficacies, to detecthidden therapeutic activities.

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GlaxoSmithKline Researchand Development, 5 MooreDrive, Research TrianglePark, North Carolina 27709,USA. e-mail:tpk1348@gsk.comDOI: 10.1038/nrd722

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LINKAGE THEORY

Based on the first law ofthermodynamics, linkage theorycreates models of protein speciesthat are connected, and whichcan interconvert eitherspontaneously or throughinteraction with other species,such as ligands and G proteins.The interconversion pathwaysbetween any species are of equalenergy, so none are preferred.

PLEIOTROPIC RECEPTOR

A receptor that couples to morethan one G protein. An exampleis the human calcitonin receptor,which couples to G

i, G

sand G

q

proteins.

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these models, every agonist-activated ternary complex isassumed to have equal propensity to produce G-proteinactivation. This idea does not agree with experimentaldata, which describe agonist-specific receptor activestates that have differing abilities to produce G-proteinactivation (reviewed in REF. 22).

At the molecular level, efficacy as a strictly receptor-based ligand property, even when it is concerned onlywith G-protein activation, can be ambiguous forPLEIOTROPIC RECEPTORS; that is, those receptors that canactivate more than one type of G protein. This is becausethe relative stoichiometry of receptors to G proteins canaffect the quantity and quality of the response in somesystems. For example, in chinese hampster ovary (CHO)cells expressing low levels of human calcitonin recep-tors, calcitonin agonists exclusively produce elevation ofcyclic AMP. However, in human embryonic kidney(HEK) cells with high expression levels of human calci-tonin receptors, the same agonists produce both cAMPelevation and transient calcium signals18,19. This isbecause the calcitonin receptor can interact with G

s, G

q

and Giproteins with differential sensitivity, and the rela-

tive affinities of the receptors for these various G proteins

the relative ability of agonists to produce a response fora given receptor occupancy was then described byFurchgott (intrinsic efficacy)10. All of these approacheswere purely empirical, in that they did not address themolecular mechanisms that lead to efficacy.

The most explicit mechanistic depiction of efficacycan be found in receptor models that are based onLINKAGE THEORY, in which the protein species are linkedthrough energy conversions that are equal with respectto the path taken to reach a given end point (in accor-dance with the first law of thermodynamics)11–14. Interms of these models, the energy that is used to getfrom a set of reactants to a set of products is independentof how the products were derived. GPCRs produceresponses by coupling to G proteins and inducing sep-aration of the G-protein subunits, which subsequentlyactivate cellular effectors15–17. The ability of ligands to dothis is one representation of efficacy. Some examples ofmodels that are used to describe GPCR behaviour aredescribed in BOX 1. Although these models go some wayto describing the mechanism of efficacy, they arerestrictive in that they do not describe the quality of thereceptor–G-protein complex, only the quantity. Within

Box 1 | Commonly used models of GPCR systems

The classical view of receptor activationdescribes agonist (A) binding to aninactive receptor (R

i) to form a complex

(ARi) that, because of the efficacy of the

agonist, isomerizes the receptor to theactive state (AR

a) (panel a). Following

the discovery of ternary complexation forG-protein-coupled receptors (GPCRs),it was proposed that the activation of thereceptor was followed by topographicallydistinct binding of the active-statereceptor to the G protein (G). Responsethen emanates from the ternary AR

aG

complex (panel b). The extended ternarycomplex model89 (panel c) allows for thespontaneous formation of an active-statereceptor (R

a) independent of the presence

of an agonist. Ra

can then interact with,and activate, a G protein. Activation ofthe receptor (from R

ito R

a), either

spontaneously or through ligand binding,modifies the affinity of the receptor forthe G protein by the factors β and γ. It isthis modified affinity of the receptor forthe G protein that is the expression ofefficacy. The cubic ternary complex(CTC) model90–92 (panel d) does the samething, but also allows the inactive-statereceptor (R

i) to form a non-signalling

complex with the G protein, with theaffinity of the receptor for the G proteinbeing modified by the factors δ, β and γ.This non-signalling complex is requiredthermodynamically as a feature of thecomplete system, but might not berelevant physiologically.

αL

L

Ka αKa αβKa

γβKg

βKg

ARi ARa ARaG ARaG

RaGRaRi

αL

γKg

L

Ka

KgαKa

ARi ARa

RaGRiG

βKg

δγβKg

δγαKa

γKa

βL

δαβLARiG

RaRi

c Extended ternary complex model d Cubic ternary complex model

a Simple binding and activation

b Simple ternary complex model

Ri Concentration of receptor in the inactive state (this species does not activate G proteins)Ra Concentration of receptor in the active state (this species activates G proteins)G Concentration of G proteins in the systemKa Equilibrium association constant for agonist and receptorKg Equilibrium association constant for receptor and G proteinL Allosteric constant denoting the ratio of receptor in the active versus inactive state (L=Ra/Ri)

γ Factor defining the differential affinity of the receptor for G proteins when the receptor is ligand bound. In the CTC model, γ defines the effect of the ligand on G protein binding to the inactive-state receptor

α Factor defining the differential affinity of the ligand for the active versus the inactive state. Also, the effect of ligand binding on receptor activationβ Factor defining the differential affinity of the receptor for G proteins when the receptor is in the active state

δ Factor defining the synergy produced by simultaneous ligand binding on the interaction of the G protein with the receptor

A + Ri ARi ARa

A + Ri ARi ARa + G ARaG

Ka αL

Ka αL γβKg

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numerous other cellular proteins41–45. Other regionsmight mediate homologous or heterologous dimer-ization, leading to effects on receptor signalling. Forexample, dimerization of angiotensin II and bradykininreceptors leads to potentiation of angiotensin IIresponses46. Formation of heterodimers betweensomatostatin and dopamine D2 receptors leads todopamine-antagonist-sensitive somatostatin binding47.Signalling need not necessarily be involved in therapeu-tically relevant receptor dimer formation. The formationof chemokine (CC) receptor 5 (CCR5) heterodimerswith a mutant form of the CCR2 receptor (CCR2V641)prevents homodimerization of CCR5 and interfereswith the infection process of human immunodeficiencyvirus 1 (HIV-1). This could be why AIDS takes longer todevelop in individuals carrying the CCR4V641mutation48. In general, evidence indicates that manypotential protein–protein interactions might lead toeffects on receptor function that could have therapeuticapplications. These interactions are mediated by unfold-ing of specific protein regions that are associated withspecific functions.

At any given instant, the various conformations in anensemble can be represented as a Gaussian distribution.FIGURE 2a shows two such ensembles. One is referred to asthe ‘reference’ ensemble (Ref), which might be a restingor inactive ensemble of receptor conformations, the bulkof which have no overt function. The other is an ensem-ble that interacts with, and activates, G proteins (G).Receptor function results from common members(intersection) of the reference ensembles and functionalG-protein signalling ensembles5,6. The intersection shownin FIG. 2a consists of common members that can spontan-eously activate G proteins. This has been observed

dictates the overall response. Similarly, it has beenshown that co-expression of G

sprotein can change the

apparent relative efficacy of calcitonin agonists in HEKcells20. Such data show that the initial concept, that effi-cacy is a molecular property that describes the ability ofa molecule to induce a physiological response, has lost itsreceptor-based definition. Efficacy is now more a func-tion of the entire GPCR system, involving both the recep-tor and its cellular environment. From this standpoint,drugs could have several intrinsic efficacies, even withinthe context of producing a physiological response21.

With increasing technology has come the ability toobserve a rich array of GPCR behaviour, which includesPLEIOTROPIC COUPLING to different G proteins, dimerization,oligomerization, internalization, desensitization andinteraction with numerous other cellular coupling pro-teins (FIG. 1). Studies of these behaviours indicate thatsome molecules can induce some of these effects but notothers, and that, occasionally, a drug might not producean overt physiological response but might, nevertheless,change receptor behaviour (that is, have efficacy). So,there are different ‘efficacies’ for different behavioursbeyond the activation of G proteins (see below). Thequestion is whether a general molecular mechanism canaccount for all of these expressions of efficacy. Oneapproach to describing the numerous other manifesta-tions of efficacy that do not necessarily produce a cellularresponse is to consider receptor proteins in terms ofENSEMBLE THEORY.

Protein ensemble theoryFor simplicity, most models of protein systems describeonly single conformations. However, in theoreticalterms, it is known that proteins exist as collections ofnumerous conformations. As proteins traverse anenergy landscape, they sample numerous conformationsaccording to thermal energy22,23. Experimental tech-niques, such as the use of nuclear magnetic resonancespectroscopy to study the rate of deuterium exchange inaqueous solution24–26,have shown that proteins constantlyexplore conformational space. The chosen conforma-tions and the rate at which they are selected depend onthe level of thermal energy imparted to the system27–32. Agiven receptor might adopt numerous conformations asdifferent regions of the protein fold and unfold in avariety of combinations. The fact that only specificregions of receptors may unfold at any instant is rele-vant to receptor function. For example, certain regionsof the third intracellular loop of GPCRs are known tointeract with G proteins to produce activation33–37, andconformational unfolding to expose these regions leadsto receptor–G-protein coupling. The conformation thatexposes this region (the ‘activating’ conformation) couldalso be viewed as a collection of conformations, inwhich other regions of the receptor are folded differently.So, a collection of conformations, referred to as an‘ensemble’, is associated with a given function — in thiscase, G-protein activation. Similarly, there could bespecific regions of a receptor that control its interactionwith auxiliary membrane proteins — such as receptor-activity-modifying proteins (RAMPs)38–40 — or with

PLEIOTROPIC COUPLING

Literally meaning ‘havingmulitple phenotypic expressions’,in this case, pleiotropic couplingrefers to the ability of somereceptors to activate more thanone G protein and thereforestimulate multiple responsepathways in cells.

ENSEMBLE THEORY

This usage refers to the study of proteins as collections ofmicroconformations, somebeing energetically preferredover others. The interaction ofthese collections ofconformations with ligandscauses them to redistribute therelative conformations into anew ensemble.

PDZ DOMAIN

(PSD-95, Dlg and ZO-1/2).Protein–protein interactiondomain that binds to carboxy-terminal polypeptides inparticular.

SH2 DOMAIN

(Src-homology domain 2).A protein motif that recognizesand binds tyrosine-phosphorylated sequences,and thereby has a key role inrelaying cascades of signaltransduction.

SH3 DOMAIN

(Src-homology domain 3.) A protein sequence of ~50amino acids that recognizes andbinds sequences that are rich inproline.

PDZ

PSH3

SH2

Gs

D

Gi

RAMPsArr GRK

Figure 1 | Schematic diagram of a hypothetical G-protein-coupled receptor. Labels denote general regions ofinteraction of the receptor with other cellular proteins, includingdifferent G proteins (Gi, Gs), PDZ-, SH2- and SH3-DOMAIN

proteins, receptor-activity-modifying proteins (RAMPs), arrestin(Arr), G-protein-coupled receptor kinase (GRK), sites fordimerization with other GPCRs (D), and phosphorylation sitesthat lead to uncoupling and internalization (P). Any one of theseactive processes could be considered a form of expression ofefficacy. The figure is a general description of various loci forprotein interactions, but does not represent accurate locations,as, in most cases, these are not well characterized at present.

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R E V I E W S

The restructuring of a collection of receptor conform-ations through selective binding affinities emphasizesthe idea that affinity is not a passive phenomenon,whereby a ligand simply binds to a protein but does notchange it. Just because we do not have the ‘experimentaleyes’ to see a change in receptor conformation, thisdoes not necessarily mean that it does not occur. Suchconformational shifts have been observed for the bind-ing of β

2-adrenoceptor antagonists, which do not pro-

duce receptor activation, to β2-adrenoceptors that are

covalently labelled with a fluorescent probe56. This sup-ports the idea that, if a ligand has MACRO-AFFINITY for areceptor, it must also be considered that the ligand haschanged the conformation of the receptor. As efficacyis the product of the effect of the ligand on the recep-tor, this indicates a more compelling relationshipbetween the properties of affinity and efficacy than pre-viously indicated by experimental data.

The formation of ligand-bound ensembles, and howthose ensembles intersect with various functionalensembles, forms the basis of the drug effect. So, if aligand produces conformations that are coincident witha G-protein activation ensemble, then agonism results(FIG. 2b). Similarly, ligands that form specific ensemblesthat are not coincident with functional ensembles (thatis, they are within the reference ensemble (FIG. 2c) or thatare in a different part of conformational space (FIG. 2d)),produce antagonism.

The spectrum of efficaciesEfficacy was first defined by Stephenson9 in terms ofresponse. It seemed that many other receptor behaviours,such as desensitization and receptor internalization, were

experimentally in recombinant systems with high levelsof receptor expression, and is referred to as constitutivereceptor activity49. It follows that there could be inter-secting ensembles for a range of GPCR behaviours, suchas pleiotropic activation of several G proteins, internal-ization, dimerization, and so on.

The conformational cafeteriaMolecules influence receptor behaviour through selectiveaffinity for the various conformations in the receptorreference ensemble. In essence, the ligand enters a confor-mational cafeteria and chooses (binds most strongly to)the conformations for which it has the highest affinity.This binding effectively removes the conformation fromthe pool of possible conformations, and it is replacedthrough mass action at the expense of a conformation forwhich the ligand has a lower affinity (the ‘non-preferred’conformation). In this way, the ligand shifts the equilib-rium towards the preferred conformation and creates a biasin the reference ensemble (see BOX 2 for a linkage-theoryexample of how this occurs).

The idea that selective affinity can change the relativeproportions of protein conformations is not new. Infact, this forms the basis of the two-state theory, inwhich the selective affinity of a ligand for an ion channelproduces a bias towards open channels and a subsequentphysiological response50–52. This concept has also beenextended to GPCRs in terms of active and inactivereceptor states53–55. Ensemble theory applies the ideabeyond just a minimal model of two receptor states, onepromoting a response and one not, and extends it to anynumber of states, some of which might mediate a physio-logical response or other behaviours of the receptor.

MACRO-AFFINITY

Although some ligands will bindpreferentially to some receptorconformations over others, theweighted average affinity that aligand has for a receptorensemble is known as the‘macro-affinity’ of the ligand forthe receptor. It is theconcentration of ligand that isbound to 50% of the receptors atany one instant.

a Native ensembles b Agonist

c Antagonist

0 50 100

GRef

150 250200 300

Freq

uenc

y

d Antagonist

0 50 150100

GRef

200 250 300

Freq

uenc

y

0 50 150100

GRef

200 250 300

Freq

uenc

y

0 50 150100

GRef

200 250 300

Freq

uenc

y

Figure 2 | GPCR ensembles. a | Different functional ensembles of conformations are expressed as Gaussian distributions ofreceptor states. It should be noted that the x axis of these distributions has no intrinsic meaning, and x values are arbitrarily arrangedto show the relationship of functional ensembles. Intersections refer to common activity. b | The ligand-bound ensemble intersectswith the G-protein signalling ensemble, so the ligand functions as an agonist. c,d | The ligand produces ensembles that do notintersect with the signalling ensemble. As the ligands create bias in the conformations, making agonist activation more difficult, theyfunction as antagonists. G, G-protein-activating ensemble; Ref, reference (inactive) ensemble.

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internalization62. Similarly, the chemokine peptideRANTES (regulated on activation, normal T-cellexpressed and secreted) produces CCR5 receptor inter-nalization as well as receptor activation63,64, whereas theRANTES analogue aminooxypentane-RANTES (AOP-RANTES) does not produce CCR5-mediated chemo-taxis65, but does promote rapid internalization ofCCR5 receptors66–68. These ligands have no efficacy forthe production of a response, but do have efficacy forreceptor internalization.

Activation of angiotensin receptors with angio-tensin II produces activation, phosphorylation andinternalization in transfected CHO-K1 cells. By con-trast, the analogue [Sar1,Ile4,Ile8]AngII does not activateor internalize the receptor, but does promote receptorphosphorylation69. Although receptor activation, phos-phorylation and internalization have been linked inmany receptor systems, discontinuities between theability of agonists to produce these effects have alsobeen clearly documented. Studies of two agonists foropioid receptors — morphine and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) — illustrate this.Whereas morphine induces phosphorylation of opioidreceptors, DAMGO does not70. The µ-opioid agonistsmethadone and l-α-acetyl methadone produce dispro-portionate desensitization and receptor phosphorylationwhen compared with their ability to produce aresponse71. Similarly, methadone and another µ-opioidreceptor agonist, buprenorphine, show desensitizingproperties that are different from those of morphine72.Enkephalins and morphine both stimulate µ- and δ-opioid receptors, but it has been shown thatenkephalins, but not morphine, induce receptor inter-nalization73. Other behaviours induced by GPCR lig-ands can also be dissociated from receptor signalling.For example, the internalization-promotingchemokine derivative AOP-RANTES also promotesdimerization of CCR5 receptors, without producingconventional chemotaxis-based agonism68.

All of these apparently diverse effects can beaccounted for in terms of ensemble theory. Within theframework of this theory, ligands produce ligand-bound ensembles that intersect with the numerousbehaviour-defining receptor ensembles. There are prac-tical applications of this idea. First, it supports the furtherstudy of ligands with known macro-affinity. The histor-ical dissociation of affinity from efficacy has led to theassumption that affinity is simply the occupation of areceptor by a ligand. Now that such affinity has beenassociated with other GPCR effects, it would be worth-while taking ligands that have known affinity and test-ing them for other activities, such as inducement ofinternalization, dimerization and association with othermembrane proteins. For example, the receptor internal-ization property of AOP RANTES (an antagonist ofCCR5) is extremely useful in preventing HIV-1 infection,as it removes the co-receptor that is needed for entry ofthe viral genome into healthy cells65,67.

The discovery of properties other than activation ofG proteins for ligands at GPCRs underscores a newtheme in pharmacological drug screening; namely that

secondary to the response and were simply follow-onconsequences57–59. However, an indication thatresponse is not a necessary prerequisite has beenobserved for opioid receptors, for which it has beenshown that, although receptor internalization correl-ates with agonist efficacy, G-protein activation per se isnot required60,61. Increasingly sophisticated technology,such as radiation inactivation, bioluminescence reso-nance energy transfer and fluorescence energy transfer,allow direct observation of these ‘secondary’ behaviours,such as dimerization, oligomerization, desensitization,internalization and interaction with other non-signallingmembrane proteins. This ability has led to the discoveryof ligands that short-circuit the system. So, some ligandscan directly produce these secondary activities withoutproducing a response at all, whereas others produce aresponse without producing the apparently obligatorysecondary effects. For example, although GPCR inter-nalization into the cell cytoplasm is often closely linkedwith receptor activation, the cholecystokinin (CCK)-receptor antagonist D-Tyr-Gly-[(Ile28,31,D-Trp30)cholecys-tokinin-26-32]-phenethyl ester does not producereceptor stimulation, but does cause profound receptor

Box 2 | Ligand binding creates biased receptor conformations

Assume that a receptor protein exists in only two conformations (R1

and R2) and that

the ligand ([A]) has an affinity of 1/KA

for R1

and α/KA

for R2.The amount of receptor

in conformation R2

as a fraction ρ of the total receptor number ([R2]/([R

1]+[R

2])), is

given as:

where L is an allosteric constant, which is defined as [Ra]/[Ri].In the absence of ligand ([A] = 0), ρ

0 = L/(1 + L), and in the presence of a maximal

concentration of ligand (saturating the receptors; [A]→∞), ρ∞ = α(1 + L) /(1 + αL).Therefore, the effect of a ligand on changing the ratio between the R

1and R

2states is

given by the ratio ρ∞/ρ0;

When this ratio is > 1, then the presence of the ligand will enrich the R2

state. If ρ∞/ρ0 < 1,

then R2

is depleted. Only if α = 1 (identical affinity for R1

and R2) will redistribution

not occur.This can also be seen in terms of thermodynamics.A given ensemble has a characteristic

Gibbs free energy.A change in this free energy will result in a change in the make-up of theensemble. The equation for the change in Gibbs free energy (Φ) for a system of two proteinconformations i and 0 is (REF. 93):

where ∆Φi0 is the free energy of an arbitrary state i in the absence of ligand, K

a,0is the

binding constant to the reference state (0) and Ka,i

is the binding constant to state i (thefavoured state). It can be seen from EQN 3 that in order for there to be no change inGibbs free energy (and, therefore, no change in the make-up of the protein ensemble),then the natural logarithmic term must equal zero. If the ligand has a preferentialaffinity for any conformation i in the ensemble, then the logarithmic term will not bezero, and the Gibbs free energy of the system will change. This, in turn, will cause achange in the relative amounts of conformations 0 and i. So, by the act of binding to thecollection of receptor conformations, a ligand would necessarily change the bias in theconformations in the ensemble.

L(1 + α[A]/KA)

[A]/KA(1 + αL) + 1 + Lρ = (1)

α(1 + L)

(1 + αL)= (2)∞ρ

0ρ

(1 + Ka,i[A])

(1 + Ka,0[A])= (3)∆Φi ∆Φi

0 – RTln

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radio-ligand probe was needed to detect interference ofthe interaction by foreign molecules. However, it is nowknown that receptors can be considered loquacious withrespect to interactions with cellular components, andthat they signal constantly. So, a new dimension inscreening can be gained by listening in on the conversa-tion. A constitutive receptor screen has the advantage ofconstant monitoring of a protein ensemble (that is, theintersection of the reference and G-protein signallingensembles; FIG. 3a). Under these circumstances, it mightbe supposed that any molecule with macro-affinityforms a new ligand-bound ensemble, and the shift inthe prevailing conformations will be detected as achange in the magnitude of the constitutive activity(either an increase or, as seen in FIG. 3b, a decrease)82,83.This idea also suggests that optimal drug screeningprocesses should use maximal numbers of methods todetect receptor conformational changes.

For example, if only one G-protein response is mon-itored for a given receptor screen, then a ligand–receptorinteraction that produces a conformational change inthe receptor but does not produce a specific change inthe monitored receptor–G-protein interaction wouldnot be detected. For a GPCR that interacts with both G

s

and Gq

proteins, an optimum system would be one thatwould detect interference with either G

sor G

qsignalling.

In this regard, Xenopus laevus melanophores, whichincrease their pigmentation with activation of either G

s

or Gq

and decrease it with activation of Gior G

o, offer an

extended spectrum of detection84–87. Constitutivescreening using melanophores as the GPCR host cellshas been shown to be stable and effective82,83. Ideally, anassay in which protein conformational changes can bedetected directly would be optimal for screening forpotential therapeutically useful molecules.

Application of ensemble theory Models can be classified in terms of their complexity,number of parameters and the ability to estimate those

if efficacy is not observed, this does not necessarilyimply its absence. Rather, it implies only that the assaydoes not have the appropriate ‘eyes’ to see it. The mostpronounced example of this is the discovery of inverseagonism. In systems that exhibit spontaneous produc-tion of receptors in the active state, with concomitantagonist-independent signalling, there are ligands thatpreferentially stabilize the inactive state of the receptor.These ligands convert the spontaneous active state intothe inactive state, and so reverse the constitutive activityof the system — they produce inverse agonism.

First discovered for GPCRs by Costa and Herz49

using the δ-opioid receptor antagonist ICI 174864([N,N′-diallyl-Tyr1,Aib2,3]Leu5-enkephalin), inverse ago-nism was considered an oddity in receptor pharmacol-ogy that was associated only with some specialized sys-tems. However, the apparent paucity of inverse agonistsin the pharmacological world was actually due to thelimited availability of constitutively active receptor sys-tems for testing at that time. As these systems becamecommonplace, it was seen that many, if not most, appar-ently simple competitive antagonists are in fact inverseagonists. This would be predicted on theoreticalgrounds, as an antagonist would require identical affin-ity for at least three (and probably more) receptor con-formational states (namely R

i, R

aand R

aG; see BOX 1) for

it not to change the conformational make-up of thereceptor system (and thus not change constitutive sig-nalling; BOX 2). The discovery of negative efficacy inantagonists (leading to inverse agonism) might indicateunique therapeutic profiles (that is, blockade of natural74

or pathological constitutive activity75), a negative propen-sity to cause TACHYPHYLLAXIS of antagonism due to receptorupregulation76–79, or alteration in G-protein levels80,81.

The idea that affinity is linked to receptor conforma-tional changes compels different views of how GPCRscould be screened for biologically active molecules.Historically, receptors have been treated as proteins that‘speak only when spoken to’; that is, either an agonist or

TACHYPHYLLAXIS

The reduction in responseduring repeated receptorstimulation by an agonist;usually ascribed to theproduction of a desensitizedstate of the receptor.

a

0 100 300 400 500

Freq

uenc

y

GRef

Real-time sustained response Loss of constitutive response

b

0 100 300 400 500

Freq

uenc

y

GRef

Add ligand

Figure 3 | Detection of ligand binding through observation of constitutive receptor activity. a | The intersection of thereference and G-protein signalling ensembles represents receptor conformations that spontaneously signal in the absence of ligand;that is, constitutive receptor activity. b | As ligands produce ligand-bound ensembles, the constitutive receptor activity is lost, andbinding of the ligand is detected.

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mathematics, as an attempt to unify apparently diversepharmacological activities.

ConclusionsThe intimate relationship between affinity and efficacyis becoming appreciated as new technologies allow thedetection of different receptor behaviours. At the veryleast, this has exposed efficacy as a property beyondbeing simply the ability to produce a physiologicalresponse. The exploitation of this relationship fortherapeutic advantage and an increased ability to detectbiologically active ligands is the next challenge forreceptor pharmacology.

parameters88. Ensemble theory represents a heuristicmodel, which can generally describe a system that hasnumerous parameters, many of which could probablynot be estimated independently. So, ensemble theorydoes not allow modelling of experimental data, and itshould be considered only as a conceptual tool.However, there are predictive elements within thisapproach that can be accessed through the mathematicsthat describe the probability of energy states and ligandinteractions between receptors and proteins — these aredescribed separately in a probabilistic model of receptorfunction5,6. In general, this review uses the concepts con-tained in the probabilistic model, without the formal

1. Waud, D. R. Pharmacological receptors. Pharmacol. Rev.20, 49–88 (1968).

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AcknowledgementsT.K. would like to thank O. Onaran, University of Ankara, Turkey, forinsightful discussions on mechanisms of efficacy.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/angiotensin II | angiotensin II receptor | bradykinin receptor | CCKreceptor | CCR2 | CCR5 | cholecystokinin | dopamine D2 receptor |RAMPs | RANTES | somatostatin receptor

FURTHER INFORMATIONEncyclopedia of Life Sciences: http://www.els.net/G proteins | G-protein-coupled receptorsAccess to this interactive links box is free online.