Download G protein-coupled receptor dimers: Functional

Pharmacology & Therapeutics 118 (2008) 359–371
Contents lists available at ScienceDirect
Pharmacology & Therapeutics
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a
Associate editor: A. Christopoulos
G protein-coupled receptor dimers: Functional consequences,
disease states and drug targets
Matthew B. Dalrymple, Kevin D.G. Pfleger ⁎, Karin A. Eidne
Laboratory for Molecular Endocrinology - GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, University of Western Australia, Nedlands,
Perth, WA 6009, Australia
A R T I C L E
I N F O
A B S T R A C T
Keywords:
GPCR
Dimerisation
Disease
Drug targets
Allosterism
Abbreviations:
A R, Adenosine receptor subtype 2a; AT R,
Angiotensin-II
receptor subtype 1; β AR, 1
2a
Beta-2 adrenergic receptor; B R, Bradykinin-2
2
receptor; BRET, Bioluminescence
resonance
2
energy transfer; Ca , Calcium ions; CaSR,
2+
Calcium-sensing receptor;
CB1, Cannabinoid receptor subtype 1; CRLR, Calcitoninreceptor-like receptor; D R, Dopamine-2
receptor; DOP, Delta-opioid
receptor; ER,
2
Endoplasmic reticulum; ERK, Extracellular
signal-regulated kinases; ECD, Extracellular domain; 5-HT, 5-hydroxytryptamine
(serotonin); FRET, Fluorescence resonance
energy transfer; GABA Rs, Gamma-aminobutyric acid receptors;
GnRHR, GonaB
dotrophin-releasing hormone receptor;
GPCRs, G protein-coupled receptors; HH, Hypogonadotropic hypogonadism; KOP, Kappaopioid receptor; MAPKs, Mitogen-activated protein kinases; mGluRs, Metabotropic glutamate
receptors; MOP, Mu-opioid receptor; ORs, Olfactory receptors; OxR1, Orexin receptor subtype 1;
RAMPs, Receptor activity-modifying proteins;
WT, Wild type.
With an ever-expanding need for reliable therapeutic agents that are highly effective and exhibit minimal
deleterious side effects, a greater understanding of the mechanisms underlying G protein-coupled receptor
(GPCR) regulation is fundamental. GPCRs comprise more than 30% of all therapeutic drug targets and it is
likely that this will only increase as more orphan GPCRs are identified. The past decade has seen a dramatic
shift in the prevailing concept of how GPCRs function, in particular the growing acceptance that GPCRs are
capable of interacting with one another at a molecular level to form complexes, with significantly different
pharmacological properties to their monomeric selves. While the ability of like-receptors to associate and
form homodimers raises some interesting mechanistic issues, the possibility that unlike-receptors could
heterodimerise in certain tissue types, producing a functionally unique signalling complex that binds specific
ligands, provides an invaluable opportunity to refine and redefine pharmacological interventions with
greater specificity and efficacy.
© 2008 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . .
G protein-coupled receptor homo/heterodimerisation
2.1.
Agonist-induced dimerisation . . . . . . . .
2.2.
Constitutive dimerisation . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
360
0
0
360
0
360
0
361
⁎ Corresponding author. Western Australian Institute for Medical Research (WAIMR), B-Block, QEII Medical Centre, Hospital Ave, Perth, Western Australia, 6009, Australia. Tel.: +61
8 9346 3591; fax: +61 8 9346 1818.
E-mail address: kpfl[email protected] (K.D.G. Pfleger).
0163-7258/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2008.03.004
360
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
3.
4.
Role of dimerisation in G protein-coupled receptor synthesis and maturation
Functional relevance of G protein-coupled receptor heterodimerisation . . .
4.1.
Chemokine receptors. . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Adenosine-2a and metabotropic glutamate-5 receptors . . . . . . .
4.3.
Opioid receptors. . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Pharmacological rescue of misrouted G protein-coupled receptors . . . . . .
6.
Allosteric modulation of G protein-coupled receptor activity . . . . . . . .
6.1.
Receptor activity-modifying proteins (RAMPs) . . . . . . . . . . . .
6.2.
Dimerisation-mediated G protein-coupled receptor allosterism . . .
7.
Dimerisation and disease pathogenesis. . . . . . . . . . . . . . . . . . .
7.1.
Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
Parkinson's disease . . . . . . . . . . . . . . . . . . . . . . . .
7.3.
Hypogonadotropic hypogonadism. . . . . . . . . . . . . . . . . .
7.4.
Schizophrenia and psychosis . . . . . . . . . . . . . . . . . . . .
8.
Heterodimer-specific drugs . . . . . . . . . . . . . . . . . . . . . . . .
9.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
GPCRs are heptahelical membrane proteins capable of transducing
extracellular signals via heterotrimeric G proteins that elicit downstream responses through second-messenger generation. While
GPCRs are classified into several different families (for a review see
Bockaert & Pin, 1999), all share the same basic structure consisting of
an extracellular N-terminus, 3 intracellular and extracellular loops, 7
transmembrane domains that are highly conserved and a cytoplasmic
C-terminus. Despite their architectural homology, GPCRs are activated
by a vast array of stimuli, including light photons, odorants,
neurotransmitters, calcium ions (Ca2+), peptides and hormones.
Analysis of the nematode Caenorhabditis elegans genome indicated
that as many as 5% of all genes code for GPCRs (Bargmann, 1998) and it
has been suggested that this could correlate with up to 5000 GPCRs in
mammals (Marchese et al., 1999). More recent studies of sequences
from the human genome project have shown that the total number of
genes encoding GPCRs exceeds 950, with at least half of these
comprised of taste and odorant receptors (Takeda et al., 2002).
In spite of the concerted efforts of numerous research groups, the
only GPCRs thus far to have a 3-dimensional crystal structure defined
are the Family A receptors rhodopsin (Palczewski et al., 2000) and the
beta-2 adrenergic receptor (Rasmussen et al., 2007). Members
belonging to Family B GPCRs lack the distinctive DRY motif (AspArg-Tyr) that characterises most Family A GPCRs, exhibit a large
extracellular N-terminus that contains numerous disulfide bridges,
and includes the corticotrophin-releasing factor and growth hormone-releasing hormone receptors. Family C GPCRs include the
metabotropic glutamate receptors (mGluRs), the γ-aminobutyric acid
receptors (GABABRs) and calcium-sensing receptor (CaSR), that again
exhibit structural properties distinct from those of the Family A GPCRs
(see Parmentier et al., 2002), most notable of which are a shortened
third intracellular loop and a large, bi-lobed N-terminus that is
thought to contain the ligand-binding domain.
The original GPCR signalling paradigm proposed a 1:1:1 stoichiometry, with a monomeric GPCR binding a single, specific ligand and
initiating a downstream signalling cascade via a single heterotrimeric
G protein subunit. While this made logical sense, it is now believed that
this model alone cannot explain the complexity of receptor signalling.
The notion of GPCR dimerisation was first conceived more than
20 years ago, with researchers utilising a bivalent antibody with a
gonadotrophin-releasing hormone (GnRH) antagonist attached at
each end. Their data implied that this ‘conjugated’ antagonist might be
capable of acting as an agonist that promoted “microaggregates” of
GnRH receptor (GnRHR) leading to biphasic regulation of receptor
surface expression (Conn et al., 1982a, 1982b). Another early study
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
361
0
362
0
362
0
363
0
363
0
364
0
0
366
366
0
0
366
0
366
367
0
367
0
0
367
0
368
0
368
368
0
0
369
0
369
indicated that GPCRs were capable of forming functional complexes,
using a series of chimeric adrenergic/muscarinic receptors to illustrate
functional rescue of receptor signalling upon coexpression by transcomplementation of mutant receptors (Maggio et al., 1993).
Regardless of these early indications that GPCRs could interact at a
molecular level, constituting a new hierarchy of receptor signalling, it
was only towards the end of the millennium that acceptance of the
concept began to expand. In fact, direct physical evidence for GPCR
dimerisation only became available in 2003, following atomic-force
microscopy analysis of the rhodopsin receptor that clearly showed
neat rows of rhodopsin dimers arranged within retinal disc membranes (Fotiadis et al., 2003; Fig. 1). The term dimer is often used
interchangeably with oligomer in the literature, as current methodologies are often unable to differentiate the two. Consequently, for the
sake of avoiding confusion during this review, the term ‘dimer’ will be
used to refer to any receptor complex consisting of more than one
GPCR. The tremendous growth of knowledge regarding GPCR
dimerisation over the past few years has been reviewed extensively
in a number of recent articles (Bouvier, 2001; Dean et al., 2001;
Kroeger et al., 2003; Bai, 2004; Milligan, 2004; Terrillon & Bouvier,
2004; Milligan, 2006).
2. G protein-coupled receptor homo/heterodimerisation
A facet of GPCR dimerisation research that remains contentious is
whether GPCRs inherently exist in dimeric complexes, or whether
dimers are induced by the presence or absence of receptor ligands.
2.1. Agonist-induced dimerisation
Several studies utilising bioluminescence resonance energy transfer
(BRET) or fluorescence resonance energy transfer (FRET) analysis of GPCR
dimerisation, have shown that the extent of dimerisation may be
dependent on ligand activation or significantly modulated in the presence
of ligand (Angers et al., 2000; Cheng & Miller, 2001; Kroeger et al., 2001).
FRET studies of GnRHR dimerisation found that the addition of agonist but
not antagonist led to a significant increase in the FRET signal intensity
(Cornea et al., 2001), indicative of a ligand-specific, protein–protein
interaction that was not observed in the absence of agonist (Horvat et al.,
2001). The reverse may also hold true in some instances, with the
addition of ligand resulting in the apparent dissociation of receptor
dimers and a loss of BRET signal (Cheng & Miller, 2001).
However, due to some of the inherent limitations of resonance
energy transfer methods, interpretation of results from such assays
requires careful consideration. A number of recent reviews address
some of the issues that must be considered when using such techniques
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
361
Fig. 1. Organisation and topography of the cytoplasmic surface of rhodopsin. (a) Topograph obtained using atomic-force microscopy, showing the paracrystalline arrangement of
rhodopsin dimers in the native disc membrane. Inset, arcs in the calculated powder diffraction pattern reflect the regular arrangement of rhodopsin in the membrane. (b) Angularly
averaged powder diffraction pattern, showing peaks at (8.4 nm)−1, (4.2 nm)−1 and (3.8 nm)−1. (c) Magnification of a region of the topograph in a, showing rows of rhodopsin dimers, as
well as individual dimers (inside dashed ellipse), presumably broken away from one of the rows, and occasional rhodopsin monomers (arrowheads). The rhodopsin molecules
protrude from the lipid bilayer by 1.4 ± 0.2 nm (n = 111). The topograph in (c) is shown in relief, tilted by 5°. Vertical brightness ranges: 1.6 nm (a and c). Scale bars: a, 50 nm; inset,
(5 nm)−1; c, 15 nm (from Fotiadis et al., 2003 with permission, © 2003 Nature).
(Bouvier, 2001; Kroeger et al., 2003; Pfleger & Eidne, 2006). Given that
both BRET and FRET are proximity-based methodologies, any change in
the structural conformation of the receptor(s) of interest could result in
movement of the donor and/or acceptor proteins that in turn leads to
alterations in the signal observed. Therefore, due diligence is required
when interpreting data when variation in resonance energy transfer is
detected (relative to control measurements). While an increase in BRET
signal may be indicative of an increase in protein–protein interactions
within a sample, it is possible that the change in signal stems from
conformational changes in the proteins of interest, which subsequently
lead to the donor–acceptor molecules being brought into greater
proximity and/or a more favourable relative orientation.
2.2. Constitutive dimerisation
It has been shown for a number of Family C GPCRs that the
endogenous receptor unit consists of either homo- or heterodimers,
which are required for receptor function and/or expression (Jones
et al., 1998; Bai et al., 1999; Tateyama et al., 2004). Co-immunoprecipitation studies of wild-type and epitope-tagged metabotropic
glutamate receptor 5 (mGluR5) revealed that the receptors existed
as covalent dimers linked by disulfide bridges within the N-terminus
(Romano et al., 1996). Similar findings were also shown for the mGluR1a,
mGluR2/3 and mGluR4 receptor subtypes. Subsequent co-immunoprecipitation work revealed that while the disulfide bridges were essential
for covalent mGluR5 dimerisation, truncated receptor mutants with
mutated cysteine residues were still capable of forming dimers,
presumably through non-covalent interactions (Romano et al., 2001).
Several years ago, BRET was utilised to indicate the existence of
constitutive β-2 adrenergic receptor (β2AR) homodimers (Angers
et al., 2000), with evidence for the specificity of this interaction
provided by coexpression of β2AR with another distantly related GPCR
that produced no BRET signal. Since this study, a number of papers
have used BRET to monitor constitutive dimer formation between
GPCRs (reviewed by Pfleger & Eidne, 2005), including the opioid and
cannabinoid receptors (Rios et al., 2006). BRET saturation experiments
have been used in an effort to demonstrate the specificity of
heterodimer interactions. By maintaining a constant amount of
donor protein and coexpressing an increasing amount of acceptor
protein, a non-specific interaction theoretically produces a linear
relationship between BRET signal and the amount of acceptor protein
relative to the donor protein. Conversely a specific dimer interaction
exhibits a different profile, with the BRET signal reaching a plateau
irrespective of ever-increasing amounts of acceptor protein (Mercier
et al., 2002; Milligan et al., 2005).
While this technique provides a useful means of assessing the
specificity of receptor dimerisation, it is not without its critics (James
et al., 2006). However, the authors of BRET studies have continually
emphasised the need for strict controls to be employed in such assays,
in order to minimise erroneous interpretation of results (Bouvier et al.,
2007). Again, the inclusion of appropriate (negative) controls in these
BRET experiments is of paramount importance when assessing the
specificity of receptor interactions. Issues such as receptor expression
levels and possible conformational limitations of certain BRET-tagged
GPCR constructs, which could hinder or limit efficient RET between
proteins of interest, must be considered when designing such assays
(Pfleger & Eidne, 2006; Pfleger et al., 2006).
There are indications that many, if not most, GPCRs are trafficked to
the plasma membrane in a dimeric complex (Bulenger et al., 2005) and
that their interaction is not directly influenced by the presence or
absence of a particular ligand. Why then should we be interested in
compounds that specifically target heterodimers? The reason is that
numerous examples already exist in the literature which demonstrate
that particular GPCR heterodimeric complexes exhibit markedly
different pharmacological profiles when compared to those of the
monomeric/homodimeric forms of the receptor following agonist
challenge (AbdAlla et al., 2000; Gomes et al., 2000; Hilairet et al.,
2003; Gomes et al., 2004; El-Asmar et al., 2005; Breit et al., 2006; Ellis
et al., 2006; Rios et al., 2006). Thus, while binding of ligand in most
instances has no effect upon GPCR dimers, the dimeric state of the
receptors themselves can impact upon the signalling pathways elicited
by the binding of ligand.
Furthermore, as will be discussed subsequently, the potential to
generate cell-permeable pharmaceutical compounds capable of acting
as ‘pharmacochaperones’ may provide a therapeutic avenue for the
treatment of deleterious conditions stemming from dimerisation
deficits. Such cases are often the result of inadequate receptor export
from the endoplasmic reticulum (ER) to the plasma membrane, due to
a dominant-negative effect of mutant receptor upon wild-type (WT)
receptor trafficking.
3. Role of dimerisation in G
protein-coupled receptor synthesis and maturation
The pathogenesis of some disorders can involve dominantnegative effects of receptor mutants that prevent signalling of normal
362
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
receptors by dimeric inhibition of receptor trafficking. Therefore, an
intriguing concept to emerge from this is the possibility that various
disease states and disorders may be addressed more effectively by
appreciating the role of particular dimeric interactions. In a number of
instances, human diseases caused by genetic mutations result from
non-functional GPCRs that have an incorrect quaternary structure and
are not properly trafficked to the plasma membrane (for reviews see
Kim & Arvan, 1998; Morello et al., 2000). These conditions, which are
sometimes referred to as ER storage diseases (Callea et al., 1992), often
involve a dominant-negative receptor mutant that effectively prevents surface expression of functional WT receptors, reducing
potential binding sites and ablating downstream signalling mechanisms as a consequence.
The notion that some receptors require dimerisation in order to be
correctly targeted to the plasma membrane is not new (for a recent
review see Prinster et al., 2005), with a series of studies during the mid
90s indicating this was the case for the GABAB receptor (GABABR).
Several groups demonstrated that the GABABR1 subtype was poorly
expressed at the cell surface in heterologous systems with little
signalling activity, but coexpression with a second subtype (GABABR2)
resulted in a fully functional heterodimer that was expressed at the
plasma membrane (Couve et al., 1998; Jones et al., 1998; Kaupmann
et al., 1998; White et al., 1998). It was later revealed that membrane
expression of GABABR1 is prevented by an ER retention motif located on the C-terminus of the receptor and that interaction with the
C-terminus of GABABR2 masks this signal, allowing proper trafficking of
the functional receptor to the cell membrane (Margeta-Mitrovic et al.,
2000). It was also shown that while heterodimerisation was crucial for
functional GABABR targeting to the plasma membrane, only intracellular
loops of the GABABR2 subtype were required for functional coupling of
the heterodimer to the G protein (Margeta-Mitrovic et al., 2001).
Although the coiled-coil interface between the C-termini of receptor
monomers is crucial for cell surface expression of functional GABABRs,
mutagenesis studies utilising C-terminally truncated GABABRs revealed
that this domain was not required for heterodimerisation of receptors per
se (Pagano et al., 2001).
Findings from co-immunoprecipitation studies of vasopressin and
oxytocin receptor dimerisation indicated that immature receptors
located in the ER could form dimers. This led the authors to suggest
that the receptors may form obligate dimers during biosynthesis
(Terrillon et al., 2003). A recent study of olfactory receptors (ORs), in
particular the mouse 71 (M71) OR, found that in a heterologous system
this OR exhibited very poor surface expression that was dramatically
improved when β2AR was coexpressed (Hague et al., 2004). The
interaction was specific, as increased surface expression of the M71 OR
did not occur when coexpressed with other adrenergic receptors.
These results imply that dimerisation of ORs may be necessary for
successful trafficking of some receptors to the cell membrane.
Indeed, co-immunoprecipitation was originally used to show that
β2AR monomers interacted with one another, and that a synthetic
peptide comprising 20 amino acid residues from transmembrane VI of
the receptor substantially inhibited the number of β2AR dimers
detected, reducing the ability of the receptor to generate second
messengers (Hebert et al., 1996). This indicated that transmembrane
VI of the receptor was crucial for protein–protein interactions.
Subsequent studies with a series of β2ARs with mutations in the
transmembrane VI domain showed that they reduced dimerisation
between WT receptors (Salahpour et al., 2004). β2AR mutants with an
ER retention motif within the C-terminus acted in a dominantnegative fashion when coexpressed with WT receptors, preventing
trafficking to the plasma membrane. This indicated that dimerisation
of β2ARs is necessary for receptor export to the plasma membrane.
Further analysis of a signalling-impaired, constitutively desensitised
β2AR mutant found that coexpression with WT β2AR had a dominantpositive effect, with heterodimerisation resulting in functional
complementation of the mutant receptor (Hebert et al., 1998).
4. Functional relevance of
G protein-coupled receptor heterodimerisation
Recently, it has become apparent that different GPCR subtypes can
interact with each other on the cell surface in a variety of complexes
with the potential for multiple permutations. The consequences of
these interactions are not well-understood, or exploited, but are
predicted to have a major impact on drug discovery (George et al.,
2000). GPCRs have proven to be very tractable drug targets and
therefore are of great interest to the pharmaceutical industry. The
mere notion that two previously unrelated GPCRs (either in terms of
familial classification or receptor subtype) could directly interact with
one another, or impact upon the other in a downstream fashion,
creates the proverbial “Pandora's box” with respect to the possibilities
of drug specificity and efficacy. Selected examples of functionally
relevant GPCR dimers are shown in Table 1 and are discussed below.
Examples of potential mechanisms for regulating receptor complexes
that have implications for disease pathogenesis, such as allosterism
and pharmacochaperones, will be discussed subsequently.
4.1. Chemokine receptors
The role chemokine receptors play in determining susceptibility to
HIV infection raises the possibility of developing therapeutic agents
that target these GPCRs. Coexpression of either CCR3 or CCR5 with the
CD4 glycoprotein facilitates HIV infection in previously resistant celllines (Choe et al., 1996). While CD4 was essential for HIV entry and
chemokine receptors acted strictly as positive co-factors for this
process, it could be shown that another chemokine receptor, CXCR4,
functioned as the primary receptor for infection by HIV-2 in CD4negative cells (Endres et al., 1996).
Subsequent studies have shown that chemokine receptors are
capable of forming both homo- and heterodimers. Using a number of
different techniques, including BRET, homo- and heterodimerisation
has been demonstrated for the CCR5 and CCR2b receptors (Mellado
Table 1
Key examples of functionally relevant GPCR heterodimers
Heterodimer
Functional relevance
Reference(s)
GABABR1 +
GABABR2
Correct trafficking of GABABR1
to plasma membrane
β2AR +
M71 OR
T1R2 + T1R3
Correct trafficking of M71 OR
to the plasma membrane
Sweet taste perception
Kaupmann et al., 1998
White et al., 1998
Jones et al., 1998
Margeta-Mitrovic et al., 2000
Hague et al., 2004
T1R1 + T1R3
AT1aR + B2R
MOP + DOP
Umami taste perception
(amino acids L-glutamate,
L-aspartate)
Hypertension/preeclampsia
Enhanced morphine analgesia
KOP + DOP
A2aR + D2R
Tissue-specific analgesia
Parkinson's disease
A2aR +
mGluR5
Drug dependence and addiction
5-HT2a +
mGluR2
Psychosis and schizophrenia
CCR2 + CXCR4
CCR2 + CCR5
HIV resistance
Nelson et al., 2001
Zhao et al., 2003
Nelson et al., 2002
Zhao et al., 2003
AbdAlla et al., 2000, 2001
Gomes et al., 2000, 2004
George et al., 2000
Daniels et al., 2005
Waldhoer et al., 2005
Ferre et al., 1991
Kanda et al., 1998
Canals et al., 2003
Fuxe et al., 2003, 2005
Pinna et al., 2005
Ferre et al., 1999, 2002
Nishi et al., 2003
Kachroo et al., 2005
Adams et al., 2007
Aghajanian & Marek, 2000
Scruggs et al., 2003
Gonzalez-Maeso et al., 2007, 2008
Mellado et al., 1999
Rodriguez-Frade et al., 2004
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
et al., 2001; Huttenrauch et al., 2002; Issafras et al., 2002). The
functional consequences of heterodimerisation between these receptor subtypes are somewhat unclear, although interactions between
the CCR5 and CCR2 receptors caused an increased sensitivity of these
receptors to chemokine exposure (Mellado et al., 2001). This is in
contrast to other studies analysing CCR5/CCR2b heterodimerisation,
which found negative binding cooperativity between the receptor
subunits for their respective ligands, suggesting that the heterodimer
is only capable of binding a single ligand at a time (El-Asmar et al.,
2005). BRET was also utilised in a study that showed that the CXCR4
receptor was unable to heterodimerise with CCR5, but forms
constitutive receptor clusters (oligomers) consisting of two or more
CXCR4 receptor monomers (Babcock et al., 2003). There remains some
conjecture in this regard as earlier studies have shown that
dimerisation of CXCR4 is not constitutive but rather inducible in the
presence of a specific chemokine, SDF-1α (Vila-Coro et al., 1999). The
development of agents that can modulate the nature of these protein–
protein interactions may thus provide a means of preventing HIV
infection, or an alternate means of treating patients with HIV. In
particular, the use of allosteric molecules that do not compete with
endogenous ligands for their binding site may provide the most
specific means of targeting deleterious signalling complexes without
the side effects seen with agonist or antagonist treatments.
Assays utilising a monoclonal antibody raised against CCR5, which
did not compete for the ligand-binding site nor initiate signalling,
were able to illustrate that by inducing dimerisation of CCR5 the
antibody was capable of preventing HIV infection both in vitro and in
vivo (Vila-Coro et al., 2000). Another research group subsequently
determined that binding of the HIV-1 envelope glycoprotein to an
allosteric site on CCR5 inhibits binding of the endogenous ligand to the
receptor (Staudinger et al., 2001). The allosteric nature of chemokine
receptor interactions has been further demonstrated by a recent study
of CCR2 and CCR5 heterodimerisation, which found negative binding
cooperativity between dimer subunits as a result of allosteric interactions (Springael et al., 2006).
A small molecule inhibitor, named Repertaxin, was recently shown
to bind the chemokine receptors CXCR1 and CXCR2 allosterically, and
in doing so prevented receptor signalling by modifying the conformation of the receptor (Bertini et al., 2004). Similarly, two allosteric
modulators of CXCR4 have been described, RSVM and ASLW, neither of
which compete for the orthosteric binding site but exhibit partial- and
super-agonist characteristics respectively (Sachpatzidis et al., 2003).
These forms of intervention present a novel, and as yet relatively
untapped source of future therapies.
4.2. Adenosine-2a and metabotropic glutamate-5 receptors
The adenosine-2a receptor (A2aR) and metabotropic glutamate-5
receptor (mGluR5) are both expressed by γ-aminobutyric acid (GABA)
striatal neurons (Schiffmann et al., 1991; Fink et al., 1992; Testa et al.,
1995; Ferre et al., 1999, 2002), a region of the brain that has been
implicated in reward-seeking behaviour commonly associated with
drugs of addiction (Adams et al., 2007). Following a report that
indicated that treatment of alcohol-conditioned mice with either
adenosine-1 receptor (A1R) or A2aR-specific agonists reduced alcohol
withdrawal symptoms (Kaplan et al., 1999), several studies have
attempted to elucidate the underlying molecular mechanisms. Using
membrane preparations from rat striatal neurons, treatment with
either A2aR or mGluR5 specific agonists decreased the affinity of
dopamine for dopamine-2 receptor (D2R), and co-administration of
these compounds caused a synergistic decline in dopamine binding
(Ferre et al., 1999). Confocal microscopy and co-immunoprecipitation
were used to demonstrate that A2aR and mGluR5, when coexpressed
in HEK293 cells, were colocalised at the plasma membrane and that
simultaneous treatment of these receptors with respective agonists
led to a synergistic increase in ERK phosphorylation (Ferre et al.,
363
2002). Furthermore, these results were replicated in an in vivo rat
model, and A2aR and mGluR5 were colocalised in rat striatal
membrane preparations. This led the authors to speculate that a
functional interaction between A2aR and mGluR5 is necessary to
overcome the inhibitory effect of dopamine upon A2aR signalling.
Another study utilising mouse striatum found that phosphorylation of
the dopaminergic-related protein DARPP-32 by agonist-occupied
mGluR5, is dependent upon activation of A2aR by endogenous adenosine (Nishi et al., 2003). Again, a synergistic effect was seen when
samples were simultaneously treated with A2aR and mGluR5 agonists
with respect to DARPP-32 phosphorylation, and related to the level of
ERK signalling.
Subsequent studies that have focussed upon the role of these two
GPCRs in the pathogenesis of behaviours associated with drug abuse,
such as heightened reward-seeking (drug dependence), tolerance,
withdrawal and relapse, produced some intriguing insights into the
nature of the physiological mechanisms underlying these associated
disorders. Using both rat and mouse models, several research groups
showed that treatment of animals with the mGluR5 antagonist MPEP
(or the highly selective mGluR5 antagonist MTEP) not only reduced
the reward-seeking behaviour correlated with substances of abuse,
including alcohol (Arolfo et al., 2004; Backstrom et al., 2004;
Schroeder et al., 2005; Besheer et al., 2006; Adams et al., 2007),
cocaine (Tessari et al., 2004; Kenny et al., 2005) and nicotine (Tessari
et al., 2004), but also minimised the occurrence of substance
tolerance, withdrawal symptoms and relapse. Similarly, blockade of
A2aR by specific antagonists also led to a decline in symptoms
associated with substance abuse (Arolfo et al., 2004; Yao et al., 2006),
with an analogous effect seen using an A2aR knockout model (Soria
et al., 2006).
Consequently, studies have investigated the potential for A2aR and
mGluR5 to interact as a heterodimeric unit, and the ramifications this
may have upon the previously discussed observations in relation to
alcohol abuse. Combined, selective antagonism of both receptors at
sub-threshold doses reduced alcohol consumption and prevented
subsequent relapse following re-presentation of cues previously
associated with alcohol availability (Adams et al., 2007; Fig. 2).
These effects were not seen in animals treated with an A1R-specific
antagonist in combination with either an A2aR or mGluR5 antagonist,
suggesting that diminished alcohol consumption and susceptibility to
relapse are mediated by interactions between A2aR and mGluR5.
Another study found that apparent heterodimerisation between
A1R and A2aR subtypes enables the precise regulation of glutamatergic
signalling by reducing the affinity of A1R for agonists, which is
dependent upon the relative concentration of endogenous adenosine
(Ciruela et al., 2006). In addition, research exploring the potential role
of A2aR and mGluR5 heterodimerisation in Parkinson's disease found
that the anti-Parkinsonian effects of mGluR5 antagonists (increased
motor control) were potentiated by an A2aR antagonist (Kachroo et al.,
2005). Furthermore, the ability of mGluR5 antagonist to enhance
locomotion was dependent upon the presence of a functional A2aR.
Interestingly, the absence of D2R expression also abrogated the
therapeutic effects of mGluR5 antagonism. Thus, the earlier proposition that multimeric complexes consisting of D2R, A2aR and mGluR5
protomers could be present in striatal neurons seems feasible (Fuxe
et al., 2003), and may provide a fascinating target for future therapies
aimed at alleviating both the symptoms of Parkinson's disease and
the behavioural defects stemming from substance abuse. The role of
A2aR/D2R heterodimerisation in the therapeutic intervention of
Parkinson's disease will be discussed shortly.
4.3. Opioid receptors
The opioid receptors are an important target for analgesia. Coimmunoprecipitation studies showed that the kappa-opioid receptor
(KOP) and delta-opioid receptor (DOP) form dimeric complexes when
364
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
Fig. 2. Effects of A2aR antagonist (SCH 58261) and mGluR5 antagonist (MTEP) on operant
ethanol self-administration in alcohol-preferring (iP) rats (n = 10). Black bars represent
ethanol responses; white bars represent water responses; ⁎ denotes significantly
different to vehicle. (a) Ethanol self-administration was not significantly altered by SCH
58261 at 1.0 mg/kg i.p., nor MTEP at 0.25 mg/kg i.p. SCH 58261 at 2.0 mg/kg i.p.
significantly reduced responding for ethanol (p b 0.001) without altering responding for
water. When co-administered, SCH 58261 and MTEP significantly reduced responding
for ethanol without affecting responding for water. Veh, Vehicle; M0.25, MTEP (0.25 mg/
kg i.p.); S1 and S2, SCH 58261 at 1.0 and 2.0 mg/kg; S0.5M0.25, SCH 58261 (0.5 mg/kg i.p.)
and MTEP (0.25 mg/kg). (b) The ethanol-lever time-course shows SCH 58261 at
2.0 mg/kg i.p. significantly reduced responding in the 5 min (p = 0.009) and 20 min
(p = 0.013) bins. SCH 58261 (0.5 mg/kg) and MTEP (0.25 mg/kg i.p.) significantly attenuated
responding for the first 10 min (p b 0.001). ■, Vehicle; ▼, SCH 58261 (2 mg/kg); ◊, SCH
58261 (0.5 mg/kg i.p.) and MTEP (0.25 mg/kg). (c) SCH 58261 at 0.5 mg/kg and MTEP at
0.25 mg/kg i.p. significantly increased (p b 0.001) the latency from the session start until the
first reinforced ethanol reward (from Adams et al., 2007 with permission, © 2007
International Journal of Neuropsychopharmacology).
In contrast, other researchers used BRET to demonstrate a specific
interaction between the MOP and KOP subtypes (Wang et al., 2005).
Previous immunocytochemical studies showed that these subtypes
were coexpressed in rat spinal tissue (Wessendorf & Dooyema, 2001)
suggesting the in vivo relevance of the receptor association. Other
studies illustrated that DOP and MOP were able to form heterodimers
(Gomes et al., 2000). Binding of selective DOP agonists to DOP
increased the binding affinity of MOP agonists and vice versa,
suggesting positive binding cooperativity may occur between receptor
monomers comprising this heterodimer. Additionally, treatment with
a combination of DOP- and MOP-specific agonists caused synergistic
potentiation of heterodimer signalling with a significant increase in
the ability of the receptor to couple to downstream effector
mechanisms.
An investigation of MOP/DOP heterodimers by BRET analysis
showed that dimerisation was independent of agonist challenge and
treatment with a DOP antagonist led to a substantial increase in MOPmediated morphine analgesia (Gomes et al., 2004). More recently,
BRET analysis of cannabinoid receptor 1 (CB1) showed specific
interactions with opioid receptors (mu, delta and kappa), independent
of ligand, but no dimerisation with CCR5 (Rios et al., 2006). Of
particular interest was the observation that binding of agonist to one
receptor of the heterodimer pair (CB1) resulted in a significant
reduction in the signalling efficacy of the second receptor (opioid)
when exposed to its endogenous ligand. This effect was reciprocal and
indicates that in certain tissue types the CB1 and opioid receptors may
be able to allosterically modulate the actions of one another, although
to what end is still unclear.
Evidence for MOP/DOP heterodimers, and observations that
cotreatment of cells with a DOP antagonist caused enhanced binding
affinity and potency of a selective mu-agonist at MOP (Gomes et al.,
2000), led to a succession of investigations into this relationship. The
MOP/DOP heterodimer was found to be a unique signalling complex
with a novel binding pocket and alternate G protein coupling, distinct
from either receptor monomer (George et al., 2002). BRET analyses
reaffirmed these findings and further suggested that cotreatment with
a DOP-specific antagonist augmented the analgesic properties of
morphine acting via MOP (Gomes et al., 2004).
Finally, the discovery of an agonist that is specific to an opioidreceptor heterodimer is particularly interesting. Waldhoer et al.
(2005) showed that an analgesic agonist, 6′-GNTI, selectively
activated DOP/KOP heterodimers in a tissue-specific manner
(Fig. 3). The formation of a unique signalling complex was further
illustrated by altered binding affinities of antagonists for their
respective receptor monomers when compared to the heterodimer
profile. This raises the possibility that GPCR heterodimers can be
specifically targeted without activating receptor monomers (or other
dimers), thereby avoiding potential side effects due to activation of
alternate opioid signalling pathways (Gupta et al., 2006). The side
effects of morphine and other opiates can be minimised when
administered intrathecally (Fairbanks, 2003), and previous studies
have demonstrated the coexpression of DOP and KOP in spinal
neurons (Wessendorf & Dooyema, 2001), so there is considerable
potential for compounds such as 6′-GNTI to be utilised in future pain
management strategies that are efficacious and exhibit negligible side
effects.
5. Pharmacological rescue of
misrouted G protein-coupled receptors
coexpressed in a heterologous system (Jordan & Devi, 1999). This
interaction resulted in altered receptor pharmacology and was
specific, as KOP did not dimerise with the mu-opioid receptor
(MOP). The finding agreed with computational modelling of opioidreceptor dimerisation, which predicted that there were no residues
between these two receptors capable of forming an interface (Filizola
et al., 2002).
A multitude of factors influence the correct trafficking of GPCRs
from the ER to the cell membrane, including motifs in the C-terminus
that act as an ER retention signal, GTPases that target receptors to
transport proteins (for review see Duvernay et al., 2005) and RAMPs
that promote the trafficking of receptors such as the CaSR from the ER
to the Golgi apparatus for receptor maturation (Bouschet et al., 2005).
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
365
Fig. 3. KOP/DOP heterodimers are specifically targeted by 6′-GNTI. (a) Coexpression of opioid-receptor types in HEK-293 cells was visualised by immunofluorescent staining of cells
stably expressing HA-tagged DOP (DOP) and FLAG-tagged KOP (KOP), with antibodies directed to the respective epitope tags (scale bar = 10 μm). (b) Ratio of opioid-receptor
heterodimers versus homodimers by serial immunoprecipitation (IP). Cells coexpressing both HA-DOP (HADOP) and FLAG-KOP (FKOP), or each individually was lysed and the
receptors were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted (IB) with anti-HA antibody to detect KOP/DOP heterodimers (lanes 5 and 7).
After the FLAG immunoprecipitation (F), the lysates were immunoprecipitated with anti-HA antibody and immunoblotted for HA to detect the DOP homodimers/monomers
remaining after immunoprecipitation of the heterodimers. Compare lanes 5 (heterodimers) and 6 (homodimers/monomers). (c) 6′-GNTI-induced Ca2+ release in HEK-293 cells
expressing one or two opioid-receptor types. Agonist-mediated intracellular Ca2+ release was measured in cells expressing the chimeric G protein Δ6-Gqi4-myr (200 ng for every
40,000 cells) and MOP (Δ), DOP (□), or KOP (○) alone or MOP/DOP (■), KOP/MOP (▲), or KOP/DOP (●). Intracellular Ca2+ release was measured in a Flex apparatus (Molecular
Devices), where relative light units (RLU) = maximum Ca2+ peak / cell number × 1000. Shown are representative curves carried out in duplicate (n = 4). (Inset) Structure of 6′-GNTI.
(d) Effects of receptor type-selective antagonists on 6′-GNTI-induced Ca2+ release in cells expressing the KOP/DOP heterodimer. Cells expressing the KOP/DOP heterodimer were
preincubated for 30 min with increasing doses of DOP-selective antagonist NTI (○) or KOP-selective antagonist NorBNI (●) and stimulated with 100 nM 6′-GNTI. Agonist-induced
Ca2+ release was assessed as described in (c). Data are mean ± SEM measured in duplicates. (e and f) Effect of 6′-GNTI (e) and KOP- or DOP-selective antagonists, (f) on competition
for [3H] diprenorphine binding to cells expressing KOP/DOP heterodimers. Whole-cell competition binding experiments were performed on cells stably expressing the KOP/DOP
heterodimers. Cells were incubated with 1.5 nM [3H] diprenorphine and increasing amounts of 6′-GNTI (■) (e), NorBNI (●) (f), or NTI (○) (f). Shown are representative curves (n = 4)
carried out in duplicate. Note that error bars in (e) are too small to be visualised (from Waldhoer et al., 2005 with permission, © 2005 PNAS).
Thus, a number of potential approaches could be used to develop
pharmaceutical intervention strategies, based upon the utilisation of
such cellular mechanisms, in order to treat conditions related to the
incorrect trafficking of GPCRs.
Hypogonadotropic hypogonadism (HH) is a disease model involving dysfunctional receptor trafficking. Fourteen naturally occurring
mutations have been described for GnRHR that result in HH of
varying severity, a disorder characterised by a lack of development at
puberty and the subsequent absence of secondary sexual development (Conn et al., 2002). Such a disease aetiology can be overcome
through the use of ‘pharmacochaperones’ engineered to correct the
quaternary structure of misfolded proteins which enable proper
export of the receptor to the plasma membrane and functional rescue
of the receptor (Leanos-Miranda et al., 2002). Design criteria for
pharmacochaperones include: specificity for the receptor of interest,
ability of the molecule to reach the target (cell permeability) and the
ability to dissociate from the receptor or at least not compete for the
same (orthosteric) binding site as the endogenous ligand (Conn et al.,
2002).
Studies utilising the ‘pharmacochaperone’ antagonist IN3, which
targets human GnRHR (Janovick et al., 2002; Leanos-Miranda et al.,
2002; Janovick et al., 2003; Leanos-Miranda et al., 2003, 2005), have
clearly demonstrated that this cell-permeable compound alleviates
the dominant-negative effect of mutant receptors upon the trafficking
of WT GnRHR. Thus therapeutic strategies do not need to directly
modulate receptor activity, but can act by modulating GPCR
expression at the plasma membrane. Such approaches are based
upon the dominant-negative effects of the dimerisation of mutant
receptors with WT receptors that underlie the disease state. This may
not always be the case, since one particular point mutation in the
366
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
human GnRHR displayed a lack of ligand-binding specificity or
second-messenger signalling, but little if any difference was observed
between WT and mutant receptors in cell surface expression (Pralong
et al., 1999). This would suggest that the lack of ligand binding was
not due to a lack of cell surface receptor expression, but rather an
inability of mutant receptors to couple to downstream signalling
machinery.
6. Allosteric modulation of G protein-coupled receptor activity
The ability of compounds, other than endogenous ligands, to bind
GPCRs and alter their functional capability is a facet of GPCR research
that is rapidly gaining impetus. Rather than binding to the receptor
within the natural binding pocket (orthosteric site), allosteric
compounds are capable of interacting with GPCRs at alternate sites
that do not compete with natural ligands for receptor binding,
modifying receptor activity through a number of mechanisms
(Christopoulos & Kenakin, 2002; May et al., 2004; Durroux, 2005;
Presland, 2005; May et al., 2007).
6.1. Receptor activity-modifying proteins (RAMPs)
The importance of RAMPs in GPCR functionality was first
established in a study of the calcitonin-receptor-like receptor
(CRLR), which revealed that the pharmacological profile of this
receptor was determined by its association with a family of novel
proteins termed RAMPs (McLatchie et al., 1998). When dimerised with
RAMP1, CRLR functions as a calcitonin-gene-related peptide receptor,
however, if associated with either RAMP2 or RAMP3, CRLR behaves as
an adrenomedullin receptor.
Studies of the calcitonin receptor show that receptor affinity for
amylin is modified by dimerisation with particular RAMP subtypes
(Hay et al., 2004). The specificity of these interactions was further
illustrated by measuring receptor/RAMP binding affinities for several
different agonists and antagonists in order to pharmacologically
distinguish receptor complexes (Hay et al., 2005). The pharmacology
of CRLR is partially affected by the manner in which the receptor is
glycosylated and its associated RAMP subtype (McLatchie et al., 1998).
Thus, the ability of different RAMPs to associate with certain GPCRs
and effectively form distinct heterodimeric protein complexes that
exhibit vastly divergent pharmacological properties, provides strong
support for the rationale that such effects could be observed in
heterodimeric structures comprised of different GPCR subtypes. The
possibility then that a single GPCR could exhibit a multitude of
pharmacological profiles depending upon the GPCR subtype with
which it is associated is plausible, and has significance for the design
of novel drugs.
6.2. Dimerisation-mediated G protein-coupled receptor allosterism
Although the role of RAMPs in altering the activity of GPCRs has
been briefly discussed, several other examples of allosteric modulation have been reported for GPCRs. Aside from chemical compounds
and small peptides, it has been proposed that GPCR dimerisation can
also facilitate modified receptor activity. Allosteric modification has
been shown between the CB1 and the orexin receptor (Hilairet et al.,
2003). When stably transfected into CHO cells, these GPCRs can exist
as heterodimers at the plasma membrane in the absence of ligand, but
exhibit dramatically different pharmacological properties to their
monomeric selves when exposed to ligands. When coexpressed with
the CB1, hypersensitisation of the orexin receptor (subtype 1; OxR1)
was observed in the presence of its endogenous ligand, with a 100fold increase in orexin potency in respect to the activity of ERK1/2. In
addition, coexpression of human OxR1 and CB1 in HEK293 cells
resulted in receptor heterodimers that failed to traffic correctly to the
plasma membrane, in contrast to expression of receptors alone (Ellis
et al., 2006). Treatment with either OxR1 or CB1 antagonist restored
expression of both receptors at the cell surface, and reciprocally
antagonised the other, unoccupied receptor's ability to activate MAPKs
following treatment with respective agonist. This effect was only seen
when OxR1 and CB1 were coexpressed in HEK293 cells.
Family C GPCRs, including mGluRs, GABABRs and the CaSR exhibit
allosterism, although the means by which this is accomplished is
diverse. While it has been shown by a number of studies that the
proper trafficking of the GABABR1 subtype to the plasma membrane
requires dimerisation with the GABABR2 subtype (Jones et al., 1998;
Kaupmann et al., 1998; White et al., 1998), a GABABR1 mutant that is
capable of trafficking to the plasma membrane in the absence of
GABABR2 was unable to activate downstream signalling pathways in
spite of ligand binding to the receptor (Margeta-Mitrovic et al., 2000;
Pagano et al., 2001).
These results indicate that interactions between different domains
of Family C GPCR subtypes (particularly the N-terminal) are necessary
for effective coupling to heterotrimeric G proteins and subsequent
production of second messengers (Pin et al., 2005). This was further
demonstrated by a series of chimeric receptors comprised of
alternately paired extracellular domains (ECD) and heptahelical
domains of the two receptor subtypes (Galvez et al., 2001). While
the ECD of GABABR1 bound ligand and the heptahelical domain of
GABABR2 was necessary for G protein coupling and specificity, the
paramount determinant of a functional receptor was the heterodimeric pairing of respective domains from each receptor subtype. Not
only does such complementation lead to an active GABAB receptor, the
binding affinity of ligand to the GABABR1 subtype is allosterically
enhanced by the presence of the GABABR2 ECD.
An interesting perspective of this concept relates to the prevalence
of orphan GPCRs considered in light of GPCR dimerisation and the
possible implications this has on the understanding of GPCR ligand
binding and activation (Levoye et al., 2006b). Although classically
defined as a GPCR for which no known ligand has been identified,
“orphan” receptors could also be a GPCR monomer in a dimeric
complex that does not bind ligand. An example of such an “orphan”
receptor would be the GABABR2 subtype, which does not bind ligand
but does increase the affinity of GABABR1 for GABA by heterodimerisation and allosteric modulation (Galvez et al., 2001; Kniazeff et al.,
2002; Pin et al., 2004).
The T1R3 taste receptor forms obligate heterodimers with either
T1R2 or T1R1 subtypes, but does not bind ligands (Nelson et al., 2001,
2002; Zhao et al., 2003; Xu et al., 2004). Further evidence from studies
involving mammalian taste receptors show that detection of specific
tastes is determined by the particular combination of T1-family
receptor subtypes comprising different heterodimers (Nelson et al.,
2001; Zhao et al., 2003; Cui et al., 2006), indicating endogenous
heterodimerisation was crucial to the functional specificity of certain
GPCRs. These observations could have a profound impact upon the
food industry, with the future development of artificial flavours such
as sweeteners and umami (amino acid) tastes based upon the
particular heterodimeric taste-receptor complex the compound is
capable of activating. Similarly, the recently discovered GPR50
receptor can, when dimerised with the melatonin receptor subtype
1 (MT1R), alter the affinity of MT1R for endogenous ligands (Levoye
et al., 2006a). Thus, a number of so-called “orphan” GPCRs may in fact
play a crucial role in the functional capacity of certain heterodimeric
complexes, and in turn could theoretically be targeted as a potential
avenue of intervention for disorders associated with non-orphan
GPCRs.
7. Dimerisation and disease pathogenesis
While evidence correlating receptor dimerisation and pathogenesis of human disease is currently limited, there are indications that
aberrant cross-talk between GPCRs can directly impact on health.
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
7.1. Preeclampsia
Studies involving the vasopressor angiotensin II and its cognate
GPCR (type I angiotensin-II receptor; AT1R) found that a significant
interaction occurred between this receptor and the bradykinin-2
receptor (B2R; AbdAlla et al., 2000). Not only was it shown that
heterodimerisation of these receptors was dependent on the relative
amount of receptors present, but that the signalling capacity and
internalisation profiles of the receptors were altered in conditions
where the receptors were coupled. Given the enhanced signalling
efficacy of the AT1R when coupled with the B2R, the authors then
carried out a study to investigate the potential effects that this may
have in vivo.
Previous studies suggested that increased expression of AT1R was
not correlated with preeclampsia (Masse et al., 1998; Pouliot et al.,
1998) and that circulating levels of angiotensin II were not
significantly different from control subjects (de Jong et al., 1991).
This indicated that hypersensitivity to angiotensin II seen in women
with preeclampsia (Abdul-Karim & Assalin, 1961; Oney & Kaulhausen,
1982) may somehow be related to heterodimerisation between AT1R
and B2R. Indeed, the study illustrated that preeclampsia was strongly
correlated with an increased level of B2R expression, and increased
AT1R/B2R heterodimerisation with elevated sensitivity to circulating
angiotensin II (AbdAlla et al., 2001). Furthermore, they showed that
heterodimerisation rendered the AT1R resistant to free-radical
inactivation that exacerbated angiotensin-II sensitivity, establishing
the first disease model associated with irregular GPCR heterodimerisation. For a recent review see Shah (2005).
7.2. Parkinson's disease
The use of L-DOPA (a dopamine precursor) in the treatment of
Parkinson's disease has been documented since the 1970s, with the
harmful side effects, particularly the uncontrolled muscle contractions
known as dyskinesia, having been described shortly thereafter (for
reviews see Sassin, 1975; Nutt, 1990; Blandini, 2003). As a consequence, much research has focussed on the understanding of the
molecular mechanisms that underlie this phenomenon, and the
development of treatments that are as efficacious as L-DOPA but
which lack the undesirable side effects.
Evidence for a functional interaction between A2aR and D2R was
first shown by Ferre et al. (1991). Studies in membrane preparations of
rat striatum indicated that activation of A2aRs by a selective agonist
resulted in a significant decrease in the affinity of D2R agonist binding
sites (see also Fuxe et al., 2003, 2005) The authors of the original study
postulated that cross-talk between receptors may contribute to the
physiological responses observed following administration of adenosine antagonists such as caffeine.
A novel A2aR antagonist (KW-6002) was examined in primates
with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced
symptoms of Parkinson's disease, to determine whether it could
enhance motor control without the side effects of L-DOPA therapy
(Kanda et al., 1998). MPTP selectively damages neurons in the
substantia nigra, a region of the mid-brain involved in motor control,
producing symptoms that are almost identical to those seen in
Parkinson's patients. Not only did the A2aR antagonist significantly
improve motor disability in a dose-dependent fashion, it did so
without causing dyskinesia or hyperactivity. Another study found that
primates subjected to chronic L-DOPA treatment developed profound
dyskinesia as a result, but also exhibited a significant increase in A2aR
mRNA levels (Zeng et al., 2000). Findings with an alternative A2aR
antagonist (MSX-3) revealed that by removing the antagonistic effect
of A2aRs on D2R function, the activity of D2Rs in the presence of
dopamine agonists was potentiated (Stromberg et al., 2000). Combination therapies for Parkinson's disease with L-DOPA and adenosine
antagonists have been proposed, with early results suggesting that
367
treatments with an A2aR antagonist to potentiate D2Rs followed by a
reduced dose of L-DOPA may alleviate motor control deficits without
inducing dyskinesia (reviewed by Pinna et al., 2005).
Perhaps the most revealing insight into A2aR and D2R interactions
came from a study that utilised dual-label immunofluorescence with
confocal microscopy to show colocalisation of these receptors at the
cell membrane in human neuroblastoma cells (Hillion et al., 2002).
These confocal studies were complemented by co-immunoprecipitation that detected the constitutive formation of heterodimeric
complexes between A2a and D2 receptors. They also found that D2Rs,
which have been shown to be largely resistant to agonist-induced
desensitisation (Ng et al., 1997), exhibited accelerated desensitisation
after prolonged exposure to both dopamine and adenosine agonists,
possibly due to their co-internalisation with the A2aRs. Further
evidence for A2aR/D2R heterodimerisation has been demonstrated
utilising BRET, with two separate studies showing a BRET signal
between A2aRs and D2Rs in living cells that was not significantly
altered in the presence of agonists for either receptor (Canals et al.,
2003; Kamiya et al., 2003).
7.3. Hypogonadotropic hypogonadism
Polymorphisms identified in the human GnRHR cause hypogonadotropic hypogonadism (HH), resulting from a lack of receptor
expression at the cell surface. Numerous studies have shown that
mutant receptors, including splice variants and C-terminal truncations, act upon their WT congeners in a dominant-negative fashion.
Examples include the vasopressin-2 receptor (Zhu & Wess, 1998), the
dopamine D3 receptor (Karpa et al., 2000) and luteinising hormone
receptor (Lee et al., 2002). A study of eight naturally occurring
mutations in the human GnRHR demonstrated that in virtually all
cases the mutant receptor acted in a dominant-negative manner when
coexpressed with WT GnRHR, and was capable of inhibiting ligand
binding, WT receptor activation and generation of second messengers
(Leanos-Miranda et al., 2005). While the authors speculated that this
may be due to reduced WT receptor expression (as a result of cotransfection with a second receptor), or competition for relevant G
proteins by mutant receptors, they considered this unlikely and
concluded that dimerisation between the WT GnRHR and traffickingdeficient mutant receptor most likely led to ablation of WT receptor
signalling and expression. Furthermore, and perhaps of greater
import, was their discovery that treatment of cells expressing both
WT and mutant GnRHR with the ‘pharmacochaperone’ antagonist IN3
was able to completely overcome the signalling and expression
inhibition observed for untreated, co-transfected cells.
In order to try and unravel the molecular basis for WT GnRHR
inhibition by coexpression with GnRHR mutants, another experimental approach was adopted. By genetically fusing a green
fluorescent protein (GFP) to the C-terminus of the WT GnRHR
(including a spacer consisting of sequence from the catfish WT
GnRHR C-tail) a chimeric receptor was generated which could be
visualised using confocal microscopy (Brothers et al., 2004). By doing
so the authors were able to demonstrate that the dominant-negative
effect of mutant GnRHRs on human WT GnRHR signalling (Conn et al.,
2002; Leanos-Miranda et al., 2002; Janovick et al., 2003; LeanosMiranda et al., 2003; Ulloa-Aguirre et al., 2004) was a direct result of
retention of the WT receptor within the ER. This inhibition of WT
GnRHR expression at the plasma membrane caused a significant
decrease in the ability of the WT receptor to initiate secondmessenger signalling following agonist treatment. While treatment
with the GnRHR antagonist IN3 was able to pharmacologically rescue
the functional capacity of WT GnRHR in the presence of two of the
mutant receptors tested, the dominant-negative effect of a third
mutant receptor (S168R) remained unperturbed.
These findings were supported by a more recent study that showed
that in the presence of the GnRHR antagonist IN3, pairs of naturally
368
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
occurring mutant GnRHRs found in compound heterozygote HH
patients could be functionally restored, enabling receptor activation of
effector mechanisms and ligand binding in the majority of instances
(Leanos-Miranda et al., 2005). However it was noted that the extent of
this functional rescue was dependent upon the specific genotype of
patients, as an improvement in receptor signalling was not applicable
to all receptor pairs when treated with IN3. This would suggest that in
some instances the pharmacochaperone is capable of stabilising the
structure of mutant receptors sufficiently to overcome ER retention,
but that in other cases the structural abnormality/instability of mutant
receptors cannot be circumvented through use of such chaperones.
7.4. Schizophrenia and psychosis
The complexity of schizophrenia and its related symptoms has led
to the development of treatment protocols that, although effective,
leave a lot to be desired with respect to efficacy and unwanted side
effects. Insight into the potential neuronal mechanisms underlying the
psychotic hallucinations associated with the condition came from
observations that indoleamine and phenethylamine psychedelics
shared a high affinity for a specific subset of serotonin (5-HT) receptors
that activated the cerebral cortex (Aghajanian & Marek, 2000). Further
analysis of this relationship revealed that the potent hallucinogen
1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) activated
serotonin 2a (5-HT2a) receptors in the somatosensory cortex (Scruggs
et al., 2003). This resulted in increased glutamate release that was
blocked by cotreatment with a 5-HT2a receptor antagonist, leading
the authors to suggest that 5-HT2a “heteroceptors” mediated the
actions of hallucinogens.
An elegant study recently published in Nature has provided
substantial evidence to support this hypothesis. A collaborative effort
amongst several research groups assessed the potential interactions of
GPCRs within the cerebral cortex using a combination of BRET, FRET
and co-immunoprecipitation methodologies, clearly demonstrating
that the 5-HT2a receptor and mGluR2 formed functional heterodimeric complexes with a distinct signalling profile (Gonzalez-Maeso
et al., 2008). In addition to the allosteric modulation of endogenous
ligand-binding affinities when receptors were dimerised, coexpression of mGluR2 significantly impaired high affinity activation of Gαq/11
by the 5-HT2a receptor while enhancing Gαi activation by the 5-HT2a
receptor. This was a fundamental observation as the authors noted the
similarities between these findings and the divergent response of
pyramidal neurons to hallucinogenic and non-hallucinogenic agonists
at the 5-HT2a receptor. While non-hallucinogenic agonists led to c-fos
induction requiring Gαq/11-dependent activation of phospholipase C,
hallucinogens including DOI induced the early growth response-2
gene (egr-2) that was dependent upon Gαi activation (GonzalezMaeso et al., 2007). It was subsequently shown that induction of the
hallucinogen-specific marker egr-2 could be prevented by co-administration of an mGluR2 agonist in cortical cultures, which also
abrogated head-twitch behaviour specific to hallucinogens (Gonzalez-Maeso et al., 2008). Finally the authors found elevated expression
of the 5-HT2a receptor and diminished levels of mGluR2 expression in
post-mortem samples from untreated schizophrenic patients. They
concluded that dysregulation of mGluR2 and 5-HT2a receptor
expression, and thereby heterodimers comprised of these receptors
within cortical tissue, may result in aberrant signalling that predisposes schizophrenic patients to psychosis.
8. Heterodimer-specific drugs
There is considerable interest in the design of drugs that
specifically target GPCR heterodimers and this area of GPCR research
already boasts approaches to determine the structural requirements
needed to produce molecules that explicitly target heterodimeric
GPCR pairs (for reviews see George et al., 2002; Milligan, 2006).
Computer modelling systems (in silico) have been employed to
design novel heterodimeric compounds using a homology-based
approach. Structural models of GPCR heterodimers are generated
based on the known structures of other GPCRs that share a degree of
homology with the receptor(s) of interest (Filizola & Weinstein, 2005a,
2005b; Filizola et al., 2006). Given that the only GPCRs for which a
complete 3D crystal structure has been determined (by X-ray
crystallography and micro-crystallography) are bovine rhodopsin
(Palczewski et al., 2000) and β2AR (Rasmussen et al., 2007), the
structure of rhodopsin has been the primary basis for the modelling of
numerous GPCR structures. Interestingly, the β2AR was crystallised as
a monomer (Rasmussen et al., 2007), however, it is unclear if this
represents the physiological scenario or if the purification process
disrupted dimerisation. There already exist a number of drugs on the
market that were designed using structure-based homology modelling, including the anti-impotence drug Viagra® (Lundstrom, 2006,
2007), and the AIDS drugs Agenerase® and Viracept®, whose
structures were based on that of the HIV proteinase.
De novo structure modelling on the other hand uses first principles
to determine receptor structure using the known amino acid sequence
of the protein (for recent reviews see Reggio, 2006; Schlyer & Horuk,
2006). One group has predicted the active conformation of rhodopsin
bound to retinal, using a de novo model sharing very high homology
with the known crystal structure (Vaidehi et al., 2002). Thus, the ability
to calculate the 3D structure of different GPCRs may anticipate how
two different GPCR subtypes interact, and what physical constraints
may be imposed as a result of such associations. This de novo method
of receptor modelling could prove extremely useful for predicting the
3D structure of receptors that do not belong to Family A GPCRs.
A third technique that has been adopted to design heterodimerspecific compounds uses bivalent molecules. These consist of two
different agonists/antagonists, or a combination of both, with amino
acid spacers of varying lengths separating the ligands. Since a DOP
antagonist enhances the analgesic properties of morphine acting at
MOP (Gomes et al., 2004), a bivalent ligand was developed consisting
of MOP agonist and DOP antagonist, linked by a spacer (Daniels et al.,
2005). This dual agonist/antagonist demonstrated that bivalent
compounds can induce analgesia while at the same time minimising
tolerance and physical dependence to the morphine-analogue
component, and that the effect was primarily determined by the
distance between the two pharmacophores (Lenard et al., 2007).
Similar efforts have also been undertaken for DOP/KOP heterodimer
studies utilising a bivalent DOP/KOP-specific antagonist (Xie et al.,
2005), and a bivalent agonist that targets a heterodimer consisting of
DOP and a sensory neuron-specific receptor (Breit et al., 2006). This
provides an intriguing opportunity to design pharmaceutical compounds that exclusively target GPCR heterodimers, with the size of the
spacer between the two pharmacophores designed according to the
structural arrangement of the GPCR monomers comprising the
protein complex.
9. Concluding remarks
There is a rapidly expanding body of evidence indicating that most,
if not all, GPCRs are present at the plasma membrane as dimeric
complexes, and a growing acceptance that such complexes exhibit
distinct pharmacological properties and functional characteristics (for
a recent review see Fredholm et al., 2007). Of profound interest is the
indication that GPCRs of different types interact with one another at
the molecular level, adding yet another level of complexity and
opening an entirely new perspective with regard to pharmacological
intervention when these receptors are coexpressed in vivo.
The concept of GPCR dimerisation provides two major new
avenues of drug discovery/development research. These include the
potential existence of the heterodimer as a drug target distinct from
that provided by monomers/homodimers of individual receptor
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
subtypes, and secondly, the development of drugs capable of targeting
each receptor component resulting in synergistic effects.
There is increasing evidence that certain disease states arise from
the incorrect trafficking of GPCRs from the ER to the plasma
membrane. Indeed some mutant receptors appear to have a dominant-negative effect on the WT receptor due to dimerisation. An
understanding of this concept will facilitate the design of therapeutic
strategies to overcome such dominant-negative effects, including the
use of cell-permeable compounds such as ‘pharmacochaperones’.
As the importance of GPCR dimerisation is acknowledged, the tools
required for drug discovery and development to exploit this understanding are rapidly being developed. Consequently, we are likely to
be on the cusp of a new generation of pharmaceuticals that promise to
exhibit improved specificity and efficacy with reduced side effects.
Acknowledgments
The authors' work has been funded by the National Health and
Medical Research Council (NHMRC) of Australia (project grant
#404087). Dr. Pfleger has been supported by an NHMRC Peter Doherty
Research Fellowship (#353709) and A/Prof. Eidne by an NHMRC
Principal Research Fellowship (#212064).
References
AbdAlla, S., Lother, H., el Massiery, A., & Quitterer, U. (2001). Increased AT(1) receptor
heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness.
Nat Med 7(9), 1003−1009.
AbdAlla, S., Lother, H., & Quitterer, U. (2000). AT1-receptor heterodimers show enhanced
G-protein activation and altered receptor sequestration. Nature 407(6800), 94−98.
Abdul-Karim, R., & Assalin, S. (1961). Pressor response to angiotonin in pregnant and
nonpregnant women. Am J Obstet Gynecol 82, 246−251.
Adams, C. L., Cowen, M. S., Short, J. L., & Lawrence, A. J. (2007). Combined antagonism of
glutamate mGlu5 and adenosine A2A receptors interact to regulate alcohol-seeking
in rats. Int J Neuropsychopharmacol, 1−13.
Aghajanian, G. K., & Marek, G. J. (2000). Serotonin model of schizophrenia: emerging
role of glutamate mechanisms. Brain Res Brain Res Rev 31(2–3), 302−312.
Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., et al. (2000). Detection
of beta 2-adrenergic receptor dimerization in living cells using bioluminescence
resonance energy transfer (BRET). Proc Natl Acad Sci U S A 97(7), 3684−3689.
Arolfo, M. P., Yao, L., Gordon, A. S., Diamond, I., & Janak, P. H. (2004). Ethanol operant
self-administration in rats is regulated by adenosine A2 receptors. Alcohol Clin Exp
Res 28(9), 1308−1316.
Babcock, G. J., Farzan, M., & Sodroski, J. (2003). Ligand-independent dimerization of
CXCR4, a principal HIV-1 coreceptor. J Biol Chem 278(5), 3378−3385.
Backstrom, P., Bachteler, D., Koch, S., Hyytia, P., & Spanagel, R. (2004). mGluR5
antagonist MPEP reduces ethanol-seeking and relapse behavior. Neuropsychopharmacology 29(5), 921−928.
Bai, M. (2004). Dimerization of G-protein-coupled receptors: roles in signal transduction. Cell Signal 16(2), 175−186.
Bai, M., Trivedi, S., Kifor, O., Quinn, S. J., & Brown, E. M. (1999). Intermolecular
interactions between dimeric calcium-sensing receptor monomers are important
for its normal function. Proc Natl Acad Sci U S A 96(6), 2834−2839.
Bargmann, C. I. (1998). Neurobiology of the Caenorhabditis elegans genome. Science
282(5396), 2028−2033.
Bertini, R., Allegretti, M., Bizzarri, C., Moriconi, A., Locati, M., Zampella, G., et al. (2004).
Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors
CXCR1 and CXCR2: prevention of reperfusion injury. Proc Natl Acad Sci U S A 101(32),
11791−11796.
Besheer, J., Stevenson, R. A., & Hodge, C. W. (2006). mGlu5 receptors are involved in the
discriminative stimulus effects of self-administered ethanol in rats. Eur J Pharmacol
551(1–3), 71−75.
Blandini, F. (2003). Adenosine receptors and l-DOPA-induced dyskinesia in Parkinson's
disease: potential targets for a new therapeutic approach. Exp Neurol 184(2), 556−560.
Bockaert, J., & Pin, J. P. (1999). Molecular tinkering of G protein-coupled receptors: an
evolutionary success. Embo J 18(7), 1723−1729.
Bouschet, T., Martin, S., & Henley, J. M. (2005). Receptor-activity-modifying proteins are
required for forward trafficking of the calcium-sensing receptor to the plasma
membrane. J Cell Sci 118(Pt 20), 4709−4720.
Bouvier, M. (2001). Oligomerization of G-protein-coupled transmitter receptors. Nat
Rev Neurosci 2(4), 274−286.
Bouvier, M., Heveker, N., Jockers, R., Marullo, S., & Milligan, G. (2007). BRET analysis of
GPCR oligomerization: newer does not mean better. Nat Methods 4(1), 3−4.
Breit, A., Gagnidze, K., Devi, L. A., Lagace, M., & Bouvier, M. (2006). Simultaneous
activation of the delta opioid receptor (deltaOR)/sensory neuron-specific receptor-4
(SNSR-4) hetero-oligomer by the mixed bivalent agonist bovine adrenal medulla
peptide 22 activates SNSR-4 but inhibits deltaOR signaling. Mol Pharmacol 70(2),
686−696.
369
Brothers, S. P., Cornea, A., Janovick, J. A., & Conn, P. M. (2004). Human loss-of-function
gonadotropin-releasing hormone receptor mutants retain wild-type receptors in
the endoplasmic reticulum: molecular basis of the dominant-negative effect. Mol
Endocrinol 18(7), 1787−1797.
Bulenger, S., Marullo, S., & Bouvier, M. (2005). Emerging role of homo- and
heterodimerization in G-protein-coupled receptor biosynthesis and maturation.
Trends Pharmacol Sci 26(3), 131−137.
Callea, F., Brisigotti, M., Fabbretti, G., Bonino, F., & Desmet, V. J. (1992). Hepatic
endoplasmic reticulum storage diseases. Liver 12(6), 357−362.
Canals, M., Marcellino, D., Fanelli, F., Ciruela, F., de Benedetti, P., Goldberg, S. R., et al.
(2003). Adenosine A2A-dopamine D2 receptor–receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy
transfer. J Biol Chem 278(47), 46741−46749.
Cheng, Z. J., & Miller, L. J. (2001). Agonist-dependent dissociation of oligomeric
complexes of G protein-coupled cholecystokinin receptors demonstrated in living
cells using bioluminescence resonance energy transfer. J Biol Chem 276(51),
48040−48047.
Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., et al. (1996). The betachemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell 85(7), 1135−1148.
Christopoulos, A., & Kenakin, T. (2002). G protein-coupled receptor allosterism and
complexing. Pharmacol Rev 54(2), 323−374.
Ciruela, F., Casado, V., Rodrigues, R. J., Lujan, R., Burgueno, J., Canals, M., et al. (2006).
Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1–
A2A receptor heteromers. J Neurosci 26(7), 2080−2087.
Conn, P. M., Leanos-Miranda, A., & Janovick, J. A. (2002). Protein origami: therapeutic
rescue of misfolded gene products. Mol Interv 2(5), 308−316.
Conn, P. M., Rogers, D. C., & McNeil, R. (1982). Potency enhancement of a GnRH agonist:
GnRH-receptor microaggregation stimulates gonadotropin release. Endocrinology
111(1), 335−337.
Conn, P. M., Rogers, D. C., Stewart, J. M., Niedel, J., & Sheffield, T. (1982). Conversion of a
gonadotropin-releasing hormone antagonist to an agonist. Nature 296(5858), 653−655.
Cornea, A., Janovick, J. A., Maya-Nunez, G., & Conn, P. M. (2001). Gonadotropin-releasing
hormone receptor microaggregation. Rate monitored by fluorescence resonance
energy transfer. J Biol Chem 276(3), 2153−2158.
Couve, A., Filippov, A. K., Connolly, C. N., Bettler, B., Brown, D. A., & Moss, S. J. (1998).
Intracellular retention of recombinant GABAB receptors. J Biol Chem 273(41),
26361−26367.
Cui, M., Jiang, P., Maillet, E., Max, M., Margolskee, R. F., & Osman, R. (2006). The
heterodimeric sweet taste receptor has multiple potential ligand binding sites. Curr
Pharm Des 12(35), 4591−4600.
Daniels, D. J., Lenard, N. R., Etienne, C. L., Law, P. Y., Roerig, S. C., & Portoghese, P. S. (2005).
Opioid-induced tolerance and dependence in mice is modulated by the distance
between pharmacophores in a bivalent ligand series. Proc Natl Acad Sci U S A 102(52),
19208−19213.
de Jong, C. L., Dekker, G. A., & Sibai, B. M. (1991). The renin–angiotensin–aldosterone
system in preeclampsia. A review. Clin Perinatol 18(4), 683−711.
Dean, M. K., Higgs, C., Smith, R. E., Bywater, R. P., Snell, C. R., Scott, P. D., et al. (2001).
Dimerization of G-protein-coupled receptors. J Med Chem 44(26), 4595−4614.
Durroux, T. (2005). Principles: a model for the allosteric interactions between ligand
binding sites within a dimeric GPCR. Trends Pharmacol Sci 26(7), 376−384.
Duvernay, M. T., Filipeanu, C. M., & Wu, G. (2005). The regulatory mechanisms of export
trafficking of G protein-coupled receptors. Cell Signal 17(12), 1457−1465.
El-Asmar, L., Springael, J. Y., Ballet, S., Andrieu, E. U., Vassart, G., & Parmentier, M. (2005).
Evidence for negative binding cooperativity within CCR5–CCR2b heterodimers. Mol
Pharmacol 67(2), 460−469.
Ellis, J., Pediani, J. D., Canals, M., Milasta, S., & Milligan, G. (2006). Orexin-1 receptorcannabinoid CB1 receptor heterodimerization results in both ligand-dependent
and -independent coordinated alterations of receptor localization and function.
J Biol Chem 281(50), 38812−38824.
Endres, M. J., Clapham, P. R., Marsh, M., Ahuja, M., Turner, J. D., McKnight, A., et al. (1996).
CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87(4), 745−756.
Fairbanks, C. A. (2003). Spinal delivery of analgesics in experimental models of pain and
analgesia. Adv Drug Deliv Rev 55(8), 1007−1041.
Ferre, S., Karcz-Kubicha, M., Hope, B. T., Popoli, P., Burgueno, J., Gutierrez, M. A., et al. (2002).
Synergistic interaction between adenosine A2A and glutamate mGlu5 receptors:
implications for striatal neuronal function. Proc Natl Acad Sci U S A 99(18), 11940−11945.
Ferre, S., Popoli, P., Rimondini, R., Reggio, R., Kehr, J., & Fuxe, K. (1999). Adenosine A2A
and group I metabotropic glutamate receptors synergistically modulate the binding
characteristics of dopamine D2 receptors in the rat striatum. Neuropharmacology
38(1), 129−140.
Ferre, S., von Euler, G., Johansson, B., Fredholm, B. B., & Fuxe, K. (1991). Stimulation of
high-affinity adenosine A2 receptors decreases the affinity of dopamine D2
receptors in rat striatal membranes. Proc Natl Acad Sci U S A 88(16), 7238−7241.
Filizola, M., Olmea, O., & Weinstein, H. (2002). Prediction of heterodimerization
interfaces of G-protein coupled receptors with a new subtractive correlated
mutation method. Protein Eng 15(11), 881−885.
Filizola, M., Wang, S. X., & Weinstein, H. (2006). Dynamic models of G-protein coupled
receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular
dynamics simulations in a POPC bilayer. J Comput Aided Mol Des 20(7–8), 405−416.
Filizola, M., & Weinstein, H. (2005). The study of G-protein coupled receptor
oligomerization with computational modeling and bioinformatics. Febs J 272(12),
2926−2938.
Filizola, M., & Weinstein, H. (2005). The structure and dynamics of GPCR oligomers: a
new focus in models of cell-signaling mechanisms and drug design. Curr Opin Drug
Discov Dev 8(5), 577−584.
370
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
Fink, J. S., Weaver, D. R., Rivkees, S. A., Peterfreund, R. A., Pollack, A. E., Adler, E. M.,
et al. (1992). Molecular cloning of the rat A2 adenosine receptor: selective coexpression with D2 dopamine receptors in rat striatum. Brain Res Mol Brain Res
14(3), 186−195.
Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., & Palczewski, K. (2003).
Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature
421(6919), 127−128.
Fredholm, B. B., Hokfelt, T., & Milligan, G. (2007). G-protein-coupled receptors: an
update. Acta Physiol (Oxf) 190(1), 3−7.
Fuxe, K., Agnati, L. F., Jacobsen, K., Hillion, J., Canals, M., Torvinen, M., et al.
(2003). Receptor heteromerization in adenosine A2A receptor signaling:
relevance for striatal function and Parkinson's disease. Neurology 61(11 Suppl 6),
S19−S23.
Fuxe, K., Ferre, S., Canals, M., Torvinen, M., Terasmaa, A., Marcellino, D., et al. (2005).
Adenosine A2A and dopamine D2 heteromeric receptor complexes and their
function. J Mol Neurosci 26(2–3), 209−220.
Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., et al. (2001). Allosteric
interactions between GB1 and GB2 subunits are required for optimal GABA(B)
receptor function. Embo J 20(9), 2152−2159.
George, S. R., Fan, T., Xie, Z., Tse, R., Tam, V., Varghese, G., et al. (2000). Oligomerization of
mu- and delta-opioid receptors. Generation of novel functional properties. J Biol
Chem 275(34), 26128−26135.
George, S. R., O'Dowd, B. F., & Lee, S. P. (2002). G-protein-coupled receptor oligomerization
and its potential for drug discovery. Nat Rev Drug Discov 1(10), 808−820.
Gomes, I., Gupta, A., Filipovska, J., Szeto, H. H., Pintar, J. E., & Devi, L. A. (2004). A role for
heterodimerization of mu and delta opiate receptors in enhancing morphine
analgesia. Proc Natl Acad Sci U S A 101(14), 5135−5139.
Gomes, I., Jordan, B. A., Gupta, A., Trapaidze, N., Nagy, V., & Devi, L. A. (2000).
Heterodimerization of mu and delta opioid receptors: a role in opiate synergy.
J Neurosci 20(22), RC110.
Gonzalez-Maeso, J., Ang, R. L., Yuen, T., Chan, P., Weisstaub, N. V., Lopez-Gimenez, J. F., et al.
(2008). Identification of a serotonin/glutamate receptor complex implicated in
psychosis. Nature 452(7183), 93−97.
Gonzalez-Maeso, J., Weisstaub, N. V., Zhou, M., Chan, P., Ivic, L., Ang, R., et al. (2007).
Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling
pathways to affect behavior. Neuron 53(3), 439−452.
Gupta, A., Decaillot, F. M., & Devi, L. A. (2006). Targeting opioid receptor heterodimers:
strategies for screening and drug development. Aaps J 8(1), E153−E159.
Hague, C., Uberti, M. A., Chen, Z., Bush, C. F., Jones, S. V., Ressler, K. J., et al. (2004).
Olfactory receptor surface expression is driven by association with the beta2adrenergic receptor. Proc Natl Acad Sci U S A 101(37), 13672−13676.
Hay, D. L., Christopoulos, G., Christopoulos, A., Poyner, D. R., & Sexton, P. M. (2005).
Pharmacological discrimination of calcitonin receptor: receptor activity-modifying
protein complexes. Mol Pharmacol 67(5), 1655−1665.
Hay, D. L., Christopoulos, G., Christopoulos, A., & Sexton, P. M. (2004). Amylin receptors:
molecular composition and pharmacology. Biochem Soc Trans 32(Pt 5), 865−867.
Hebert, T. E., Loisel, T. P., Adam, L., Ethier, N., Onge, S. S., & Bouvier, M. (1998). Functional
rescue of a constitutively desensitized beta2AR through receptor dimerization.
Biochem J 330(Pt 1), 287−293.
Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., et al. (1996). A
peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits
both receptor dimerization and activation. J Biol Chem 271(27), 16384−16392.
Hilairet, S., Bouaboula, M., Carriere, D., Le Fur, G., & Casellas, P. (2003). Hypersensitization of the orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by
the specific CB1 antagonist, SR141716. J Biol Chem 278(26), 23731−23737.
Hillion, J., Canals, M., Torvinen, M., Casado, V., Scott, R., Terasmaa, A., et al. (2002).
Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors
and dopamine D2 receptors. J Biol Chem 277(20), 18091−18097.
Horvat, R. D., Roess, D. A., Nelson, S. E., Barisas, B. G., & Clay, C. M. (2001). Binding of
agonist but not antagonist leads to fluorescence resonance energy transfer between
intrinsically fluorescent gonadotropin-releasing hormone receptors. Mol Endocrinol
15(5), 695−703.
Huttenrauch, F., Nitzki, A., Lin, F. T., Honing, S., & Oppermann, M. (2002). Beta-arrestin
binding to CC chemokine receptor 5 requires multiple C-terminal receptor
phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif. J Biol
Chem 277(34), 30769−30777.
Issafras, H., Angers, S., Bulenger, S., Blanpain, C., Parmentier, M., Labbe-Jullie, C., et al.
(2002). Constitutive agonist-independent CCR5 oligomerization and antibodymediated clustering occurring at physiological levels of receptors. J Biol Chem
277(38), 34666−34673.
James, J. R., Oliveira, M. I., Carmo, A. M., Iaboni, A., & Davis, S. J. (2006). A rigorous
experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods 3(12), 1001−1006.
Janovick, J. A., Goulet, M., Bush, E., Greer, J., Wettlaufer, D. G., & Conn, P. M. (2003).
Structure–activity relations of successful pharmacologic chaperones for rescue of
naturally occurring and manufactured mutants of the gonadotropin-releasing
hormone receptor. J Pharmacol Exp Ther 305(2), 608−614.
Janovick, J. A., Maya-Nunez, G., & Conn, P. M. (2002). Rescue of hypogonadotropic
hypogonadism-causing and manufactured GnRH receptor mutants by a specific
protein-folding template: misrouted proteins as a novel disease etiology and
therapeutic target. J Clin Endocrinol Metab 87(7), 3255−3262.
Jones, K. A., Borowsky, B., Tamm, J. A., Craig, D. A., Durkin, M. M., Dai, M., et al. (1998).
GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1
and GABA(B)R2. Nature 396(6712), 674−679.
Jordan, B. A., & Devi, L. A. (1999). G-protein-coupled receptor heterodimerization
modulates receptor function. Nature 399(6737), 697−700.
Kachroo, A., Orlando, L. R., Grandy, D. K., Chen, J. F., Young, A. B., & Schwarzschild, M. A.
(2005). Interactions between metabotropic glutamate 5 and adenosine A2A
receptors in normal and parkinsonian mice. J Neurosci 25(45), 10414−10419.
Kamiya, T., Saitoh, O., Yoshioka, K., & Nakata, H. (2003). Oligomerization of adenosine
A2A and dopamine D2 receptors in living cells. Biochem Biophys Res Commun 306(2),
544−549.
Kanda, T., Jackson, M. J., Smith, L. A., Pearce, R. K., Nakamura, J., Kase, H., et al. (1998).
Adenosine A2A antagonist: a novel antiparkinsonian agent that does not provoke
dyskinesia in parkinsonian monkeys. Ann Neurol 43(4), 507−513.
Kaplan, G. B., Bharmal, N. H., Leite-Morris, K. A., & Adams, W. R. (1999). Role of adenosine
A1 and A2A receptors in the alcohol withdrawal syndrome. Alcohol 19(2), 157−162.
Karpa, K. D., Lin, R., Kabbani, N., & Levenson, R. (2000). The dopamine D3 receptor
interacts with itself and the truncated D3 splice variant d3nf: D3–D3nf interaction
causes mislocalization of D3 receptors. Mol Pharmacol 58(4), 677−683.
Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P., et al. (1998).
GABA(B)-receptor subtypes assemble into functional heteromeric complexes.
Nature 396(6712), 683−687.
Kenny, P. J., Boutrel, B., Gasparini, F., Koob, G. F., & Markou, A. (2005). Metabotropic
glutamate 5 receptor blockade may attenuate cocaine self-administration by
decreasing brain reward function in rats. Psychopharmacology (Berl) 179(1), 247−254.
Kim, P. S., & Arvan, P. (1998). Endocrinopathies in the family of endoplasmic reticulum
(ER) storage diseases: disorders of protein trafficking and the role of ER molecular
chaperones. Endocr Rev 19(2), 173−202.
Kniazeff, J., Galvez, T., Labesse, G., & Pin, J. P. (2002). No ligand binding in the GB2
subunit of the GABA(B) receptor is required for activation and allosteric interaction
between the subunits. J Neurosci 22(17), 7352−7361.
Kroeger, K. M., Hanyaloglu, A. C., Seeber, R. M., Miles, L. E., & Eidne, K. A. (2001).
Constitutive and agonist-dependent homo-oligomerization of the thyrotropinreleasing hormone receptor. Detection in living cells using bioluminescence
resonance energy transfer. J Biol Chem 276(16), 12736−12743.
Kroeger, K. M., Pfleger, K. D., & Eidne, K. A. (2003). G-protein coupled receptor
oligomerization in neuroendocrine pathways. Front Neuroendocrinol 24(4),
254−278.
Leanos-Miranda, A., Janovick, J. A., & Conn, P. M. (2002). Receptor-misrouting: an
unexpectedly prevalent and rescuable etiology in gonadotropin-releasing hormone
receptor-mediated hypogonadotropic hypogonadism. J Clin Endocrinol Metab 87(10),
4825−4828.
Leanos-Miranda, A., Ulloa-Aguirre, A., Janovick, J. A., & Conn, P. M. (2005). In vitro
coexpression and pharmacological rescue of mutant gonadotropin-releasing
hormone receptors causing hypogonadotropic hypogonadism in humans expressing compound heterozygous alleles. J Clin Endocrinol Metab 90(5), 3001−3008.
Leanos-Miranda, A., Ulloa-Aguirre, A., Ji, T. H., Janovick, J. A., & Conn, P. M. (2003).
Dominant-negative action of disease-causing gonadotropin-releasing hormone
receptor (GnRHR) mutants: a trait that potentially coevolved with decreased
plasma membrane expression of GnRHR in humans. J Clin Endocrinol Metab 88(7),
3360−3367.
Lee, C., Ji, I., Ryu, K., Song, Y., Conn, P. M., & Ji, T. H. (2002). Two defective heterozygous
luteinizing hormone receptors can rescue hormone action. J Biol Chem 277(18),
15795−15800.
Lenard, N. R., Daniels, D. J., Portoghese, P. S., & Roerig, S. C. (2007). Absence of
conditioned place preference or reinstatement with bivalent ligands containing
mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores.
Eur J Pharmacol 566(1–3), 75−82.
Levoye, A., Dam, J., Ayoub, M. A., Guillaume, J. L., Couturier, C., Delagrange, P., et al.
(2006). The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor
function through heterodimerization. Embo J 25(13), 3012−3023.
Levoye, A., Dam, J., Ayoub, M. A., Guillaume, J. L., & Jockers, R. (2006). Do orphan
G-protein-coupled receptors have ligand-independent functions? New insights from
receptor heterodimers. EMBO Rep 7(11), 1094−1098.
Lundstrom, K. (2006). Latest development in drug discovery on G protein-coupled
receptors. Curr Protein Pept Sci 7(5), 465−470.
Lundstrom, K. (2007). Structural genomics and drug discovery. J Cell Mol Med 11(2),
224−238.
Maggio, R., Vogel, Z., & Wess, J. (1993). Coexpression studies with mutant muscarinic/
adrenergic receptors provide evidence for intermolecular “cross-talk” between
G-protein-linked receptors. Proc Natl Acad Sci U S A 90(7), 3103−3107.
Marchese, A., George, S. R., Kolakowski, L. F., Jr., Lynch, K. R., & O'Dowd, B. F. (1999). Novel
GPCRs and their endogenous ligands: expanding the boundaries of physiology and
pharmacology. Trends Pharmacol Sci 20(9), 370−375.
Margeta-Mitrovic, M., Jan, Y. N., & Jan, L. Y. (2000). A trafficking checkpoint controls
GABA(B) receptor heterodimerization. Neuron 27(1), 97−106.
Margeta-Mitrovic, M., Jan, Y. N., & Jan, L. Y. (2001). Function of GB1 and GB2 subunits
in G protein coupling of GABA(B) receptors. Proc Natl Acad Sci U S A 98(25),
14649−14654.
Masse, J., Forest, J. C., Moutquin, J. M., Degrandpre, P., & Forest, V. I. (1998). A prospective
longitudinal study of platelet angiotensin II receptors for the prediction of
preeclampsia. Clin Biochem 31(4), 251−255.
May, L. T., Avlani, V. A., Sexton, P. M., & Christopoulos, A. (2004). Allosteric modulation of
G protein-coupled receptors. Curr Pharm Des 10(17), 2003−2013.
May, L. T., Leach, K., Sexton, P. M., & Christopoulos, A. (2007). Allosteric modulation of
G protein-coupled receptors. Annu Rev Pharmacol Toxicol 47, 1−51.
McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., et al. (1998).
RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like
receptor. Nature 393(6683), 333−339.
Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., de Ana, A. M., & Martinez, A. C.
(1999). Chemokine control of HIV-1 infection. Nature 400(6746), 723−724.
M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371
Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., Fernandez, S., Martin de Ana, A.,
Jones, D. R., et al. (2001). Chemokine receptor homo- or heterodimerization
activates distinct signaling pathways. Embo J 20(10), 2497−2507.
Mercier, J. F., Salahpour, A., Angers, S., Breit, A., & Bouvier, M. (2002). Quantitative
assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization
by bioluminescence resonance energy transfer. J Biol Chem 277(47), 44925−44931.
Milligan, G. (2004). G protein-coupled receptor dimerization: function and ligand
pharmacology. Mol Pharmacol 66(1), 1−7.
Milligan, G. (2006). G-protein-coupled receptor heterodimers: pharmacology, function
and relevance to drug discovery. Drug Discov Today 11(11–12), 541−549.
Milligan, G., Wilson, S., & Lopez-Gimenez, J. F. (2005). The specificity and molecular
basis of alpha1-adrenoceptor and CXCR chemokine receptor dimerization. J Mol
Neurosci 26(2–3), 161−168.
Morello, J. P., Petaja-Repo, U. E., Bichet, D. G., & Bouvier, M. (2000). Pharmacological
chaperones: a new twist on receptor folding. Trends Pharmacol Sci 21(12), 466−469.
Nelson, G., Chandrashekar, J., Hoon, M. A., Feng, L., Zhao, G., Ryba, N. J., et al. (2002). An
amino-acid taste receptor. Nature 416(6877), 199−202.
Nelson, G., Hoon, M. A., Chandrashekar, J., Zhang, Y., Ryba, N. J., & Zuker, C. S. (2001).
Mammalian sweet taste receptors. Cell 106(3), 381−390.
Ng, G. Y., Varghese, G., Chung, H. T., Trogadis, J., Seeman, P., O'Dowd, B. F., et al. (1997).
Resistance of the dopamine D2L receptor to desensitization accompanies the upregulation of receptors on to the surface of Sf9 cells. Endocrinology 138(10), 4199−4206.
Nishi, A., Liu, F., Matsuyama, S., Hamada, M., Higashi, H., Nairn, A. C., et al. (2003).
Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling. Proc
Natl Acad Sci U S A 100(3), 1322−1327.
Nutt, J. G. (1990). Levodopa-induced dyskinesia: review, observations, and speculations.
Neurology 40(2), 340−345.
Oney, T., & Kaulhausen, H. (1982). The value of the angiotensin sensitivity test in the early
diagnosis of hypertensive disorders in pregnancy. Am J Obstet Gynecol 142(1), 17−20.
Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., et al. (2001).
C-terminal interaction is essential for surface trafficking but not for heteromeric
assembly of GABA(b) receptors. J Neurosci 21(4), 1189−1202.
Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., et al. (2000).
Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289(5480),
739−745.
Parmentier, M. L., Prezeau, L., Bockaert, J., & Pin, J. P. (2002). A model for the functioning
of family 3 GPCRs. Trends Pharmacol Sci 23(6), 268−274.
Pfleger, K. D., & Eidne, K. A. (2005). Monitoring the formation of dynamic G-proteincoupled receptor–protein complexes in living cells. Biochem J 385(Pt 3), 625−637.
Pfleger, K. D., & Eidne, K. A. (2006). Illuminating insights into protein–protein
interactions using bioluminescence resonance energy transfer (BRET). Nat Methods
3(3), 165−174.
Pfleger, K. D., Seeber, R. M., & Eidne, K. A. (2006). Bioluminescence resonance energy
transfer (BRET) for the real-time detection of protein–protein interactions. Nat
Protoc 1(1), 337−345.
Pin, J. P., Kniazeff, J., Binet, V., Liu, J., Maurel, D., Galvez, T., et al. (2004). Activation
mechanism of the heterodimeric GABA(B) receptor. Biochem Pharmacol 68(8),
1565−1572.
Pin, J. P., Kniazeff, J., Liu, J., Binet, V., Goudet, C., Rondard, P., et al. (2005). Allosteric
functioning of dimeric class C G-protein-coupled receptors. Febs J 272(12), 2947−2955.
Pinna, A., Wardas, J., Simola, N., & Morelli, M. (2005). New therapies for the treatment of
Parkinson's disease: adenosine A2A receptor antagonists. Life Sci 77(26),
3259−3267.
Pouliot, L., Forest, J. C., Moutquin, J. M., Coulombe, N., & Masse, J. (1998). Platelet angiotensin
II binding sites and early detection of preeclampsia. Obstet Gynecol 91(4), 591−595.
Pralong, F. P., Gomez, F., Castillo, E., Cotecchia, S., Abuin, L., Aubert, M. L., et al. (1999).
Complete hypogonadotropic hypogonadism associated with a novel inactivating
mutation of the gonadotropin-releasing hormone receptor. J Clin Endocrinol Metab
84(10), 3811−3816.
Presland, J. (2005). Identifying novel modulators of G protein-coupled receptors via
interaction at allosteric sites. Curr Opin Drug Discov Dev 8(5), 567−576.
Prinster, S. C., Hague, C., & Hall, R. A. (2005). Heterodimerization of g protein-coupled
receptors: specificity and functional significance. Pharmacol Rev 57(3), 289−298.
Rasmussen, S. G., Choi, H. J., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., et al.
(2007). Crystal structure of the human beta2 adrenergic G-protein-coupled receptor.
Nature 450(7168), 383−387.
Reggio, P. H. (2006). Computational methods in drug design: modeling G proteincoupled receptor monomers, dimers, and oligomers. Aaps J 8(2), E322−E336.
Rios, C., Gomes, I., & Devi, L. A. (2006). Mu opioid and CB1 cannabinoid receptor
interactions: reciprocal inhibition of receptor signaling and neuritogenesis. Br J
Pharmacol 148(4), 387−395.
Rodriguez-Frade, J. M., del Real, G., Serrano, A., Hernanz-Falcon, P., Soriano, S. F., Vila-Coro,
A. J., et al. (2004). Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in
trans on the CCR2 chemokine receptor. Embo J 23(1), 66−76.
Romano, C., Miller, J. K., Hyrc, K., Dikranian, S., Mennerick, S., Takeuchi, Y., et al. (2001).
Covalent and noncovalent interactions mediate metabotropic glutamate receptor
mGlu5 dimerization. Mol Pharmacol 59(1), 46−53.
Romano, C., Yang, W. L., & O'Malley, K. L. (1996). Metabotropic glutamate receptor 5 is a
disulfide-linked dimer. J Biol Chem 271(45), 28612−28616.
Sachpatzidis, A., Benton, B. K., Manfredi, J. P., Wang, H., Hamilton, A., Dohlman, H. G., et al.
(2003). Identification of allosteric peptide agonists of CXCR4. J Biol Chem 278(2),
896−907.
Salahpour, A., Angers, S., Mercier, J. F., Lagace, M., Marullo, S., & Bouvier, M. (2004).
Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell
surface targeting. J Biol Chem 279(32), 33390−33397.
Sassin, J. F. (1975). Drug-induced dyskinesia in monkeys. Adv Neurol 10, 47−54.
371
Schiffmann, S. N., Jacobs, O., & Vanderhaeghen, J. J. (1991). Striatal restricted adenosine
A2 receptor (RDC8) is expressed by enkephalin but not by substance P neurons: an
in situ hybridization histochemistry study. J Neurochem 57(3), 1062−1067.
Schlyer, S., & Horuk, R. (2006). I want a new drug: G-protein-coupled receptors in drug
development. Drug Discov Today 11(11–12), 481−493.
Schroeder, J. P., Overstreet, D. H., & Hodge, C. W. (2005). The mGluR5 antagonist MPEP
decreases operant ethanol self-administration during maintenance and after
repeated alcohol deprivations in alcohol-preferring (P) rats. Psychopharmacology
(Berl) 179(1), 262−270.
Scruggs, J. L., Schmidt, D., & Deutch, A. Y. (2003). The hallucinogen 1-[2,5-dimethoxy-4iodophenyl]-2-aminopropane (DOI) increases cortical extracellular glutamate
levels in rats. Neurosci Lett 346(3), 137−140.
Shah, D. M. (2005). Role of the renin–angiotensin system in the pathogenesis of
preeclampsia. Am J Physiol Renal Physiol 288(4), F614−F625.
Soria, G., Castane, A., Ledent, C., Parmentier, M., Maldonado, R., & Valverde, O. (2006).
The lack of A2A adenosine receptors diminishes the reinforcing efficacy of cocaine.
Neuropsychopharmacology 31(5), 978−987.
Springael, J. Y., Le Minh, P. N., Urizar, E., Costagliola, S., Vassart, G., & Parmentier, M.
(2006). Allosteric modulation of binding properties between units of chemokine
receptor homo- and hetero-oligomers. Mol Pharmacol 69(5), 1652−1661.
Staudinger, R., Wang, X., & Bandres, J. C. (2001). Allosteric regulation of CCR5 by guanine
nucleotides and HIV-1 envelope. Biochem Biophys Res Commun 286(1), 41−47.
Stromberg, I., Popoli, P., Muller, C. E., Ferre, S., & Fuxe, K. (2000). Electrophysiological and
behavioural evidence for an antagonistic modulatory role of adenosine A2A
receptors in dopamine D2 receptor regulation in the rat dopamine-denervated
striatum. Eur J Neurosci 12(11), 4033−4037.
Takeda, S., Kadowaki, S., Haga, T., Takaesu, H., & Mitaku, S. (2002). Identification of
G protein-coupled receptor genes from the human genome sequence. FEBS Lett
520(1–3), 97−101.
Tateyama, M., Abe, H., Nakata, H., Saito, O., & Kubo, Y. (2004). Ligand-induced
rearrangement of the dimeric metabotropic glutamate receptor 1alpha. Nat Struct
Mol Biol 11(7), 637−642.
Terrillon, S., & Bouvier, M. (2004). Roles of G-protein-coupled receptor dimerization.
EMBO Rep 5(1), 30−34.
Terrillon, S., Durroux, T., Mouillac, B., Breit, A., Ayoub, M. A., Taulan, M., et al. (2003).
Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and
heterodimers during biosynthesis. Mol Endocrinol 17(4), 677−691.
Tessari, M., Pilla, M., Andreoli, M., Hutcheson, D. M., & Heidbreder, C. A. (2004).
Antagonism at metabotropic glutamate 5 receptors inhibits nicotine- and cocainetaking behaviours and prevents nicotine-triggered relapse to nicotine-seeking.
Eur J Pharmacol 499(1–2), 121−133.
Testa, C. M., Standaert, D. G., Landwehrmeyer, G. B., Penney, J. B., Jr., & Young, A. B.
(1995). Differential expression of mGluR5 metabotropic glutamate receptor mRNA
by rat striatal neurons. J Comp Neurol 354(2), 241−252.
Ulloa-Aguirre, A., Janovick, J. A., Leanos-Miranda, A., & Conn, P. M. (2004). Misrouted cell
surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism. Hum Reprod Updat 10(2), 177−192.
Vaidehi, N., Floriano, W. B., Trabanino, R., Hall, S. E., Freddolino, P., Choi, E. J., et al. (2002).
Prediction of structure and function of G protein-coupled receptors. Proc Natl Acad
Sci U S A 99(20), 12622−12627.
Vila-Coro, A. J., Mellado, M., Martin de Ana, A., Lucas, P., del Real, G., Martinez, A. C., et al.
(2000). HIV-1 infection through the CCR5 receptor is blocked by receptor
dimerization. Proc Natl Acad Sci U S A 97(7), 3388−3393.
Vila-Coro, A. J., Rodriguez-Frade, J. M., Martin De Ana, A., Moreno-Ortiz, M. C., Martinez,
A. C., & Mellado, M. (1999). The chemokine SDF-1alpha triggers CXCR4 receptor
dimerization and activates the JAK/STAT pathway. Faseb J 13(13), 1699−1710.
Waldhoer, M., Fong, J., Jones, R. M., Lunzer, M. M., Sharma, S. K., Kostenis, E., et al. (2005).
A heterodimer-selective agonist shows in vivo relevance of G protein-coupled
receptor dimers. Proc Natl Acad Sci U S A 102(25), 9050−9055.
Wang, D., Sun, X., Bohn, L. M., & Sadee, W. (2005). Opioid receptor homo- and
heterodimerization in living cells by quantitative bioluminescence resonance
energy transfer. Mol Pharmacol 67(6), 2173−2184.
Wessendorf, M. W., & Dooyema, J. (2001). Coexistence of kappa- and delta-opioid
receptors in rat spinal cord axons. Neurosci Lett 298(3), 151−154.
White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., et al. (1998).
Heterodimerization is required for the formation of a functional GABA(B) receptor.
Nature 396(6712), 679−682.
Xie, Z., Bhushan, R. G., Daniels, D. J., & Portoghese, P. S. (2005). Interaction of bivalent
ligand KDN21 with heterodimeric delta–kappa opioid receptors in human
embryonic kidney 293 cells. Mol Pharmacol 68(4), 1079−1086.
Xu, H., Staszewski, L., Tang, H., Adler, E., Zoller, M., & Li, X. (2004). Different functional
roles of T1R subunits in the heteromeric taste receptors. Proc Natl Acad Sci U S A
101(39), 14258−14263.
Yao, L., McFarland, K., Fan, P., Jiang, Z., Ueda, T., & Diamond, I. (2006). Adenosine A2a
blockade prevents synergy between mu-opiate and cannabinoid CB1 receptors and
eliminates heroin-seeking behavior in addicted rats. Proc Natl Acad Sci U S A 103(20),
7877−7882.
Zeng, B. Y., Pearce, R. K., MacKenzie, G. M., & Jenner, P. (2000). Alterations in preproenkephalin
and adenosine-2a receptor mRNA, but not preprotachykinin mRNA correlate with
occurrence of dyskinesia in normal monkeys chronically treated with l-DOPA. Eur J
Neurosci 12(3), 1096−1104.
Zhao, G. Q., Zhang, Y., Hoon, M. A., Chandrashekar, J., Erlenbach, I., Ryba, N. J., et al.
(2003). The receptors for mammalian sweet and umami taste. Cell 115(3), 255−266.
Zhu, X., & Wess, J. (1998). Truncated V2 vasopressin receptors as negative regulators of
wild-type V2 receptor function. Biochemistry 37(45), 15773−15784.