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

MINIREVIEW
The impact of G-protein-coupled receptor
hetero-oligomerization on function and pharmacology
Roberto Maggio
1
, Francesca Novi
1
, Marco Scarselli
2
and Giovanni U. Corsini
1
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 receptor
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:
(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.
FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2939
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, b
2
-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 G
s
-coupled dopamine D
1
recep-
tors and adenylate cyclase inhibition by the G
i
-coupled
dopamine D
2
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 G
i
-cou-

pled and G
q
-coupled receptors [6] and by G
s
-coupled
and G
q
-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 D
2
and somato-
statin SSTR
5
receptors results in synergistic inhibition
of adenylate cyclase [8]. Similarly, coexpression of
angiotensin I and bradykinin B

2
receptors 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 A
1
and D
1
receptors with both
adenosine and dopamine agonists, but not with either
of them separately, has been shown to reduce the
signaling efficacy of D
1
receptors on subsequent stimu-
lation [10]. In COS-7 cells cotransfected with dopamine
D
2
and D
3
receptors, highly selective D
3
agonists inhi-
bit adenylate cyclase, but remain ineffective in cells
transfected with D
3
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 G
i
⁄ G
o
-proteins
to pertussis-insensitive (probably G
z
) proteins, in transi-
ently cotransfected COS-7 cells [13]; (b) chemokine
CCR
2b
and CCR
5
receptors that gained coupling selec-
tivity for G
11
-protein in cotransfected HEK-293 cells
[14]; (c) dopamine D
1
and D
2
receptors that gained
coupling selectivity for G
q

-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 G
i
and G
s
is
not altered, whereas simultaneous dopamine stimulation
of both receptors results in the activation of G
q
. 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 B
4
(LTB
4
) receptor
BLT1 dimer (2·BLT1.LTB
4
) so as to form a stoichio-
metrically defined (2·BLT1.LTB
4
)Ga
i2
b
1
c
2
pentameric
assembly. They suggested that receptor dimerization
Function and pharmacology of hetero-oligomers R. Maggio et al.
2940 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS
may play a crucial role in transducing the LTB
4
-
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 b
2
or dopamine D
2
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 a
1a
and a
1b
receptors [21], neurokinin NK
1
and l opioid
receptors [22], and the b
2
-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 b
2
-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 b
2
-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
b
2
-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 M
2
receptors.
Muscarinic cholinergic receptors can appear to be
more numerous when labeled with [
3
H]quinuclidinyl-
benzilate than with N-[
3
H]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-[
3
H]methylscopolamine and the noncompetitive effect
of N-methylscopolamine on [
3
H]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 signaling 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 b
2
-adrenergic receptors leads to rapid
R. Maggio et al. Function and pharmacology of hetero-oligomers
FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2941
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
b
2

-adrenergic receptors is inhibited on coexpression of
b
1
-adrenergic receptors. They suggested that hetero-
oligomerization between b
1
and b
2
receptors may inhi-
bit the agonist-promoted b
2
ability to activate the
ERK1 ⁄ 2 signaling pathway. In another paper, Breit
et al. [32] showed that hetero-oligomerization of
b
3
-adrenergic receptors with b
2
-receptors modified
their effect on ERK1 ⁄ 2 phosphorylation. Novi et al.
[6,33] showed that a mutant muscarinic M
3
receptor
that is incapable of binding to b-arrestin-1 impairs
completely the ability of wild-type M
3
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).
Function and pharmacology of hetero-oligomers R. Maggio et al.
2942 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS
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 LTB
4
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 LTB
4
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
AB

C
No effect No effect
ERK activation
ERK activation
ERK activation
H
HH HH
HH
H
H
HH
H
H
HH
HH
H
GPCR
β-arrestin β-arrestin
β-arrestin β-arrestin
β-arrestin
β-arrestin β-arrestin
GPCR GPCR GPCR GPCR
GPCR GPCR GPCR GPCR
GPCR GPCR GPCR GPCR
GPCR GPCR
GPCR 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.
R. Maggio et al. Function and pharmacology of hetero-oligomers
FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2943
from Saveanu and coworkers [46]. The chimeric agonist
they synthesized comprises a somatostatin–dopamine
molecule (BIM-23A387) directed against the dopamine
D
2
and somatostatin SST
2
receptors. 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
D
2
and somatostatin SST
2
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 receptors 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.
References
1 Lefkowitz RJ (2004) Historical review: a brief history

and personal retrospective of seven-transmembrane
receptors. Trends Pharmacol 25, 413–422.
2 Agnati LF, Ferre S, Lluis C, Franco R & Fuxe K
(2003) Molecular mechanisms and therapeutical implica-
tions of intramembrane receptor ⁄ receptor interaction
among heptahelical receptors with examples from stri-
atopallidal GABA neurons. Pharmacol Rev 55, 509–550.
3 Angers S, Salahpour A & Bouvier M (2002) Dimeriza-
tion: an emerging concept for G protein-coupled
receptor ontogeny and function. Annu Rev Pharmacol
Toxicol 42, 409–435.
4 Jordan BA, Trapaidze N, Gomes I, Nivarthi R & Devi
LA (2001) Oligomerization of opioid receptors with
beta2-adrenergic receptors: a role in trafficking and
mitogen-activated protein kinase activation. Proc Natl
Acad Sci USA 98, 343–348.
5 Lee SP, So CH, Rashid AJ, Varghese G, Cheng R,
Lanca AJ, O’Dowd BF & George SR (2004) Dopamine
D1 and D2 receptor co-activation generates a novel
phospholipase C-mediated calcium signal. J Biol Chem
279, 35671–35678.
6 Novi F, Scarselli M, Corsini GU & Maggio R (2004)
The paired activation of the two components of the
muscarinic M3 receptor dimer is required for induction
of ERK1 ⁄ 2 phosphorylation. J Biol Chem 279, 7476–
7486.
7 Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub
MA, Taulan M, Jockers R, Barberis C & Bouvier M
(2003) Oxytocin and vasopressin V1a and V2 receptors
form constitutive homo- and heterodimers during bio-

synthesis. Mol Endocrinol 17, 677–691.
8 Rocheville M, Lange DC, Kumar U, Patel SC, Patel
RC & Patel YC (2000) Receptors for dopamine and
somatostatin: formation of hetero-oligomers with
enhanced functional activity. Science 288, 154–157.
9 AbdAlla S, Lother H & Quietterer U (2000) AT
1
-recep-
tor heterodimers show enhanced G-protein activation
and altered receptor sequestration. Nature 407, 94–98.
10 Gine
´
s S, Hillion J, Torvinen M, LeCrom S, Casado V,
Canela E, Rondin S, Lew J, Watson S, Zoli M, et al.
(2000) Dopamine D1 and adenosine A1 receptors
assemble into functionally interacting heteromeric com-
plexes. Proc Natl Acad Sci USA 97, 8606–8611.
11 Scarselli M, Novi F, Schallmach E, Lin R, Baragli A,
Colzi A, Griffon N, Corsini GU, Sokoloff P, Levenson
R, et al. (2001) D
2
⁄ D
3
dopamine receptor heterodimers
exhibit unique functional properties. J Biol Chem 276,
30308–30314.
12 Maggio R, Scarselli M, Novi F, Millan MJ & Corsini
GU (2003) Potent activation of dopamine D3 ⁄ D2
heterodimers by the antiparkinsonian agents, S32504,
pramipexole and ropinirole. J Neurochem 87, 631–641.

13 George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G
& O’Dowd BF (2000) Oligomerization of l- and d-opi-
oid receptors. Generation of novel functional properties.
J Biol Chem 275, 26128–26135.
14 Mellado M, Rodriguez-Frade JM, Vila-Coro AJ,
Fernandez S, Martin de Ana A, Jones DR, Toran JL &
Martinez AC (2001) Chemokine receptor homo- or
heterodimerization activates distinct signaling pathways.
EMBO J 20, 2497–2507.
15 Allouche S, Polastron J, Hasbi A, Homberger V &
Jauzac P (1999) Differential G-protein activation by
alkaloid and peptide opioid agonists in the human
Function and pharmacology of hetero-oligomers R. Maggio et al.
2944 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS
neuroblastoma cell line SK-N-BE. Biochem J 342,
71–78.
16 Baneres JL & Parello J (2003) Structure-based analysis
of GPCR function: evidence for a novel pentameric
assembly between the dimeric leukotriene B4 receptor
BLT1 and the G-protein. J Mol Biol 329 , 815–829.
17 Chinault SL, Overton MC & Blumer KJ (2004) Sub-
units of a yeast oligomeric G protein-coupled receptor
are activated independently by agonist but function in
concert to activate G protein heterotrimers. J Biol Chem
279, 16091–16100.
18 Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet
DG, Barret C & Bouvier M (1996) A peptide derived
from a beta2-adrenergic receptor transmembrane
domain inhibits both receptor dimerization and activa-
tion. J Biol Chem 271, 16384–16392.

19 George SR, Ng GY, Lee SP, Fan T, Varghese G, Wang
C, Deber CM, Seeman P & O’Dowd BF (2003) Block-
ade of G protein-coupled receptors and the dopamine
transporter by a transmembrane domain peptide: novel
strategy for functional inhibition of membrane proteins
in vivo. J Pharmacol Exp Ther 307, 481–489.
20 Terrillon S, Barberis C & Bouvier M (2004) Hetero-
dimerization of V1a and V2 vasopressin receptors deter-
mines the interaction with beta-arrestin and their
trafficking patterns. Proc Natl Acad Sci USA 101,
1548–1553.
21 Stanasila L, Perez JB, Vogel H, Cotecchia S, Stanasila
L, Perez JB, Vogel H & Cotecchia S (2003) Oligomeri-
zation of the alpha 1a- and alpha 1b-adrenergic receptor
subtypes. Potential implications in receptor internaliza-
tion. J Biol Chem 278, 40239–40251.
22 Pfeiffer M, Kirscht S, Stumm R, Koch T, Wu D,
Laugsch M, Schroder H, Hollt V & Schulz S (2003)
Heterodimerization of substance P and mu-opioid recep-
tors regulates receptor trafficking and resensitization.
J Biol Chem 278, 51630–51637.
23 Ramsay D, Kellett E, McVey M, Rees S & Milligan G
(2002) Homo- and hetero-oligomeric interactions
between G-protein-coupled receptors in living cells moni-
tored by two variants of bioluminescence resonance
energy transfer (BRET): hetero-oligomers between
receptor subtypes form more efficiently than between
less closely related sequences. Biochem J 365, 429–440.
24 Park PS, Sum CS, Pawagi AB & Wells JW (2002)
Cooperativity and oligomeric status of cardiac muscari-

nic cholinergic receptors. Biochemistry 41 , 5588–5604.
25 Luttrell LM & Lefkowitz RJ (2002) The role of beta-
arrestins in the termination and transduction of G-pro-
tein-coupled receptor signals. J Cell Sci 115, 455–465.
26 Luttrell LM, Ferguson SSG, Daaka Y, Miller WE,
Maudsley S, Della Rocca GJ, Lin FT, Kawakatsu H,
Owada K, Luttrell DK, et al. (1999) Beta-arrestin-
dependent formation of beta2 adrenergic receptor-Src
protein kinase complexes. Science 283, 655–661.
27 McDonald PH, Chow C-W, Miller WE, Laporte SA,
Field ME, Lin F-T, Davis RJ & Lefkowitz RG (2000)
Beta-arrestin 2: a receptor-regulated MAPK scaffold for
the activation of JNK3. Science 290, 1574–1577.
28 Tohgo A, Pierce KL, Choy EW, Lefkowitz RJ &
Luttrell LM (2002) beta-Arrestin scaffolding of the
ERK cascade enhances cytosolic ERK activity but
inhibits ERK-mediated transcription following
angiotensin AT1a receptor stimulation. J Biol Chem 277,
9429–9436.
29 DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins
RD & Bunnett NW (2000) beta-arrestin-dependent
endocytosis of proteinase-activated receptor 2 is
required for intracellular targeting of activated
ERK1 ⁄ 2. J Cell Biol 148, 1267–1281.
30 DeFea KA, Vaughn ZD, O’Bryan EM, Nishijima D,
Dery O & Bunnett NW (2000) The proliferative and
antiapoptotic effects of substance P are facilitated by
formation of a beta-arrestin-dependent scaffolding com-
plex. Proc Natl Acad Sci USA 97, 11086–11091.
31 Lavoie C, Mercier JF, Salahpour A, Umapathy D, Breit

A, Villeneuve LR, Zhu WZ, Xiao RP, Lakatta EG,
Bouvier M, et al. (2002) Beta 1 ⁄ beta 2-adrenergic recep-
tor heterodimerization regulates beta 2-adrenergic recep-
tor internalization and ERK signaling efficacy. J Biol
Chem 277, 35402–35410.
32 Breit A, Lagace M & Bouvier M (2004) Hetero-oligo-
merization between beta2- and beta3-adrenergic recep-
tors generates a beta-adrenergic signaling unit with
distinct functional properties. J Biol Chem 279, 28756–
28765.
33 Novi F, Stanasila L, Giorgi F, Corsini GU, Cotecchia S
& Maggio R (2005) Paired activation of the two compo-
nents within muscarinic M3 receptor dimers is required
for recruitment of b-arrestin-1 to the plasma membrane.
J Biol Chem 280 , 19768–19776.
34 Han M, Gurevich VV, Vishnivetskiy SA, Sigler PB &
Schubert C (2001) Crystal structure of beta-arrestin at
1.9 A
˚
: possible mechanism of receptor binding and
membrane translocation. Structure 9, 869–880.
35 Liang Y, Fotiadis D, Filipek S, Saperstein DA,
Palczewski K & Engel A (2003) Organization of the
G protein-coupled receptors rhodopsin and opsin in
native membranes. J Biol Chem 278, 21655–21662.
36 Granzin J, Wilden U, Choe HW, Labahn J, Krafft B &
Buldt G (1998) X-ray crystal structure of arrestin from
bovine rod outer segments. Nature 391, 918–921.
37 Hirsch JA, Schubert C, Gurevich VV & Sigler PB
(1999) The 2.8 A

˚
crystal structure of visual arrestin: a
model for arrestin’s regulation. Cell 97, 257–269.
38 Jordan BA & Devi LA (1999) G-protein-coupled recep-
tor heterodimerization modulates receptor function.
Nature 399, 697–700.
39 Cheng ZJ, Harikumar KG, Holicky EL & Miller LJ
(2003) Heterodimerization of type A and B
R. Maggio et al. Function and pharmacology of hetero-oligomers
FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2945
cholecystokinin receptors enhance signaling and
promote cell growth. J Biol Chem 278, 52972–52979.
40 Xu J, He J, Castleberry AM, Balasubramanian S, Lau
AG & Hall RA (2003) Heterodimerization of alpha
2A- and beta 1-adrenergic receptors. J Biol Chem 278,
10770–10777.
41 Grant M, Patel. RC & Kumar U (2004) The role of
subtype-specific ligand binding and the C-tail domain
in dimer formation of human somatostatin receptors.
J Biol Chem 279 , 38636–38643.
42 Mesnier D & Baneres JL (2004) Cooperative conforma-
tional changes in a GPCR dimer, the leukotriene B4
receptor BLT1. J Biol Chem September 9 [Epub ahead
of print].
43 Maggio R, Vogel Z & Wess J (1993) Coexpression studies
with mutant muscarinic ⁄ adrenergic receptors provide evi-
dence for intermolecular ‘cross-talk’ between G-protein-
linked receptors. Proc Natl Acad Sci USA 90, 3103–3107.
44 Monnot C, Bihoreau C, Conchon S, Curnow KM,
Corvol P & Clauser E (1996) Polar residues in the trans-

membrane domains of the type 1 angiotensin II receptor
are required for binding and coupling. Reconstitution
of the binding site by co-expression of two deficient
mutants. J Biol Chem 271, 1507–1513.
45 Bakker RA, Dees G, Carrillo JJ, Booth RG, Lopez-
Gimenez JF, Milligan G, Strange PG & Leurs R (2004)
Domain swapping in the human histamine H1 receptor.
J Pharmacol Exp Ther 311, 131–138.
46 Saveanu A, Lavaque E, Gunz G, Barlier A, Kim S,
Taylor JE, Culler MD, Enjalbert A & Jaquet P (2002)
Demonstration of enhanced potency of a chimeric
somatostatin-dopamine molecule, BIM-23A387, in sup-
pressing growth hormone and prolactin secretion from
human pituitary somatotroph adenoma cells. J Clin
Endocrinol Metab 87, 5545–5552.
47 Bhushan RG, Sharma SK, Xie Z, Daniels DJ & Por-
toghese PS (2004) A bivalent ligand (KDN-21) reveals
d spinal and j opioid receptors are organized as hetero-
dimers that give rise to d1 and j2 phenotypes. Selective
targeting of d–j heterodimers. J Med Chem 47, 2969–
2972.
Function and pharmacology of hetero-oligomers R. Maggio et al.
2946 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS