Allosteric activation of plasma membrane receptors—physiological implications and structural...

Progress in Biophysics & Molecular Biology 81 (2003) 219–240

Review

Allosteric activation of plasma membranereceptors—physiological implications and structural origins

Arthur D. Conigrave*, Alison H. Franks

School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia

Abstract

Allosteric modulation of receptors has physiological not just pharmacological significance. Thus, thechemical context in which an agonist signal is received can have a major impact on the nature ofthe physiological response by modifying receptor sensitivity and/or maximal activity—even the nature ofthe signalling response. In addition, recognising that an endogenous activator is the allosteric modulatorof a known receptor, rather than the agonist of a novel receptor, has the potential to solve, in dramaticfashion, key physiological questions. What is an allosteric modulator and why are allosteric effects onreceptors so diverse and frequently complex? What is the scope of allosteric effects? Can the existence ofendogenous modulators be predicted from a receptor’s amino acid sequence? How should screening forendogenous allosteric modulators be undertaken? These questions form the framework of this mini-reviewon physiological and structural aspects of receptor allostery.r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Plasma membrane; Receptor; Metabotropic receptor; G-protein-coupled receptor; Allosteric modulator;

Agonist; Conformational induction; Conformational selection

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

2. Specifying an agonist signal by chemical context—significance of allosteric modulation of

receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

3. Receptors as proteins—a structural view of allosteric modulation . . . . . . . . . . . . . . 221

3.1. Functional receptor modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

*Corresponding author. Tel.: +61-2-9351-3883; fax: +61-2-9351-4726.

E-mail address: [email protected] (A.D. Conigrave).

0079-6107/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0079-6107(03)00020-8

1. Introduction

The appreciation that conformationally specific receptor surfaces represent potential target sitesfor allosteric modulators is a major theme in modern pharmacology (review: Christopoulos andKenakin, 2002). Thus, many new and established drug classes are allosteric modulators that bindat receptor surfaces that are distinct from the orthosteric (i.e., agonist-binding) site. These surfacesrepresent potential target sites for drug development.The idea that allosteric modulation may also operate physiologically is less well appreciated

but of considerable significance. Thus, the surfaces of many receptors may conceal currentlyunrecognised physiological secrets. This mini-review attempts to bring together threedistinct aspects of the allosteric modulation of plasma membrane receptors: a continuouslyexpanding pharmacology, a new physiology, and the structure–function relationships thatunderpin them. It begins with a statement of a physiological problem—how is the cellularresponse to an agonist specified when receptor sub-types are co-expressed and how is the balancebetween different receptors and signalling pathways controlled? Consideration is then givento receptors as proteins, to the functional modules within receptor proteins and to the impact ofallosteric modulators, whether endogenous biochemical species or xenobiotics, on receptorfunction.

3.2. Impact of allosteric modulators on receptor function . . . . . . . . . . . . . . . . . . 223

3.3. Metabotropic receptors as coenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 223

4. Theoretical concepts in receptor activation and its allosteric modulation . . . . . . . . . . . 224

4.1. Early examples of receptor allostery . . . . . . . . . . . . . . . . . . . . . . . . . . 225

5. Diversity of allosteric effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

5.1. Effects on potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

5.2. Effects on efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

5.3. Complex effects on potency and efficacy . . . . . . . . . . . . . . . . . . . . . . . . 229

5.4. Effects on the selection of signalling pathways . . . . . . . . . . . . . . . . . . . . . 229

6. Impact of molecular cloning on the study of receptor allostery . . . . . . . . . . . . . . . . 231

6.1. Site-directed mutagenesis and chimeric receptors . . . . . . . . . . . . . . . . . . . . 231

6.2. Protein expression and crystallisation . . . . . . . . . . . . . . . . . . . . . . . . . . 232

7. Mechanisms of allosteric modulation of G-protein-coupled receptors . . . . . . . . . . . . 232

7.1. Mechanism of G-protein activation . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

8. Endogenous allosteric modulators of plasma membrane receptors . . . . . . . . . . . . . . 233

8.1. Endogenous modulation of NMDA receptors . . . . . . . . . . . . . . . . . . . . . 234

8.2. Inorganic allosteric modulators of G-protein-coupled receptors . . . . . . . . . . . . 234

8.3. Organic endogenous modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

9. Conservation of endogenous modulators according to biochemical class . . . . . . . . . . . 235

10. Approaches to the identification of endogenous allosteric modulators . . . . . . . . . . . . 235

10.1. Criteria required for the identification of novel allosteric modulators . . . . . . . . . 236

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

A.D. Conigrave, A.H. Franks / Progress in Biophysics & Molecular Biology 81 (2003) 219–240220

2. Specifying an agonist signal by chemical context—significance of allosteric modulation of

receptors

From the perspective of an agonist-releasing cell, the ability to specify a particular receptor-dependent response would appear to be limited. This follows because many agonists activatereceptors of distinct classes (e.g., ionotropic and metabotropic receptors) leading to the activationof multiple and functionally unrelated signalling pathways in a cell that co-expresses them. Theproblem is compounded by the existence of receptor sub-types.Where distinct receptors for the same biochemical species are co-expressed, the biophysicist is

often left to ponder what determines the physiological significance of an agonist signal. Is it, forexample, entirely dependent upon gene expression in the target cell, thus controlling the absoluteand relative levels of plasma membrane receptors or perhaps the cellular levels of key signallingcomponents? This review considers an alternative perspective that returns the initiative,somewhat, to cells ‘‘upstream’’ of the target—the physiological significance of an agonist signalmay also depend upon the chemical context in which it is received.The appreciation that receptors, like enzymes, can be regulated allosterically by endogenous

molecules, of chemical classes distinct from the agonist, is one powerful instance of signalspecification by chemical context. Thus, precise physiological control may arise, not only byvariations in gene expression in the target cell (a relatively slow process requiring hours for fulleffect), but via regulation of the levels of sub-type specific receptor modulators (a process that cantake place within seconds). Viewed in this way, the agonist signal, although necessary for receptoractivation, may not in itself specify the physiological response—signal specification in many casesmay depend on the presence or absence of modulators. Thus, the apparently unrelated secretoryactivities of neighbouring or distant cells, dietary influences, metabolic state, even diurnal rhythmsmay play significant roles in determining physiological outcomes.The appreciation that receptors can be regulated allosterically also has implications for the

matching of agonists to their target receptors. Some long-sought-after receptors for ‘‘orphanagonists’’, e.g., receptors for amino acids or fatty acids in the gastrointestinal tract, may havealready been cloned with well-recognised agonists and readily available in vitro systems to studythem. Thus, some physiologically recognised ‘‘agonists’’ may not, in pharmacological terms, beagonists at all—but rather allosteric activators.

3. Receptors as proteins—a structural view of allosteric modulation

One important outcome of the molecular cloning efforts of the last 20 years has been therealisation that the vast majority of receptors—whether ionotropic (ligand-gated ion channels) ormetabotropic (receptors that couple to cytoplasmic enzymes)—are proteins. This has immediateimplications for function. First, proteins are organised into discrete structural units calleddomains—typically taking the form of helices, sheets and barrels connected by loops and turns.Second, adoption of three-dimensional structure (‘‘conformation’’) is dependent upon the ways inwhich a protein’s constituent domains interact with one another leading to the assembly of muchlarger structural/functional elements. Third, a protein can adopt multiple three-dimensionalstructures depending upon its ability to undergo rearrangements of its larger structural elements.

A.D. Conigrave, A.H. Franks / Progress in Biophysics & Molecular Biology 81 (2003) 219–240 221

3.1. Functional receptor modules

Receptor function—arising from the binding of an agonist molecule to a discrete surface, theorthosteric binding site—implies a change in state: a structural rearrangement of major elementswithin the protein. At its simplest, receptor function arises from interactions between distinctsensing and effectormodules. The binding of a receptor agonist to a structurally discrete surface ofthe receptor’s sensing module is sufficient for receptor activation—sufficient to either drive thestructural rearrangement that leads to the adoption of an ‘‘active’’ conformation state of theeffector module or stabilise an otherwise short-lived active conformation. The study of certain G-protein-coupled receptors (GPCRs) especially of sub-class C (Fig. 1) makes it clear that there isalso a third type of functional module, a signal transmission module that serves to couple agonistbinding to activation of the effector unit.

SENSOR

EFFECTOR

STM

GDP

GDP GTP

GTP

GTP

Fig. 1. Role of signal transmission module in GPCRs belonging to Group C. The bilobed Venus Fly Trap domain at

the N-terminus (approximately 500 residues) acts as the sensing module for Group C receptors and interacts with a

second VFT domain subunit to form a dimer. Agonist binding stabilises domain closure (Kunishima et al., 2000). The

effector module, as in other GPCRs, involves domains formed by the intracellular loops (especially loops 2 and 3) and

the cytoplasmic aspects of the transmembrane helices (Chang et al., 2000). Signalling via these receptors also requires a

100 amino acid cysteine-rich domain (Hu et al., 2000), which does not participate in either agonist binding or G-protein

coupling. This domain contributes to a signal transmission module (STM) which, as shown in the figure, presumably

also includes elements of the transmembrane region (shown as rectangles in Fig. 1). It physically couples agonist

binding to the VFT sensor domains to the activation of the G-protein binding effector domain. The mechanism implied

by the figure: VFT domain closure induces approximation of the dimeric transmembrane regions leading to G-protein

binding and activation is speculative. Signal transmission modules in GPCRs from sub-groups that lack extracellular

sensing modules are necessarily restricted to the transmembrane region being packed into the same restricted space as

the sensing and effector modules.


3.2. Impact of allosteric modulators on receptor function

According to its classical definition, an allosteric activator has no effect on receptor behaviourin the absence of agonist. However, in the presence of an allosteric activator, the impact of theagonist is enhanced. This may occur either by an effect on potency, i.e., receptor activation ismore pronounced at lower agonist concentrations, or by an effect on efficacy, i.e., the amplitudeof the maximal response is magnified or by an effect on signal pathway selection. The impact of l-amino acids on the human calcium-sensing receptor (Fig. 2) is an example of an effect on potency;the impact of oleamide on the 5HT2A receptor (Fig. 3) is an example of an effect on efficacy. Theinverse effects of oleamide on 5-HT2A receptors, via which it promotes PI-PLC activity, and 5-HT7A receptors, via which it suppresses adenylyl cyclase activity (Thomas et al., 1997) is anexample of allosterically specified effector switching. Note that effector switching may occur eitherwithin the same cell if the receptors are co-expressed or between target cells if the receptors areselectively expressed on distinct but neighbouring cell-types.An allosteric activator binds to a distinct surface on the receptor protein acting to stabilise one or

more active conformations of the receptor’s sensing, transmission or effector modules. Allostericinhibitors, unlike antagonists, do not compete with the agonist for binding. Instead, they act tostabilise a conformation that is either unfavourable for agonist binding or unfavourable for signaltransmission. Under this latter circumstance, agonist binding can be preserved.

3.3. Metabotropic receptors as coenzymes

The majority of plasma membrane receptor proteins activate enzyme-driven pathways or, moreappropriately, enzyme-driven cascades since each step in the signalling pathway typically has a

Fig. 2. Impact of l-amino acids on the calcium-sensing receptor. l-Phenylalanine stereoselectively enhances the

sensitivity of the calcium-sensing receptor to its physiological agonist, Ca2+ (redrawn from Conigrave et al., 2000b).

The calcium-sensing receptor was stably expressed in HEK-293 cells and exposed to increasing concentrations of

extracellular Ca2+ in the presence or absence of l-phenylalanine. Receptor response in the form of Ca2+ mobilisation

was detected by fura-2 fluorescence. The concentrations of l-phenylalanine were as follows: 0mM (J), 1mM (n),

3mM (&), 10mM (K), 30mM (m) and 100mM (’). Similar data were obtained with other aromatic and aliphatic l-

amino acids as well as plasma-like l-amino acid mixtures. d-Phenylalanine and other d-amino acids were much less

effective (not shown).


substantial amplifying effect. For the majority of metabotropic receptors, one or more enzymeshave been identified that represent the starting points for specific signal transduction cascades.Viewed in this way, metabotropic receptors are coenzymes that regulate enzyme activity in aligand-dependent fashion. The co-enzyme complex includes receptor, agonist and allostericmodulator(s).In the case of GPCRs, the plasma membrane-embedded receptor complex acts to control the

distribution of a second, cytoplasmically restricted, co-enzyme between two molecular forms(Fig. 1). These heterotrimeric guanine nucleotide binding proteins (or G-proteins) undergo areceptor-dependent transition between an inactive, GDP-bound, associated form and an active,GTP-bound, dissociating form which activates the first biochemically recognisable enzymes in thesignalling cascade.G-protein activation, release and dissociation not only triggers downstream signalling events

but has a well-recognised impact on the structure and function of the receptor protein itself—acting in many cases to reduce the affinity of agonist binding (review: Christopoulos and Kenakin,2002).

4. Theoretical concepts in receptor activation and its allosteric modulation

The theoretical framework developed originally for the analysis of enzymes and adapted for thepharmacological analysis of receptor allostery—regulation by chemical context—has beenreviewed in detail elsewhere (Christopoulos and Kenakin, 2002). One important distinction hasbeen drawn between allosteric interactions between distinct domains on a single protein subunit,e.g., between the agonist binding site and G-protein binding site on a GPCR and allosterictransitions in receptor conformation arising from interactions between protein subunits, e.g., inionotropic receptors. In this respect, many plasma membrane receptors, including GPCRs

Fig. 3. Impact of oleamide on the 5HT2A receptor. Oleamide enhanced the maximal response of 5HT2A receptors

with no apparent effect on agonist potency. 5HT2A-transfected HeLa cells were exposed to various concentrations of

5HT in the absence (J) or presence (m) of 100 nM oleamide. Receptor response was detected by release of [3H]-inositol

phosphates. The figure has been redrawn from (Thomas et al., 1997).


(review: Rios et al., 2001), operate functionally as dimers or oligomers. Some endogenousGPCRs, e.g., the calcium-sensing receptor (Bai et al., 1999), are homodimers; others, e.g., themetabotropic GABA(B) receptor (Kaupmann et al., 1998) are, in fact, heterodimers. Thus,allosteric transitions—involving the transfer of structural information from one subunit to theother in a receptor dimer (or oligomer)—have important functional consequences for manydifferent receptors.An important distinction has also been drawn between conformational induction—in which ligand

binding provides the driving force for conformation change and conformational selection—in whichligand binding selectively stabilises one or a limited number of conformational states (Fig. 4; Getherand Kobilka, 1998). Agonists, by binding at the orthosteric site, appear to operate in many cases viathe conformational induction that follows the release of intrinsic structural constraints in thereceptor protein. This view is supported by recent analysis of bovine rhodopsin for which crystalstructure data is now available (review: Okada et al., 2001). However, agonists may also operate viaconformational selection, e.g., glutamate activation of the rat mGluR1 (Kunishima et al., 2000) andinverse agonists, which suppress resting activity, appear to stabilise inactive conformations (Getherand Kobilka, 1998). Allosteric activators may be incapable of conformational induction since theyare, in general, unable to activate their receptor targets in the absence of agonist. This considerationsuggests that allosteric activators operate by conformational selection.

4.1. Early examples of receptor allostery

Early examples of allosteric modulation of receptors by small molecules include ionotropicreceptors—including GABA(A) receptors, some molecular forms of which are modulated bybenzodiazepines (Costa et al., 1975), as well as N-methyl-d-aspartate receptors for glutamate,some forms of which are modulated by glycine (Johnson and Ascher, 1987). NMDA receptorsalso exhibit allosteric binding sites for polyamines (e.g., spermine; Ransom and Stec, 1988) andother polyvalent cations (Lu et al., 1998) and thus represent one of the earliest examples of

(A)

(B)

Ri Ri Ra

Ri RaRa

Fig. 4. Conformational induction and conformational selection models of receptor activation. Agonist-dependent

receptor activation via (A) conformational induction in which the binding of the agonist (K) releases inhibitory

restraints in the receptor molecule, or (B) conformational selection in which the receptor exchanges spontaneously

between inactive (depicted as large squares) and active forms (triangles). Note that binding of the agonist stabilises the

active form.


regulation via multiple ligand binding sites. In addition, several receptors, which weresubsequently identified as GPCRs, exhibit allosteric modulation. These early examples includedseveral muscarinic receptor sub-types especially the muscarinic M2 receptor which is modulatedby a large number of xenobiotic muscle relaxants including gallamine and alcuronium (Ellis andSeidenberg, 1987; Nedoma et al., 1986). Two other early examples from the field of GPCRsinclude some sub-classes of opioid receptor (Pert and Snyder, 1974) and the a2-adrenergicreceptor both of which are modulated by cytoplasmic Na+ concentration (Michel et al., 1980;Motulsky and Insel, 1983; Tsai and Lefkowitz, 1978).The examples described above suggest a number of general points. Firstly, allosteric

modulation of receptors is not restricted to one class of receptors, leading to an expectationthat it will ultimately be demonstrated for many, if not all, receptor classes and sub-types.Secondly, allosteric modulation of receptors can be exerted by compounds that are neithersynthesised endogenously nor naturally encountered (gallamine, benzodiazepines) as well asendogenous biochemical species (glycine, Na+ and spermine). Finally, allosteric modulation ofreceptors can be exerted via actions on extracellular (glycine), transmembrane (benzodiazepines)and intracellular (Na+) sites.

5. Diversity of allosteric effects

GPCRs are drawn upon as the principal examples in this review. This reflects, in part, theauthors’ research interests as well as the extraordinary diversity of this receptor class and theburgeoning interest in their allosteric modulation (Christopoulos, 2002; Christopoulos andKenakin, 2002). However, reference is also made below to examples of allostery affectingreceptors belonging to other structural and functional classes.The diversity of allosteric effects described for various receptors is extraordinary. The literature

abounds with reports of positive or negative effects on agonist potency, as well as positive ornegative effects on receptor efficacy. Many allosteric modulators support a combination of effectson potency and efficacy—the most obvious being the allosteric enhancement of both potency andefficacy as described recently for the rat and human mGluR1 receptor (Knoflach et al., 2001).However, some modulators work via much more complex effects, e.g., allosteric activators thatalso act as antagonists, agents that lower potency but increase efficacy, even modulators thatconvert some, but not other, agents from agonists to antagonists (see Table 1). The findingthat some allosteric modulators also interact with the orthosteric site, e.g., the antagonistic actionof imidazoquinolines on adenosine A3 receptors (Gao et al., 2002) implies an overlap between theallosteric and orthosteric binding sites. In the transmembrane regions of receptors in which thesensing, signal transmission and effector modules are packed together in close relation, it isperhaps not surprising that complex allosteric effects such as these have been observed.It should also be noted that complex allosteric effects appear to arise, in general, from

xenobiotics rather than endogenous modulators. This is compatible with the idea that the effectsof endogenous modulators are physiologically relevant, e.g., by modifying agonist potency orefficacy. The diversity and complexity of allosteric effects can be appreciated, at least in part, by aconsideration of the potential sites of action of allosteric modulators in the receptor activationsequence (Fig. 5).


Table1

DiversityofeffectsofallostericmodulatorsonGprotein-coupledreceptors

Receptor

Modulator

Effecton

affinityof

agonist

binding

Effecton

potencyof

agonists

Effecton

affinityof

antagonists

Effecton

potencyof

antagonists

Effecton

efficacy

Effecton

orthostericsite

References

AdenosineA1

2-amino-3-aroyl-

thiophenes

Enhanced

Enhanced

Enhanced

Nil

Bhattacharyaand

Linden(1995),

BrunsandFergus

(1990)

AdenosineA3

Imidazoquinolines

enhanced

Enhanced

Unchanged

Enhanced

Antagonist

Gaoetal.(2002)

Alpha2A-

adrenergic

Na+

Reduced

Enhanced

Enhanced

Motulskyand

Insel(1983)

Beta2-

adrenergic

Zn2+

Enhanced:

micromolar

concentrations

Enhanced

Complex:

reduced

affinity;

reduced

dissociation

Biphasic

Uncouples

receptorfrom

Gs

Nil

DopamineD2

Na+

Reduced

Enhanced

Enhanced

Nil

DopamineD4

Amilorideanalogs

Reduced

Yes

HoareandStrange

(1996)

Calcium-

sensing

l-aminoacids

Enhanced

Unchanged

Conigraveetal.

(2000a,b)

Phenylalkylamines

Enhanced

Enhanced

Nil

Nemethetal.(1998)

Metabotropic

Glutamate

Phenyltoluene-

pyrrolidines;

Diphenylacetyl-

carbamic

acidesters

Enhanced;

speciesspecific

Enhanced

Nil

Knoflachetal.(2001)

Muscarinic

M2

Alcuronium

Reduced

Reduced;

reverseda

Enhanced

Antagonist

(highconcen-

trations)

Maasetal.(1995),

Tuceketal.(1990),

Zahnetal.(2002)

5HT2A

receptor

Oleamide(cis-9,10-

octadecenoamide)

Unchanged

Enhanced

Nil

Thomasetal.(1997)

5HT7receptor

Oleamide

Unchanged

Reduced

Thomasetal.(1997)

5HT7A

receptor

Oleate

Biphasic:

enhanced

atlow

concentration

Enhanced

(atlow

concentration)

Reduced

Albertsetal.(2001)

aPilocarpineconvertedfromagonisttoantagonist.


5.1. Effects on potency

Most obviously, enhanced potency might arise from modulator-induced enhancement ofagonist binding affinity. Stabilisation of the agonist-bound sensing module, for example, mightlead to a reduced rate of agonist dissociation. Provided that the time spent by the effector modulein its active conformation remains unchanged and that signal transmission from the sensing unit isintact, the effect of the modulator would be confined to an effect on potency. Examples ofputative physiological modulators that modulate potency but not efficacy include the actions of

Fig. 5. Potential impact of allosteric modulators on the conformational induction and selection models of receptor

activation. The allosteric modulator (O) is shown binding to either the inactive (squares) or active (triangles) forms of

the receptor and/or to the free or agonist(K)-bound receptor. (A) Potential impacts of an allosteric modulator at

various points in the conformational induction model. Binding to (i) the free-inactive receptor may stabilise the inactive

form or, alternatively, promote agonist binding (and potentially enhance receptor potency). Binding to (ii) the agonist-

bound inactive receptor may prevent or promote the agonist-dependent transition to the active state. Finally, binding to

(iii) the agonist-bound activated receptor may either stabilise the active form or divert the receptor to an inactive

conformation. (B) Potential impacts of an allosteric modulator at various points in the conformational selection model.

Binding to (i) the inactive receptor may prevent or promote its conversion to the active form. Binding to (ii) the active

receptor may prevent or promote agonist binding thereby influencing receptor stability. Note that the impact of an

allosteric modulator binding to (iii) the agonist-bound, active receptor cannot be distinguished from the impact of

modulators on the conformational induction model.


Zn2+ on the b-adrenergic receptor and l-amino acids on the calcium-sensing receptor (see Table2). Note also that an allosteric modulator can potentially modulate binding affinity and agonistpotency independently, e.g., an allosteric modulator that stabilises the agonist-bound sensingmodule but interferes with signal transmission.

5.2. Effects on efficacy

Other allosteric modulators reported in the literature have no detectable effect on receptorbinding properties or agonist potency but greatly enhance (or suppress) efficacy. Examples ofputative physiological modulators that enhance receptor efficacy include cis-9,10-octadeceno-amide (oleamide) which activates 5-HT2A receptor-induced phospholipase C activity and,interestingly, also inhibits 5-HT7 receptor-induced cAMP formation (Thomas et al., 1997). Thus,variations in the CNS level of oleamide have the potential to modulate the balance between thesetwo receptor sub-types and their associated signalling pathways thereby influencing 5-HT-dependent physiological processes including appetite and sleep. Consistent with this idea, sleepdeprivation elevates CNS levels of oleamide and intraperitoneal injections of synthetic oleamideinduce sleep in rats (Cravatt et al., 1995).Enhanced efficacy implies a conformation state in which the effector unit interacts with a larger

number of signalling molecules, e.g., by remaining active for longer upon receipt of theintramolecular message from the signal transmission unit or by enhancing the rate of access ofsignalling molecules to the effector site. In keeping with this concept, efficacy might also beenhanced or reduced if the receptor is rendered, respectively, resistant or hypersensitive todesensitisation. For GPCRs, resistance to desensitisation might arise from a failure ofphosphorylation by GPCR kinases or from a failure of arrestin binding. The positive effects ofsome modulators on the efficacy of ionotropic receptors appears to work in this fashion, e.g.,cyclothiazide stabilises subunit interactions in oligomeric AMPA ionotropic glutamate receptorsthat limit receptor desensitisation (Sun et al., 2002).

5.3. Complex effects on potency and efficacy

As might be expected, in the absence of precise structural information, there is no predictiverelationship between binding affinity, potency and efficacy. Thus, allosteric modulators thatpromote (or suppress) potency may promote, reduce or have no effect on efficacy. One well-studied example is the impact of cytoplasmic Na+ concentration on a2-adrenergic receptors (seeTables 1 and 2). Elevated cytoplasmic Na+ concentration reduces agonist (epinephrine) bindingaffinity and enhances epinephrine-induced efficacy (Motulsky and Insel, 1983; Tsai andLefkowitz, 1978). This represents an example of an allosteric effect that stabilises the activatedeffector module and promotes dissociation of agonists from the orthosteric site perhaps viaretrograde information flow from the receptor bound G-protein (Ceresa and Limbird, 1994).

5.4. Effects on the selection of signalling pathways

This potentially exciting area is currently in its infancy. The inverse effects of oleamide on 5-HT2A and 5-HT7A receptors which signal via distinct pathways (PI-PLC and adenylyl cyclase,


Table2

BiochemicalspeciesthatareputativeendogenousallostericmodulatorsofG-protein-coupledreceptors

Modulator

Receptor

Effectsonagonistbinding

and/orpotency

Effectonefficacy

References

Cations:Na+

a2-adrenergicreceptors

Reducedbindingaffinity

(epinephrine)

Enhanced

Limbirdetal.(1982),

Micheletal.(1980),

MotulskyandInsel(1983)

DopamineD2receptor

Reduced

Enhanced

Neveetal.(1991)

DopamineD4receptor

Reduced

SchetzandSibley(2001)

Opioid(d)receptor

Reduced

Na+isrequired

Blumeetal.(1979),Pert

andSnyder(1974)

Zn2+

Beta2-adrenergicreceptor

Enhanced

Nil

Swaminathetal.(2002)

DopamineD2receptor

Schetzetal.(1999)

DopamineD4receptor

SchetzandSibley,2001

Fattyacid:oleate

5HT7Areceptor

Enhancedbindingand

potencyatlow

concentration(o15mM)

Unchanged

Albertsetal.(2001)

Fattyacidmetabolite:

oleamide

5HT2Areceptor

Nil

Enhanced

Thomasetal.(1997)

5HT7receptor

Reducedbinding

Reduced

l-aminoacids:

Aromatics,aliphatics

Calcium-sensingreceptor

Affectonbinding

unknown;Potency

enhanced

Unchanged

Conigraveetal.(2000b)


respectively) has been discussed above. It should be noted that the physiological outcome in thiscase necessarily depends on the pattern of receptor expression. Three distinct situations can bereadily envisaged with respect to the CNS. In the first, in which 5-HT2A and 5-HT7A receptors areexpressed on distinct cell-types in distinct organs of the CNS, the outcomes would be confined toeffects on maximal signalling responses—enhanced for 5-HT2A and suppressed for 5-HT7A. In asecond, in which 5-HT2A and 5-HT7A receptors are expressed on distinct cells in the same organ,oleamide would act to promote signalling via a cell-type that expresses 5-HT2A and suppresssignalling via a cell-type that expresses 5-HT7A—a form of switching between cell-types. In athird, in which 5-HT2A and 5-HT7A are co-expressed on the same cell, oleamide would act toswitch the effector output—radically altering the behaviour of the affected cell.Minor modifications of the agonist peptide, parathyroid hormone can alter the selectivity of the

PTH type-1 receptor for the PI-PLC/PKC and adenylyl cyclase signalling pathways (Janulis et al.,1993; Jouishomme et al., 1992; Takasu et al., 1999). In addition, the phosphorylation of GPCRscan induce switching between distinct G-proteins and their associated signalling pathways (Daakaet al., 1997; Lawler et al., 2001; Liang et al., 2001). These findings indicate that signal pathwayselection may be altered at the receptor protein level and regulated physiologically in someinstances by allosteric modulators. Examples of allosteric modulation of this type are eagerlyawaited.

6. Impact of molecular cloning on the study of receptor allostery

Molecular cloning has led to a completely new approach to the study of receptors and, in turn,their allosteric regulation. Thus, the organisation of receptors in membranes can now be predictedwith reasonable accuracy from the primary amino acid sequence according to a facile set of rules.These rules are based upon analyses of hydrophobicity to identify membrane-spanning helices, N-linked glycosylation sites to identify extracellular sequences and total charge distribution to predictthe orientation of receptor proteins in the presence of a physiological (i.e., internal negative)transmembrane potential difference. More sophisticated web-based analyses of functionaldomains in proteins are now widely available (e.g. http://kr.expasy.org/prosite/). In addition,cloned receptors can be expressed and studied in controlled heterologous cell culture systems totest whether interactions between agonists and putative allosteric modulators in endogenoussystems (cells and tissues) are retained.

6.1. Site-directed mutagenesis and chimeric receptors

Site-directed mutagenesis has been employed to identify amino acid residues involved in ligandbinding or action. Thus, D79 has been identified as a critical residue for the allosteric action ofNa+ on the a2-adrenergic receptor and is presumed to contribute to the intracellular Na+ bindingsite (Ceresa and Limbird, 1994). Homologous aspartate/glutamate residues in other Na+-modulated receptors appear to act in a similar way (see below). Site-directed mutagenesis has alsobeen used to implicate an EDGE sequence in the second extracellular loop of the muscarinic M2receptor in the allosteric action of gallamine (Leppik et al., 1994). More recently a site-directedmutagenesis-based approach has been used to implicate a triple serine sequence in the Venus Fly


http://kr.expasy.org/prosite/

Trap domain in the allosteric action of l-amino acids on the calcium-sensing receptor (Zhanget al., 2002).Chimeric receptors have also been used successfully to probe for receptor regions involved in

the binding or action of allosteric modulators. Thus, for example, in experiments in whichdomains were swapped between the homologous human calcium-sensing receptor and ratmetabotropic glutamate-1 receptor, the binding site of the phenylalkylamine calcimimetic, NPSR-568 was found to lie in the transmembrane region of the calcium-sensing receptor (Hauacheet al., 2000). Furthermore, a chimeric receptor approach based on domain swapping between thealcuronium- and gallamine-sensitive M2 muscarinic receptor and modulator-insensitive M5muscarinic receptor has implicated the second extracellular loop in the action of alcuronium andthe third extracellular loop in the action of gallamine (Ellis and Seidenberg, 2000).

6.2. Protein expression and crystallisation

Finally, cloned receptors or sub-domains of cloned receptors can be expressed, purified and,potentially, crystallised for determination of protein conformation and identification of ligandbinding sites. In the GPCR family, bovine rhodopsin (Palczewski et al., 2000) and the ligand-binding Venus Fly Trap domain of the rat metabotropic glutamate receptor-1 (Kunishima et al.,2000) have recently been crystallised leading to new insights into receptor activation mechanisms.In the case of the rat mGluR1, a potential allosteric site for divalent/polyvalent cations has beenidentified at the dimer interface (Tsuchiya et al., 2002).

7. Mechanisms of allosteric modulation of G-protein-coupled receptors

Evidence from a number of GPCRs supports the concept that there is a generic activationmechanism that arises from the destabilisation of transmembrane helices. Two well-studiedGPCRs, rhodopsin and the b2-adrenergic receptor, for example, appear to undergo similaragonist-dependent movements of their transmembrane helices that include relative movementsbetween TMH 3 and 6 (Farrens et al., 1996; Gether et al., 1997; Han et al., 1996). More recentwork indicates that relative movements also occur between other helices, e.g., between TMH 1and 7 in rhodopsin (Altenbach et al., 2001) which contribute to another of the hydrogen bondnetworks in the resting structure (review: Menon et al., 2001). Furthermore, mutations of GPCRsthat interfere with intermolecular interactions between neighbouring helices have been shown torender several receptors constitutively active. Thus, the loss of structural stability at the level ofthe seven transmembrane domain region appears to be a critical element of agonist-dependentreceptor activation (review: Jensen et al., 2001).Site-directed mutagenesis of residues that normally act to stabilise resting conformations of

GPCRs yield constitutively active receptors in many cases, e.g., of F199 in transmembrane helix(TMH) 5 or W279 of TMH 6 of the thyrotropin-releasing hormone receptor (Colson et al., 2001).Hydrophobic and polar interactions involving TMHs 5, 6 and 7 of the luteinising hormonereceptor are also required to stabilise the normal inactive conformation (review: Gershengorn andOsman, 2000). Thus, allosteric inhibitors that bind in the transmembrane region of GPCRs, (e.g.,the calcium-sensing receptor inhibitor NPS-2143; Nemeth et al., 2001) might act by stabilising the


resting structure, preventing the movement of transmembrane helices. On the other hand,allosteric activators that bind in the transmembrane region (e.g., the calcium-sensing receptoractivator NPS R-467; Hammerland et al., 1996) might facilitate receptor activation by stabilisingan active conformation. Given the propensity for this receptor to operate functionally as a dimer(Bai et al., 1999), it might be wondered whether the dimer interface or helices that secure the dimerinterface might represent potential targets for allosteric modulators.

7.1. Mechanism of G-protein activation

Loss of transmembrane stability upon agonist binding and signal transmission is predicted toinduce the localised cytoplasmic exposure of a previously masked peptide domain in TMH 6 thatpromotes signal transduction (Schulz et al., 2000). According to this view, binding ofheterotrimeric G-proteins to this newly exposed domain initiates GTP–GDP exchange and G-protein dissociation. Consistent with this idea, sequence-independent nine amino acid deletions ofthe third intracellular loop of the TSH receptor or TMH 6 insertions, either of which inducemovement of the N-terminus of TMH 6 towards the cytoplasm in molecular models, promoteconstitutive receptor activation (Wonerow et al., 2001). Constitutive receptor activation was alsoobserved upon non-conservative point mutations affecting D619 at the interface between the thirdintracellular loop (i3) and TMH 6 junction of this receptor. Conservation of either an Asp or Gluresidue at this junction across the superfamily of GPCRs appears to be required to maintain theinactive state and may act to cap the resting helical structure (Schulz et al., 2000). Furthermore,structural rearrangements at the cytoplasmic (N-terminal) end of TMH 6 have been detected uponagonist binding in both the a2-adrenergic receptor, using a fluorescence labelling strategy (Jensenet al., 2001), and in the muscarinic M3 receptor, using a strategy based on in situ crosslinking(Ward et al., 2002). The interaction observed between E247 in TMH 6 and R135 (a highlyconserved Arg in GPCRs) in TMH 3 of the X-ray crystal structure of bovine rhodopsin alsopoints to a key role for the cytoplasmic terminus of TMH 6 in stabilising the inactive state(Palczewski et al., 2000).A receptor’s G-protein binding surface is clearly a potential target for allosteric modulation.

Thus molecular species that stabilise the resting conformation or prevent access of G-proteins tothe binding site are potential inhibitors. Arrestins, which bind to the intracellular surfaces ofreceptors that have been phosphorylated by G-protein receptor kinases and prevent further accessof G-proteins to the effector module represent physiological examples of such an inhibitor(review: Pierce and Lefkowitz, 2001). Some small, cell permeant molecules may mimic, at least inpart, this action of the arrestins. Polyanions, for example, uncouple several GPCRs from theircorresponding G-proteins. In the case of the a2 and b2 adrenergic receptors, several suchpolyanion inhibitors of receptor activation interfere with agonist binding without interfering withthe binding of antagonists (Huang et al., 1990).

8. Endogenous allosteric modulators of plasma membrane receptors

Progress on discovering the identities of endogenous allosteric modulators lags behind thediscovery of new xenobiotic modulators of plasma membrane receptors. This is perhaps not


surprising since there are currently few useful rules for the identification of endogenous allostericmodulators. The NMDA ionotropic glutamate receptor is given as one example below. SeveralGPCRs of sub-family C, whose extracellular bilobed Venus Fly Trap binding domains arehomologous to the agonist binding sites on NMDA receptors are also discussed.

8.1. Endogenous modulation of NMDA receptors

Glutamate acts as the agonist at ionotropic NMDA receptors. However, these receptors areoligomers formed from distinct receptor subunits including NR1, NR2 and NR3. The glutamatebinding site has been located in the NR2 (A–D) subunit (Laube et al., 1997). The related NR1subunit provides a high affinity binding site for glycine (Hirai et al., 1996). Glycine and glutamateare now viewed as co-agonists of these receptor heterodimers. Heterodimers formed by NR1 andeither of the newly cloned NR3A or NR3B subunits, however, behave as excitatory glycinereceptors with no requirement for glutamate or response to NMDA (Chatterton et al., 2002). Theendogenous polyamine, spermine may also act physiologically to modulate NMDA receptorfunction in the brain (Anderson et al., 1975; Kroiggard et al., 1992).Examples of endogenous allosteric modulators of GPCRs are relatively limited but several are

worth noting here.

8.2. Inorganic allosteric modulators of G-protein-coupled receptors

As noted above, intracellular Na+ acts as an allosteric modulator of various GPCRs includinga-adrenergic (Michel et al., 1980; Motulsky and Insel, 1983; Tsai and Lefkowitz, 1978),dopaminergic D2 (Neve et al., 1991) and D4 (Schetz and Sibley, 2001), opioid (Paterson et al.,1986; Pert and Snyder, 1974) and A1 adenosine (Goodman et al., 1982) receptors. Surprisingly, ithas been commonly reported that Na+ suppresses agonist binding and promotes the binding ofantagonists—similar to the ‘‘retrograde’’ impact of G-proteins on agonist/antagonist binding atthe orthosteric site (review: Christopoulos and Kenakin, 2002). In the case of the platelet a2-adrenergic receptor, cytoplasmic Na+ also appears to selectively enhance the efficacy of agonists(Michel et al., 1980; Motulsky and Insel, 1983) and Na+ is necessary for d-opioid receptordependent suppression of adenylyl cyclase (Blume et al., 1979). As described above mutation of ahighly conserved acidic residue (D79) in TMH 2 of the a-adrenergic receptor, to yield theuncharged species Asn or Gln, resulted in complete loss of the Na+-dependent effect (Ceresa andLimbird, 1994). The homologous residue D80 in the dopamine D2 receptor serves a similarpurpose (Neve et al., 1991). More recently, Zn2+ has also been identified as an allostericmodulator of the D2 and D4 dopaminergic receptors (Schetz et al., 1999; Schetz and Sibley, 2001)and the b2-adrenergic receptor upon which it acts at potentially physiological, low micromolar,concentrations to promote isoproterenol-induced cAMP formation (Swaminath et al., 2002).

8.3. Organic endogenous modulators

Several organic endogenous allosteric modulators are also recognised. The fatty acidmetabolite, oleamide (cis-9,10-octadecenoamide), for example, acts as an allosteric modulatorat 5-hydroxytryptamine receptors. Interestingly, it promotes maximal 5HT-induced inositol


phosphate production at 5-HT2 receptors and non-competitively inhibits agonist-induced cAMPproduction at 5HT7 receptors (Table 2). In addition, fatty acids including oleate and stearate,have been found to modulate 5HT7A receptors (Alberts et al., 2001). This finding suggests thehypothesis that one or more of the 5HT receptors may act as the fatty acid sensors in thegastrointestinal tract (Sidhu et al., 2000).Recently, l-amino acids including especially members of the aromatic, aliphatic and polar

subclasses have been identified as allosteric activators of the calcium-sensing receptor (Conigraveet al., 2000b) apparently binding in the bilobed Venus Fly Trap domain (Zhang et al., 2002) whichshares homology with nutrient-sensing bacterial periplasmic binding proteins (Brauner-Osborneet al., 1999). Physiologically, this implies interactions between protein and calcium metabolismand the calcium-sensing receptor is a candidate amino acid sensor for stimulated gastric acidsecretion (Conigrave et al., 2002; Conigrave et al., 2000a).

9. Conservation of endogenous modulators according to biochemical class

In several cases, homologous receptors are regulated by endogenous allosteric modulators fromthe same biochemical class. If this phenomenon holds true more generally, the identification of anallosteric modulator for one receptor sub-type may permit the identification of biochemicallyrelated modulators of other receptors belonging to the same class. Thus, 5HT receptors aremodulated by fatty acids and fatty acid derivatives (Alberts et al., 2001; Thomas et al., 1997).Group C GPCRs, on the other hand, including metabotropic glutamate receptors, the GABA(B)receptor, the calcium-sensing receptor (Conigrave et al., 2000b) and taste receptors (Nelson et al.,2002) are sensitive to amino acids or amino acid metabolites either as agonists or allostericmodulators. Sensitivity to monovalent (especially Na+) and divalent cations, on the other hand, isconserved more broadly across a large number of GPCRs—especially belonging to Group A.These examples indicate that the binding sites for endogenous allosteric modulators are conservedamong sub-groups of GPCRs. Indeed, binding sites for endogenous modulators would appear tobe conserved in some cases across receptor classes. Thus, the NMDA ionotropic receptor and theligand-binding VFT domain of the calcium-sensing GPCR are homologous at the amino acidlevel. Moreover, both exhibit binding sites for polyamines (Quinn et al., 1997; Ransom and Stec,1988), other polycations (Brown et al., 1993; Lu et al., 1998), and amino acids (Conigrave et al.,2000a; Conigrave et al., 2000b; Johnson and Ascher, 1987). As the allosteric binding sites forspecific receptors are identified by structural and molecular techniques, predictions regarding thepresence or absence of allosteric sites on other receptors and their likely biochemical selectivitiesmay be possible.

10. Approaches to the identification of endogenous allosteric modulators

Non-biased, systematic analyses appear to be necessary before a complete picture ofphysiologically relevant receptor modulation is obtained. Even a complete analysis of simplebinary systems (i.e., in which there is one additional ligand binding site in addition to the agonistbinding site) involving all encoded human receptor genes (including primary transcripts and splice


variants) represents a massive undertaking. The minimal conditions for such an undertakingwould appear to include:

* a complete library of receptors in a convenient form for screening in tandem with majorsignalling pathways,

* a complete library of biochemical species in a convenient form for screening.

Desirable additional information includes:

* a complete description of tissue-specific receptor expression,* measurement of physiologically relevant concentration ranges of putative modulators inspecific tissue environments.

In the meantime, a physiologically driven analysis of specific cases is feasible, making use of theavailable infrastructure in basic science research laboratories. Simple conditions which shouldnormally be met prior to screening for allosteric regulation include the following:

* An unsolved problem in physiological sensing should have been identified.* The candidate receptor should be expressed in tissues that are relevant to the physiologicalphenomenon.

* The candidate receptor should have been cloned.* The candidate receptor should be readily expressed in heterologous cell culture systems.* The receptor’s endogenous agonist should have been identified.

10.1. Criteria required for the identification of novel allosteric modulators

It is also worth considering the standard of proof required to establish the existence of a novelallosteric modulator. Ideally, the following criteria should be satisfied:

* The candidate modulator should have no effect on untransfected cells.* The effect of the candidate modulator on transfected cells should be reproducible in differentcell lines.

* The effect of the candidate modulator on transfected cells should mimic its effect on cells thatexpress the receptor endogenously.

* The candidate modulator should have no effect on the levels of the receptor’s physiologicalagonist.

Acknowledgements

The authors’ research is supported by the National Health and Medical Research Council ofAustralia.

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