Báo cáo khoa học: The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology pptx

A large body of evi-dence has led us to question this classical view of hormone–receptor interaction, for it is now widely accepted that GPCRs may exist as either homo-dimers or even hig

The impact of G-protein-coupled receptor

hetero-oligomerization on function and pharmacology

Roberto Maggio1, Francesca Novi1, Marco Scarselli2and Giovanni U Corsini1

1 Department of Neurosciences, University of Pisa, Italy

2 National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA

G-protein coupled receptors (GPCRs) constitute the

largest family of seven-transmembrane receptors Their

evolutionary success is due to their extreme versatility

in binding a variety of signaling molecules such as

hor-mones and neurotransmitters The ubiquitous

distribu-tion in the human body, along with the capacity to

regulate virtually all known physiological processes,

has made this family of receptors the most important

target for drug research [1]

According to the classical view of

hormone–recep-tor interaction, a hormone binds to one recephormone–recep-tor

protein and, in turn, the hormone–receptor complex

activates the effector pathway A large body of

evi-dence has led us to question this classical view of

hormone–receptor interaction, for it is now widely

accepted that GPCRs may exist as either

homo-dimers or even higher-order homo-oligomers, besides

being capable of interacting with distantly related

receptor subtypes to form hetero-oligomers (reviewed

in [2,3])

The huge interest generated by this phenomenon among biologists in the last 10 years has led many groups to study the mechanism(s) by which GPCR dimerization occurs This has contributed to the dem-onstration that many, if not all, GPCRs can form homo-oligomers and hetero-oligomers, but it has also generated much pharmacological and functional evi-dence that is difficult to reconcile with a unique mechan-istic model of GPCR dimerization A key problem that still remains controversial is how dimerization affects G-protein coupling Although GPCR homo-oligo-merization can be accounted for by a simple receptor⁄ G-protein stoichiometry, GPCR hetero-oligomerization raises the problem of how two different receptors can influence the coupling of each other and determine the ultimate function of the complex

Keywords

bivalent ligand; G-protein; mitogen-activated

protein kinase (MAPK); oligomerization;

b-arrestin

Correspondence

R Maggio, Department of Neurosciences,

University of Pisa, Via Roma 55,

56100 Pisa, Italy

Fax: +39 050 2218717

Tel: +39 050 2218707

E-mail: r.maggio@drugs.med.unipi.it

(Received 16 February 2005, revised 7 April

2005, accepted 21 April 2005)

doi:10.1111/j.1742-4658.2005.04729.x

Although highly controversial just a few years ago, the idea that G-pro-tein-coupled receptors (GPCRs) may undergo homo-oligomerization or hetero-oligomerization has recently gained considerable attention The recognition that GPCRs may exhibit either dimeric or oligomeric structures

is based on a number of different biochemical and biophysical approaches Although much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains elusive An additional source of uncertainty stems from the realization that homo-oligomerization and hetero-oligomerization

of GPCRs may affect receptor binding and activity in different ways, depending on the type of interacting receptors In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding and cell signaling are critically analyzed

Abbreviations

GPCR, G-protein-coupled receptor; LTB 4 , leukotriene B 4 ; MAPK, mitogen-activated protein kinase.

Effect of hetero-oligomerization on G-protein

coupling and function

To make the issue even more complicated,

hetero-oligo-merization has been shown to occur between pairs of

receptors that couple with either the same G-protein

or different G-proteins On the assumption that each

receptor in the hetero-oligomer may bind only to a

sin-gle G-protein, it follows that its coupling selectivity in

the complex should, in large part, be conserved As a

matter of fact, several reports that deal with receptor

hetero-oligomerization indicate that stimulation of one

receptor in cotransfected cells is often sufficient to

activate a G-protein, leaving their coupling efficacy

unchanged For instance, b2-adrenergic receptors,

which are coupled with stimulatory G-proteins, and

d-opioid and j-opioid receptors, which are coupled with

inhibitory G-proteins, are both known to form

hetero-meric complexes, but hetero-oligomerization in this

case does not significantly alter their ligand-binding

capacity or coupling properties [4] Likewise, adenylate

cyclase stimulation by Gs-coupled dopamine D1

recep-tors and adenylate cyclase inhibition by the Gi-coupled

dopamine D2 receptors are not altered in cells

coex-pressing both receptors, even though they may form

hetero-oligomers [5] The same phenomenon has been

shown to occur in hetero-oligomers formed by Gi

-cou-pled and Gq-coupled receptors [6] and by Gs-coupled

and Gq-coupled receptors [7] One of the limitations

implied in these experiments is the actual impossibility

of establishing with certainty how many receptors

undergo hetero-oligomerization compared with those

that give rise to homo-oligomeric complexes or even

remain in a monomeric form Under these conditions,

if the molar ratio between hetero-oligomers and

homo-oligomers (or monomers) falls below a certain

thresh-old, the effect of hetero-oligomerization would remain

undetected, and the occurrence of any functional

change in the target cells would be difficult to ascertain

experimentally At odds with the above examples are

other reports describing how changes in function or

coupling efficacy may simply result from the

stimula-tion of one or both receptors of the hetero-oligomer

For example, coexpression of dopamine D2and

somato-statin SSTR5 receptors results in synergistic inhibition

of adenylate cyclase [8] Similarly, coexpression of

angiotensin I and bradykinin B2receptors in HEK-293

cells increases the efficacy and potency of angiotensin

II, but it also reduces the ability of bradykinin to

sti-mulate inositol phosphate production [9] In

fibro-blasts, pretreatment of A1 and D1 receptors with both

adenosine and dopamine agonists, but not with either

of them separately, has been shown to reduce the

signaling efficacy of D1receptors on subsequent stimu-lation [10] In COS-7 cells cotransfected with dopamine

D2 and D3receptors, highly selective D3agonists inhi-bit adenylate cyclase, but remain ineffective in cells transfected with D3 alone [11,12] However, the role played by hetero-oligomerization in each of these func-tional changes remains speculative, as the same effects may also be induced by cross-talk between the signa-ling pathways, downstream of receptor activation The acquisition of new coupling selectivity by coex-pressed receptors is perhaps one of the most intri-guing aspects of GPCR hetero-oligomerization Three major studies have shown this phenomenon clearly: (a) l-receptors and d-receptors that changed their coup-ling selectivity from pertussis-sensitive Gi⁄ Go-proteins

to pertussis-insensitive (probably Gz) proteins, in transi-ently cotransfected COS-7 cells [13]; (b) chemokine CCR2band CCR5receptors that gained coupling selec-tivity for G11-protein in cotransfected HEK-293 cells [14]; (c) dopamine D1 and D2 receptors that gained coupling selectivity for Gq-proteins in transiently cotransfected COS-7 cells [5] In the last instance, if each dopamine receptor is stimulated separately with select-ive agonists, their coupling selectivity for Giand Gs is not altered, whereas simultaneous dopamine stimulation

of both receptors results in the activation of Gq This observation can be taken to mean that cells may gain

a new coupling selectivity when the two components

of the receptor hetero-oligomer are activated simulta-neously

Assuming that each receptor in the hetero-oligomer can bind only to single G-proteins, then any new coup-ling selectivity gained by the hetero-oligomer is likely

to depend on a newly acquired spatial rearrangement

of the intracellular domain(s) that binds to these G-proteins This conclusion is not unexpected, as seve-ral studies have shown that receptors that activate spe-cific G-proteins can be induced to expose distinct intracellular domains if stimulated by different agonists [15] The conformational changes that result from receptor–receptor interactions may in fact cause vari-ation in the exposure of certain intracellular domains and, in doing so, alter the specificity of their inter-action Another possible explanation of this change in coupling selectivity comes from recent work with receptor homodimers Using a combination of mass spectrometry after chemical cross-linking and neutron scattering in solution, Baneres & Parello [16] have been able to establish unambiguously that only one G-pro-tein trimer binds to a leukotriene B4 (LTB4) receptor BLT1 dimer (2·BLT1.LTB4) so as to form a stoichio-metrically defined (2·BLT1.LTB4)Gai2b1c2 pentameric assembly They suggested that receptor dimerization

may play a crucial role in transducing the LTB4

-induced signal Similar conclusions were drawn in a

recent paper by Chinault et al [17], who demonstrated

that yeast oligomeric a-factor receptors function in

concert to activate G-proteins Further support for the

2 : 1 receptor⁄ G-protein coupling stoichiometry comes

from experiments performed with receptor fragments

Single transmembrane regions of b2 or dopamine D2

receptors prevent dimerization and stop functioning

when cotransfected with their cognate wild-type

recep-tors, indicating that disruption of their dimeric

com-plex impedes these receptors to couple with G-proteins

[18,19] If these results prove valid for heterodimers as

well, they could explain how heterodimerization affects

receptor coupling selectivity Whereas homodimers

provide pairs of identical intracellular domains,

het-erodimers have unique combinations of intracellular

domains This could confer a different coupling

effi-ciency and selectivity on the heterodimers compared

with that expressible by the homodimers of their

respective receptors

Hetero-oligomerization affects b-arrestin coupling

and internalization

GPCR activation promotes recruitment of b-arrestin

to the receptor site This leads to signal termination by

blocking G-protein interaction and it triggers receptor

internalization by endocytosis A large amount of

evidence has now accumulated indicating that

hetero-oligomerization influences b-arrestin binding and

receptor internalization This is clearly described in the

excellent paper by Terrillon et al [20] V1a and V2

vasopressin receptors are internalized by way of the

b-arrestin-dependent process However, whereas V1a

receptors are rapidly recycled to the plasma membrane

after dissociation from b-arrestin, V2 receptors do not

dissociate from b-arrestin and consequently accumulate

in the endosomes In their paper, Terrillon et al [20]

demonstrated that, in cotransfected HEK cells, V1a

and V2 receptors are endocytosed as stable

hetero-olig-omers Upon activation with nonselective agonists, the

V1a⁄ V2 hetero-oligomer follows the endocytic ⁄

recyc-ling pathway of the V2 receptor up to the endosomes

Conversely, the hetero-oligomer is targeted to the

endo-cytic⁄ recycling pathway of V1a receptor if activated

with a selective V1a agonist In the latter case, the

hetero-oligomer is rapidly recycled to the plasma

mem-brane This work clearly indicates that it is the identity

of the activated promoter within the hetero-oligomer

that determines the fate of the internalized receptors

Other examples of the reciprocal influence of receptors

in the internalization process are the adrenergic a1a

and a1b receptors [21], neurokinin NK1 and l opioid receptors [22], and the b2-adrenergic and d-opioid receptors [4] In all these studies, selective stimulation of

a single receptor component of the hetero-oligomer is sufficient to cause internalization of the entire complex

As discussed in the previous section, a critical issue

in assessing the effects of hetero-oligomerization is to establish the extent by which GPCRs tend to hetero-oligomerize To account for the above results one would have to suppose that a large fraction of the receptors expressed in the plasma membrane are already

in a hetero-oligomeric form However, this is made unlikely by the observation that j-opioid receptors exhibit a higher propensity to form homo-oligomers than b2-adrenergic and j-opioid receptors to form hetero-oligomers [23] Based on this observation, it may be reasonable to think that, in cells coexpressing two receptors, most of them are in a homo-oligomeric form In spite of this indication, however, internalizat-ion of b2-adrenergic receptors, as induced by isopro-terenol, is impeded in the presence of j-opioid receptors [4]

To explain these puzzling data, j-opioid and

b2-receptor homo-dimers could be assumed to be part

of a larger hetero-oligomeric array such as even the smallest fraction of this receptor complex could actu-ally affect functioning of the entire cluster The idea that receptors may co-operate within larger aggregates has been put forward by Park et al [24] on the basis

of radioligand binding to muscarinic M2 receptors Muscarinic cholinergic receptors can appear to be more numerous when labeled with [3 H]quinuclidinyl-benzilate than with N-[3H]methylscopolamine Binding

at near-saturating concentrations of [3 H]quinuclidinyl-benzilate was blocked fully by unlabeled N-methyl-scopolamine, which therefore appeared to inhibit noncompetitively at sites inaccessible to N-[3 H]methyl-scopolamine Both the shortfall in capacity for N-[3H]methylscopolamine and the noncompetitive effect

of N-methylscopolamine on [3H]quinuclidinylbenzilate has been described quantitatively in terms of co-opera-tive interactions within a receptor that is at least tetra-valent

Besides their effects on dampening receptor–G-pro-tein coupling and on receptor internalization, b-arres-tin also plays a major role in GPCR activation of mitogen-activated protein kinase (MAPK) In this con-text it may act as an adaptor or scaffolding for recruit-ing signalrecruit-ing molecules into a complex along with the agonist-occupied receptors (for a review see [25]) The first evidence of this effect has been provided by Luttrell et al [26], who showed that agonist phos-phorylation of b2-adrenergic receptors leads to rapid

recruitment of b-arrestin-1, carrying the activated

receptor c-Src with it Subsequent reports showed that

b-arrestins can also interact directly with component

kinases of the ERK1⁄ 2 and c-Jun N-terminal kinase 3

MAPK cascades b-Arrestins have been shown to

form complexes with angiotensin II type 1A receptor,

cRaf-1 and ERK1⁄ 2 [27,28], with protease-activated

receptor type 2, Raf-1 and ERK1⁄ 2 [29], and with

neurokinin-1 receptor, c-Src and ERK1⁄ 2 [30]

That hetero-oligomerization may interfere with

b-arrestin-mediated signaling is demonstrated by the

observation of Lavoie et al [31] that activation of

b-arrestin-mediated ERK1⁄ 2 phosphorylation by

b2-adrenergic receptors is inhibited on coexpression of

b1-adrenergic receptors They suggested that

hetero-oligomerization between b1 and b2receptors may

inhi-bit the agonist-promoted b2 ability to activate the

ERK1⁄ 2 signaling pathway In another paper, Breit

et al [32] showed that hetero-oligomerization of

b3-adrenergic receptors with b2-receptors modified

their effect on ERK1⁄ 2 phosphorylation Novi et al

[6,33] showed that a mutant muscarinic M3 receptor

that is incapable of binding to b-arrestin-1 impairs

completely the ability of wild-type M3 receptors to

recruit b-arrestin-1 to the plasma membrane and to

stimulate ERK1⁄ 2 phosphorylation All these data

indicate quite clearly that homo-oligomerization and

hetero-oligomerization have a pivotal role in defining

the GPCR–b-arrestin specificity, and consequently in

determining the receptor fate and b-arrestin-mediated

MAPK activation

As discussed above for G-proteins, the mechanism

and the stoichiometry by which GPCR and b-arrestin

interact is not known Conventionally, GPCRs and

b-arrestins are assumed to interact in a 1 : 1 molar

ratio However, this conventional view should be

reconsidered on the basis of more recent GPCR

hetero-oligomerization data and be replaced by a more

complex model of interaction between the two

pro-teins For example, two b-arrestin molecules may bind

to a receptor dimer, and this may in turn cause more

efficient sequestration and signaling of the GPCR–

b-arrestin complex Han et al [34] proposed a

mechan-istic model of b-arrestin–receptor interaction in which

the initial binding of the first b-arrestin molecule to

the receptor is followed by displacement of its terminal

C-tail and dimerization with another b-arrestin

mole-cule They speculated that b-arrestin dimerization may

help the b-arrestin–receptor complexes to cope with

the internalization machinery of the coated pits

How-ever, the possibility that dimerization of b-arrestin also

acts as a scaffold for MAPK was left open, given the

molecular dimensions of the complexes containing

both b-arrestin and MAPK [27,29,30] Another possi-bility yet to be considered is that a b-arrestin monomer may bind to a receptor dimer so that the resulting receptor combination reinforces the bond strength of the heterodimers This hypothesis is based on a recent study on the organization of rhodopsin in native plasma membranes [35] Arrestin, the cognate b-arres-tin of the visual system, has a bipartite structure with two structurally homologous seven-stranded b-sand-wiches forming two putative rhodopsin-binding grooves separated by 3.8 nm [36,37] This spatial arrangement may mean that the rhodopsin dimer sur-face matches perfectly the arrestin molecule by charge complementarity A cartoon showing the hypothetical mechanisms of receptor–b-arrestin interaction and ERK1⁄ 2 signaling is shown in Fig 1

Regardless of the mechanism by which b-arrestins bind to GPCRs, the signaling pathway activated by these proteins is another way by which GPCR hetero-oligomerization can influence cell physiology In view

of the fact that MAPK plays a pivotal role in such cell processes as cell growth, division, differentiation and apoptosis, it is likely that, in the near future, the phar-macology of GPCR hetero-oligomers can be exploited

to gain control of these cellular events

Pharmacological diversity

In the last 6 years, a growing number of receptors have been shown to behave as hetero-oligomers and to exhibit an unexpected level of pharmacological diver-sity Jordan & Devi [38] presented the first evidence that the pharmacology of interacting receptors is dif-ferent from that of the constituent monomers (or homodimers) They showed that j–d-opioid hetero-oligomers had no significant affinity for either j-selective or d-selective agonists or antagonists in cotransfected cells, even though the hetero-oligomers had a stronger affinity for the partially selective lig-ands Following this pivotal work, many other researchers have shown how ligand affinity changes on receptor coexpression [39–41] In all these studies, the extent by which ligand affinity changes is accounted for by a single parameter determined by competition binding analysis It should be clear that this parameter does not provide a realistic measure of the ligand affin-ity for the hetero-oligomer At most, it may represent

an average measure of all the different affinities that the ligand expresses for the binding site(s) of both het-ero-oligomers and homo-oligomers Quite often, in the absence of detailed analyses, it is not possible to estab-lish which binding fractions can be attributed to the hetero-oligomer and which to the homo-oligomer(s)

Under these circumstances, if two GPCRs exhibit an

equivalent propensity to form either homo-oligomer or

hetero-oligomer, only 50% of the receptor population

would be in the hetero-oligomeric form This

percent-age would be even lower if the tendency to form

hetero-oligomers is significantly different

The pharmacological changes that occur within the

hetero-oligomers are most likely due to allosteric

rear-rangements induced by the interacting receptor

mono-mers Ligand binding to half of the dimer may

somehow modify the affinity for the other half This

view is supported by the work of Mesnier & Baneres

[42] with the LTB4 receptor BLT1 homodimer By

studying how fluorescence properties of

5-hydroxy-tryptophan vary, these authors have been able to show

that agonist binding to part of the LTB4 receptor

BLT1 homodimers induces conformational changes in

the remaining part of the homodimer Although not

generally accepted, another possible explanation for

how receptor pharmacology may change, at least

among receptor subtypes, is domain swapping [43–45]

According to this model of interaction, two receptors

may interact in such a way as to induce rearrangement

of their transmembrane domains, and this would

even-tually result in the formation of two novel binding

sites So far, domain swapping has been shown to

occur only among functionally impaired receptors and

never with wild-type receptors This may be because of

the technical complexity of devising experiments to

observe the effect of domain swapping when both

receptors are functional

The oligomeric nature of GPCRs can be exploited to improve drug specificity by developing dimeric ligands capable of acting as bivalent ligands The first publica-tion showing the feasibility of constructing a bivalent ligand directed to heterodimeric receptors has come

C

ERK activation

ERK activation

ERK activation

H

H

H GPCR

β-arrestin β-arrestin

β-arrestin β-arrestin β-arrestin

β-arrestin β-arrestin

GPCR GPCR

Fig 1 Alternative models of

GPCR–b-arres-tin interaction (A) The sequential binding

of the ligands to each half of the receptor

dimer induces the recruitment of two

molecules of b-arrestin and then the

activation of ERK (B) Only one molecule

of b-arrestin binds to the ligand-saturated

receptor dimer and activates ERK.

(C) A dimer of b-arrestin binds all at once

to a receptor dimer and activates ERK.

Fig 2 Proposed models of association of bivalent ligands with GPCR hetero-oligomers (A) Bivalent ligands bind pairs of receptor hetero-dimers (B) Bivalent ligands bridge two different subtypes of neighboring receptor homodimers.

from Saveanu and coworkers [46] The chimeric agonist

they synthesized comprises a somatostatin–dopamine

molecule (BIM-23A387) directed against the dopamine

D2and somatostatin SST2receptors They claimed that

this agonist suppresses secretion of both growth

hor-mone and prolactin in human pituitary somatotrophic

adenoma cells (each cell coexpressing both dopamine

D2 and somatostatin SST2 receptors) much more

powerfully than either of the two pharmacophores

given all at once or separately Portoghese’s group [47]

has more recently synthesized the ligand KDN-21,

which belongs to a series of bivalent ligands containing

d-opioid and j-opioid antagonist pharmacophores

attached to variable-length spacers This compound has

been shown to have substantially greater affinity for

d-opioid and j-opioid receptors than the univalent

ana-logs Furthermore, this compound had 200-fold higher

affinity for cotransfected d-opioid and j-opioid

recep-tors than for the same receprecep-tors transfected separately

and then allowed to interact To understand the

mech-anism by which these bivalent ligands work, the

struc-tural organization of GPCR oligomers in the plasma

membrane needs to be clarified The models of bivalent

ligand–receptor interaction proposed in Fig 2 foresee

two possible oligomeric organizations for GPCRs

The possibility of developing ligands that are

select-ive for hetero-oligomeric GPCRs is the most promising

strategy yet for targeting different tissues of the human

body Screening for drugs that would be so selective as

to restrict binding to hetero-oligomer receptors in the

presence of the corresponding homo-oligomers is the

real challenge for scientists working in this field in

the near future With these selective drugs at hands,

we will be able to shed new light on the physiological

role played by receptor hetero-oligomerization

Acknowledgements

We thank Dr Franco Giorgi for advice and helpful

discussion

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