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

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

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

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

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

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

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-

-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

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

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

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]

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