However, at least in part because expression of a single GPCR cDNA in heterologous cell systems generally resulted in production of a ligand-binding site with the expected pharmacology a
Trang 1Methods to monitor the quaternary structure of
G protein-coupled receptors
Graeme Milligan1and Michel Bouvier2
1 Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, UK
2 Biochemistry, Universite de Montreal, Quebec, Canada
Introduction
In recent times, the view that G protein-coupled
recep-tors (GPCRs) are single polypeptides that function as
isolated monomers has been challenged, and largely
supplanted, by results consistent with the existence of
GPCRs as dimers or higher-order oligomers Data in
support of GPCRs being able to form dimers or
oligo-mers had been scattered throughout the literature [1]
However, at least in part because expression of a single
GPCR cDNA in heterologous cell systems generally
resulted in production of a ligand-binding site with the
expected pharmacology and the capacity to activate
G proteins and hence initiate signal transduction, little
thought was given to potential quaternary structure
This is despite the fact that dimerization of proteins,
as a route to function, is one of the most common
themes in biology [2] This had been well established
previously for other classes of transmembrane recep-tors Key to a broad appreciation of the potential of dimerization for GPCR function was the demonstra-tion that the c-aminobutyric acid (GABA)B-receptor, long recognized as a GPCR, was an obligate hetero-dimer [3,4] Even though the GABABR1 polypeptide
is a seven transmembrane protein able to bind both GABA and a GABAB receptor antagonist [5], when expressed alone this polypeptide is not transported to the cell surface, does not have the expected characteris-tics and affinity for binding of agonists and cannot activate G protein signalling Cell surface delivery and function requires the coexpression of a second, closely related seven transmembrane polypeptide, the GABABR2, which although unable to bind GABAB receptor ligands, allows cell surface delivery of the GABABR1 The heterodimeric complex then functions with pharmacology akin to the native receptor
Subse-Keywords
dimerization; functional reconstitution;
G protein coupled receptor;
immuno-precipitation; resonance energy transfer
Correspondence
G Milligan, Davidson Building, University of
Glasgow, Glasgow, G12 8QQ Scotland, UK
Fax: +44 141330 4620
Tel: +44 141330 5557
E-mail: g.milligan@bio.gla.ac.uk
(Received 16 February 2005, accepted
4 April 2005)
doi:10.1111/j.1742-4658.2005.04731.x
A wide range of approaches has been applied to examine the quaternary structure of G protein-coupled receptors, the basis of such protein–protein interactions and how such interactions might modulate the pharmacology and function of these receptors These include coimmunoprecipitation, var-ious adaptations of resonance energy transfer techniques, functional com-plementation studies and the analysis of ligand-binding data Each of the available techniques has limitations that restrict interpretation of the data However, taken together, they provide a coherent body of evidence indica-ting that many, if not all, G protein-coupled receptors exist and function
as dimer⁄ oligomers Herein we assess the widely applied techniques and discuss the relative benefits and limitations of these approaches
Abbreviations
BRET, bioluminescence resonance energy transfer; DOP, delta opioid peptide; eYFP, enhanced yellow fluorescent protein; FRET,
fluorescence resonance energy transfer; GPCRs, G protein-coupled receptors; G(C,Y)FP, green (cyan, yellow) fluorescent protein;
NEM, N-ethyl maleimide.
Trang 2quently, it has become clear that sweet and umami
taste responses are also generated by obligate
hetero-dimer pairs of T1 taste receptors [6] and both
homo-and heterodimerization of other family C receptors is
firmly established This topic is covered in the
accom-panying review by Pin and colleagues [7] Analysis of
the importance of quaternary structure for function of
the rhodopsin-like family A GPCRs has been more
recalcitrant to analysis, as examples of obligate
hetero-dimerization akin to the GABAB receptor have not
been noted, although there are reports, for example, of
the requirement for heterodimeric interactions to allow
cell surface delivery of certain a1-adrenoceptor
sub-types [8] and olfactory receptors [9] Nevertheless, it is
now generally accepted that family A GPCRs can exist
as dimers and⁄ or higher order oligomers [10–12] A
growing database on the formation of GPCR dimers
prior to membrane delivery [13–15] and the potential
that GPCRs can bind to single heterotrimeric G
pro-teins as dimers [16] is certainly consistent with such
a receptor dimer model However, the importance of
dimerization for receptor function and regulation in
physiological systems remains to be demonstrated
unambiguously A significant number of recent reviews
have covered these topics [11,12,17] and the purpose of
the current piece is to critically evaluate the
approa-ches used to explore dimerization of class A GPCRs
Coimmunoprecipitation
The ability to differentially epitope-tag GPCRs has
been central to the widespread use of
coimmunoprecipi-tation following coexpression of two forms of the same
GPCR in heterologous expression systems Initially
Hebert et al [18] coexpressed c-myc- and HA-tagged
forms of the b2-adrenoceptor in insect Sf9 cells and
demonstrated interaction between the two forms of the
receptor as both were present in immunoprecipitates
generated using either anti-HA or anti-c-myc Igs These
studies also provided a series of key controls (a) in a
lack of cross-reaction between the antibodies and the
alternately tagged form of the receptor and (b) in a lack
of coimmunoprecipitation of the HA-tagged b2
-adreno-ceptor when coexpressed with a c-myc-tagged form of
the M2muscarinic acetylcholine receptor These studies
also noted the resistance to monomerization during
SDS⁄ PAGE of at least a proportion of the
coimmuno-precipitated receptors; this is a commonly observed
feature in many such studies Despite such apparent
receptor dimerization (and indeed multimerization)
being detected following SDS⁄ PAGE resolution of
simple membrane preparations of transfected cells and
even native tissues [19], detection of SDS-resistant
higher-order structures was reminiscent of protein aggregation, a well-appreciated potential artefact resulting from membrane protein solubilization There-fore, the hydrophobicity of the transmembrane domains of GPCRs raised concerns that apparent coimmunoprecipitation might reflect little more than nonspecific aggregation following detergent extraction
of proteins from cells and membranes This resulted in widespread incorporation of mixed samples into co-immunoprecipitation studies to counter this concern
In such experiments detergent extracts of cells or membranes, each expressing only one of the tagged GPCRs, are combined prior to the immunoprecipitation step This is generally considered a sound means to elim-inate nonspecific aggregation as a potential explanation
of spurious coimmunoprecipitation (Fig 1)
Coimmunoprecipitation has been of particular use in efforts to identify heterodimeric interactions between GPCRs [20] However, coimmunoprecipitation studies such as that of Salim et al [21] intimated widespread interactions between coexpressed GPCRs and, assu-ming that interaction between different GPCRs is not
a purely stochastic process, raised further questions about the use of coimmunoprecipitation in the absence
of further supporting evidence of protein–protein
inter-Fig 1 Differentially epitope-tagged forms of the a1b-adrenoceptor have to be coexpressed to be coimmunoprecipitated N-terminally Flag- or c-myc-tagged forms of the hamster a 1b -adrenoceptor were expressed individually (flag, myc) or coexpressed (flag + myc) in HEK293 cells Samples expressing each form of the receptor were also mixed (mix) Lysates of cells were resolved by SDS ⁄ PAGE and immunoblotted to detect expression of each form, or the samples were immunoprecipitated using anti-Flag Ig, resolved by SDS ⁄ PAGE and then immunoblotted to detect c-myc immunoreactivity Coimmunoprecipitation was only observed from samples co-expressing the two forms Data are modified from [35].
Trang 3actions A key issue that remains overlooked in many
published coimmunoprecipitation studies is whether
the samples are fully solubilized prior to the
immuno-precipitation step In many studies, centrifugation after
detergent extraction is limited to a short period in a
bench top microcentrifuge, with force in the region of
15 000 g This is far too limited to ensure production
of a fully soluble fraction and it is suggested that, as
in many other protocols, centrifugation should be for
at least 60 min and > 100 000 g As an alternative,
passage of the detergent-generated extract through a
0.22 lm filter would ensure removal of membrane
frag-ments that might contain both coexpressed forms of a
GPCR that are not actually physically in contact The
addition of alkylating agents such as NEM and
iodo-acetamide in the lysis and solubilization buffers is also
recommended in order to prevent the formation of
spurious intermolecular disulfide bonds that could be
favoured by the oxidizing condition associated with
cell lysis Notwithstanding these caveats, there is no
doubt that coimmunoprecipitation remains as the
starting point and, indeed at the heart, of many GPCR
dimerization studies Indeed, despite the general
pau-city of specific and high affinity anti-GPCR sera,
care-fully controlled coimmunoprecipitation studies remain
the most practical means to explore, in particular,
GPCR heterodimerization in native tissues and can
provide key information to support GPCR
dimeriza-tion⁄ oligomerization
Coimmunoprecipitation of GPCRs, subsequent to
cross-linking with cell-impermeant cross-linking agents
has also provided a useful means to detect GPCR
dimers⁄ oligomers in the plasma membrane The
intro-duction of cysteine residues into specific locations in
transmembrane domains of class A GPCRs, that then
allow cross-linking by bi-functional reagents, is an
extension of the basic cross-linking and
immunopre-cipitation strategy but has the added advantage of
assisting identification of potential protein–protein
interaction sites in GPCR dimers [22,23] and
determin-ing whether ligand-binddetermin-ing modulates the
organiza-tional structure of a GPCR dimer⁄ oligomer Although
the concept of coimmunoprecipitation can be extended
easily to studies that employ cotransfection with three
or more forms of the same GPCR, each incorporating
a different tag, this has only been employed to date in
a limited manner In one such example, where c-myc-,
FLAG- and HA-tagged forms of the M2 muscarinic
receptor were coexpressed in insect Sf9 cells, Park and
Wells [24] were able to immunoprecipitate complexes
containing all three epitope tags, hence providing
evidence for complexes containing at least three GPCR
monomers
Resonance energy transfer studies Fluorescence resonance energy transfer The green fluorescent protein (GFP) from Victoria aequoria has found widespread use in cell biology to monitor the expression and distribution of proteins tagged with this polypeptide Mutation of this protein has produced a range of variants with altered spectral characteristics, and pairs of these are able to function
as fluorescence resonance energy transfer (FRET) part-ners The most widely used pairing is a cyan fluores-cent protein (CFP) as energy donor and a yellow fluorescent protein (YFP) as an energy acceptor, but other pairings are also practical Obvious attractions in the use of FRET for studies of GPCR dimerization⁄ oligomerization include the capacity to monitor pro-tein–protein interactions in intact living cells both in cell populations and single cells It can be combined with cell imaging and photo-bleaching protocols to examine the cellular location of such interactions in specific subcellular compartments [25–27] Photo-bleaching of the energy acceptor construct also provides an important control to demonstrate that FRET-signals actually reflect energy transfer Photo-bleaching of the acceptor should result in increased fluorescence output from the energy donor as well as destruction of signal from the acceptor as no energy transfer can then occur This is a particularly import-ant control as direct excitation of the energy acceptor
by the exogenous light source in sensitization FRET experiments can generate background signals that can
be mistakenly interpreted as real FRET Also, the spectral overlap between the donor and acceptor emis-sion spectra needs to be properly monitored and con-trolled to determine the legitimate FRET signal These are not trivial issues as the contribution of the back-ground signals can vary considerably depending on the relative expression levels of the FRET partners and the band pass width of the fluorescence excitation and emis-sion detection systems
Although some wild-type GFPs have the tendency
to oligomerize, a characteristic that could promote artefactual oligomerization of the proteins attached to them, variants such as CFP and YFP that have lower affinity for one another, have been used successfully to monitor the quaternary structure of several GPCRs FRET between CFP and YFP covalently fused to dis-tinct GPCRs should occur only when the FRET part-ners are brought within 100 A of each other; a distance that falls within the dimensions anticipated for GPCR dimers⁄ oligomers [28] The occurrence of FRET between CFP- and YFP-fused GPCRs has
Trang 4therefore been interpreted as strong evidence for the
existence of dimers or oligomers in living cells As with
all approaches based on Fo¨rsters’ principles of
reson-ance energy transfer, the efficiency of transfer is
deter-mined both by the orientation of the energy transfer
partners and by the distance between them [28] The
fact that the efficiency of transfer varies with the
6th-power of the distance between the fluorophores
presents both opportunities and limitations for the
analysis of data (Fig 2) For instance, although the
FRET assays can be extremely sensitive detectors of
conformational changes that can reflect ligand-promoted
alteration in GPCR quaternary structure, the extent of
the D FRET observed for a given change is highly
dependent on the initial distance between the energy
donor and acceptor For example, if constitutive
dime-rization of two GPCR FRET-tagged constructs brings
the energy transfer partners into very close proximity,
the energy transfer efficiency should be close to
maxi-mal and thus, a ligand-induced alteration in distance
between the energy-transfer partners would be poorly
reported (Fig 2) In contrast, if the initial distance
between energy donor and acceptor is within the
region of the FRET R0 (i.e.: the distance between the
donor and acceptor leading to 50% of the maximal
transfer efficiency) where the Denergy transfer ⁄
Ddis-tance is maximal, the same conformational change
would lead to largeDFRET (Fig 2) A range of
fluor-escence-based techniques, including intra- and
inter-molecular FRET, have been used to demonstrate conformational changes in GPCR monomers upon agonist binding [29,30] and recent studies have expan-ded this to show that an agonist bound protomer of the leukotriene B4, BLT1 receptor produces a confir-mation change in the partner GPCR of the homodimer [31] It follows that reports of agonist-induced altera-tions in GPCR quaternary structure based only on res-onance energy transfer studies may simply reflect very small conformational alterations in the GPCR that are amplified by the high sensitivity of the resonance energy transfer responses to distance and orientation [32] (Fig 2) Thus, a large increase or decrease in FRET values observed upon ligand-binding cannot be interpreted, as is too often the case, as an indication of ligand-promoted dimer formation or dissociation The occurrence of FRET between fluorophore-tagged GPCRs could result both from the formation
of dimers or of higher oligomeric structures Unfortu-nately, the currently available FRET techniques do not permit one to distinguish easily between these two possibilities However, recent studies employing atomic force microscopy to visualize the organizational struc-ture of rhodopsin in murine rod outer segment discs have shown higher order arrays of GPCRs [33,34] and analysis of the results of GPCR transmembrane inter-face mapping for both the complement C5a receptor [23] and the a1b-adrenoceptor [35] have suggested ways
in which these GPCRs may organize into higher order structures Although not yet reported in relation to GPCR quaternary structure, three component FRET systems based on serial energy transfer from cyan to yellow to red fluorescent proteins are available [36] These and related systems may be invaluable to explore the potential of higher-order GPCR oligomeri-zation They are, however, likely to suffer from low sensitivity and thus, at least in the short term, the need
to express rather high levels of each component may raise concerns in relation to the specificity of inter-actions observed
Quantitation of the fraction of a GPCR existing as monomer vs dimer or higher-order oligomer remains
a challenging task Efforts to assess this for homo-dimers of neuropeptide Y receptor subtypes were based on FRET efficiency of appropriately tagged GPCRs compared to the FRET signal produced by a positive control generated by fusing together the two FRET partner fluorescent proteins [37] A similar attempt was made for the b2-adrenoceptor homodimer using another resonance energy transfer approach known as bioluminescence resonance energy transfer (BRET, see below) and using theoretical maximal transfer values as the 100% dimer reference [38]
How-Fig 2 Variation in resonance energy transfer efficiency as a
func-tion of distance between energy donor and acceptor Relafunc-tionship
between the energy transfer efficiency (Y-axis) and the distance
between the energy donor and acceptor (X-axis) The distance is
expressed as the ratio of the distance ‘r’ separating the donor and
the acceptor over the distance resulting in 50% of the maximal
efficacy (R0) As indicated by the Fo¨rster equation, the efficiency of
transfer varies inversely with the 6th-power of the distance The
extreme steepness of the relationship results in large changes in
RET for relatively small modifications in distance around the R0 In
contrast, large changes in the distance may not result in any RET
change when the distance is much smaller than the R 0
Trang 5ever, the values obtained, which were in the region of
20–40% as dimers for the first study and greater than
85% in the dimeric form for the latter, required a
range of assumptions, including the fact that the
recep-tors existed as dimers and not larger oligomers
There-fore, such estimates must be viewed as extremely
uncertain and require further experimental
confirma-tion As indicated above, pictures of the structural
organization of rhodopsin in the disks from rod outer
segments from mice suggest a much higher level of
dimerization⁄ oligomerization [33] However, the high
concentration of rhodopsin in rods may impose order
and structural organization that is not required for
other class A GPCRs Attempts to explore this by
employing discs from heterozygote rhodopsin
knock-out mice simply resulted in rod knock-outer segments with
reduced volume rather than an alteration in the pattern
of interactions between rhodopsin monomers [34]
Time-resolved FRET
The capacity to image FRET signals in single cells and
in specific cellular compartments, including the plasma
membrane, provides a means to confirm interactions
between GPCR monomers at the surface of individual
cells [25–27] A distinct FRET-based approach to
mon-itor interactions between GPCRs at the cell surface in
cell populations is based on time-resolved FRET using
anti-epitope tag Igs labelled with suitable FRET
accep-tor and donors [39] N-terminally epitope-tagged forms
of GPCRs should only be accessible to such antibodies
in intact cells if they have been delivered successfully
and inserted into the plasma membrane The use of
antibodies labelled with Europium chelates as energy
donor allows the use of ‘time-resolved’ protocols,
where short-lived fluorescence (up to some 50 ls)
derived from excitation of endogenous fluorophores is
allowed to decay before the long lived time-resolved
FRET signal is monitored over the ensuing 100–
200 ls This generates improved signal-to-noise ratios
Although employed in a limited number of studies to
date [40–43], limiting signals to GPCRs at the cell
sur-face using this approach has particular advantages for
analysis of GPCRs expressed in heterologous systems
where a significant fraction of the expressed GPCR(s)
can often be shown to be inside the cell, presumably
within the endoplasmic reticulum or as protein that
has failed to pass cellular quality control mechanisms
and is destined for degradation Time-resolved FRET
pairings have also been used in membrane fractions
generated by sucrose density sedimentation to detect
dimers⁄ oligomers of a1a-adrenoceptors in all
mem-brane fractions containing this GPCR as measured by
[3H]antagonist binding studies [43] As this receptor displays a high level of constitutive, agonist-independ-ent internalization and recycling [44,45], the FRET data are certainly consistent with at least a fraction of the population of this receptor existing as a dimer⁄ oligomer at all stages in its life history The major potential caveats for time-resolved FRET studies relates to the obligatory use of antibodies Appropriate controls need to be performed to make sure that oligo-merization of the partners is not promoted by the bivalent nature of the antibodies In studies assessing the effect of ligand-binding on the quaternary structure
of the receptors, the possibility that the antibodies could inhibit binding to the receptor also needs to
be controlled Finally, the presence of an antibody between the fluorophores and the proteins of interest, increases the uncertainty concerning the minimum and maximum FRET permissive distance
Bioluminescence resonance energy transfer Conceptually similar to FRET, except that energy is donated to a fluorescent protein energy acceptor by luciferase-mediated oxidation of a substrate, biolumi-nescence resonance energy transfer (BRET) has become almost as popular an approach as FRET The various benefits of BRET compared to FRET, partic-ularly the lack of requirement of a light source to excite the energy donor, have been discussed previ-ously in more specialized articles [28] In particular, we should note that the lack of potential direct excitation
of the energy acceptor that can occur in FRET experi-ments (see above) greatly simplifies controlling for the background signals However, a clear limitation of BRET is that currently it is not sufficiently sensitive to allow high resolution, single cell imaging and analysis and therefore cannot usefully report on the subcellular location of the signal Although single cell BRET signals resulting from the homodimerization of the
b2-adrenoceptor and melatonin MT1 and MT2 homodimers could be detected [32], obtaining subcellu-lar resolution will require the development of more sensitive cameras or of luminescence donors with higher light output In initial BRET studies, the energy transfer pairing was Renilla luciferase and enhanced YFP (eYFP) with h-coelenterazine acting as luciferase substrate However, the overlap between the spectrum generated via this enzymic activity of luciferase and the emission of light from eYFP subsequent to energy transfer is substantial, resulting in a relat-ively high background signal that needs to be carefully controlled As a means to improve this, the Renilla luciferase substrate DeepBlueC, which results in
Trang 6emis-sion of light that is substantially blue-shifted in
com-parison to the oxidation of h-coelenterazine, has been
used The wavelength of the light emitted by the
oxida-tion of DeepBlueC is not well aligned for energy
trans-fer to eYFP but is suited for energy transtrans-fer to the
modified fluorescent protein GFP2 and the output
from GFP2 is relatively well resolved from the Renilla
luciferase⁄ DeepBlueC oxidation spectrum [46] (Fig 3)
This results in improved signal-to-noise ratios when
employing BRET2 The major limitation of BRET2 is
the poor quantum efficiency associated with oxidation
of DeepBlueC resulting in much lower absolute signals
that thus necessitates higher GPCR expression levels
With both their limitations and advantages, BRET1 or
BRET2 can be best suited to study specific proteins of
interest Taking advantage of the distinct spectral
char-acteristics of the two BRET generations in the same
experiment, Perroy et al [47] were able to monitor the
interaction between three partners simultaneously in
the same cell population Such combinations between
different types of resonance energy transfer will cer-tainly be increasingly used to examine the stoichio-metry of GPCR complexes
Following the initial report employing BRET that examined interactions between Renilla luciferase- and eYFP-tagged forms of the b2-adrenoceptor [48], BRET has been used, to date, predominantly in ‘single point’ assays In these, single amounts of Renilla luciferase and fluorescent protein-tagged forms of a single GPCR, to study homodimerization⁄ oligomerization, or pairs of GPCRs, to study potential heterodimeriza-tion⁄ oligomerization are cotransfected into host cells However, as with coimmunoprecipitation and FRET studies, the intensity of the signal obtained does not provide a measure of absolute, or even relative, inter-action affinities between the GPCRs linked to the BRET partners, because the extent of BRET signal is dependent upon both distance between the energy donor and acceptor and their orientation The most significant advance in this area since then has been the introduction of ‘saturation’ BRET by Mercier and colleagues [38] Taking advantage of the possibility of expressing differing amounts of energy donor and acceptor-linked GPCRs in heterologous cells, these studies demonstrated that an asymptotic approach to
a maximal BRET signal was obtained with increasing energy acceptor⁄ energy donor ratios Saturation curves
so generated resemble ligand⁄ GPCR binding curves and, as such, the ratio of energy acceptor⁄ energy donor that generates half-maximal BRET signal can provide a useful measure of the relative interaction affinities of the GPCRs linked to the BRET reagents For example, although measurable BRET signals can be recorded following coexpression of a DOP opioid receptor-Renilla luciferase⁄ a1A -adrenoceptor-GFP2 BRET pair in HEK293 cells that is commensur-ate with the signals obtained for each of these two GPCR homodimers, saturation BRET studies indica-ted that the interaction affinity of this heterodimer⁄ oligomer pairing was some 75-fold lower than for the
a1A-adrenoceptor homodimer⁄ oligomer [43] This sug-gests that such a heteromeric pairing would be unlikely
to have physiological significance, even if interactions can be monitored in heterologous expression systems Although it was noted in early studies that hetero-dimeric interactions between structurally homologous receptors were of higher affinity than interactions between more distantly related class A GPCRs [46] it
is not inevitable that homodimer interactions will be of higher affinity than heterodimeric interactions Indeed,
in the studies of Ramsay et al [46] hetero interactions between the closely related KOP and DOP opioid receptors were reported to be at least as high affinity
Fig 3 Spectral properties of the energy donor and acceptor for the
two generations of BRET Schematic representation of the
emis-sion spectra for Renilla luciferase (Rluc) using coelenterazine H
(BRET 1 ) or DeepBlue Coelenterazine (BRET 2 ) as substrates and of
the overlap between the emission spectrum of Rluc and the
excita-tion spectrum of the fluorescent energy acceptors YFP for BRET 1
and GFP 2 for BRET 2 The overlap is more complete for BRET 2 than
BRET1favouring a better transfer Also shown are the emission
spectra of GFP 2 and YFP A better separation from the RLuc
emis-sion is obtained with GFP 2 in BRET 2 leading to a better
signal-to-noise ratio Not shown on this figure is the fact that the output
of light is much smaller for DeepBlue Coelenterazine than for
coelenterazine H leading to a lower detection sensitivity for BRET 2
than BRET1[46].
Trang 7as KOP receptor homo interactions More recent
studies are also consistent with melatonin MT1⁄ MT2
heterodimers being generated with higher affinity than
MT2 receptor homodimers [49] Such saturation BRET
studies have also been combined with deliberate
varia-tion in expression levels to demonstrate that potential
artefacts arising from physical crowding of GPCRs
and the production of so called ‘bystander’ BRET
(Fig 4) that does not reflect quaternary structure
inter-actions, is only produced with levels of expression in
the region of 25 pmolÆmg)1membrane protein [38] and
thus well beyond the range of expression normally
used in such studies
In the available reports employing saturation BRET, the energy acceptor⁄ energy donor ratio is generally reported simply as a fluorescence : luminescence ratio However, when well validated GPCR radioligands are available, attempts have been made to generate stand-ard curves of GPCR ligand-binding site number against fluorescence or luminescence signals to allow absolute quantitatation of energy acceptor⁄ energy donor ratio [38,43] Although luminescence and fluor-escence signals increase in a linear fashion with ligand-binding site number, surprisingly, the signal per GPCR binding site has not been equal for different GPCRs [38,43] It is not currently possible to establish the
A
B
Fig 4 BRET 2 levels as a function of energy donor and acceptor concentrations (A) BRET2saturation performed in cells coex-pressing a constant level of a GPCR fused
to the energy donor Rluc and increasing concentration of a GPCR fused to the energy acceptor GFP 2 The BRET levels are plotted as a function of the GPCR– GFP : GPCR–Rluc ratio In the case of speci-fic dimerization between the two GPCRs, a classical hyperbolic increase in the BRET signal that rapidly saturates is observed BRET 50 represents the GPCR–
GFP : GPCR–Rluc ratio leading to 50% of the maximal BRET and reflects the relative affinity of the BRET partners for one another In the case of random collisions between the BRET partners, bystander BRET leads to a quasi-linear relationship that will eventually saturate only at very high GPCR-GFP ⁄ GPCR-Rluc ratios (B) BRET 2
measurements performed in cells expres-sing increaexpres-sing concentrations of GPCR– Rluc and GPCR–GFP 2 while maintaining a constant GPCR–GFP 2 : GPCR–Rluc ratio of
1 In the case of specific GPCR dimers,
BRET levels are constant for a wide range
of total concentration since a constant pro-portion of GPCR–GFP2are engaged by a GPCR–Rluc In the case of random collisions between the BRET partners, the bystander BRET should increase as a function of the total receptor concentration for the entire range The increase in BRET observed for very high receptor expression levels > 45 pmolÆmg)1of protein most likely results from random collisions between GPCR dimers Adapted from [38].
Trang 8basis for this but it seems surprising that the enzyme
activity of Renilla luciferase or, even more so, the
fluorescence of variants of GFP should vary markedly
depending on which GPCR they are linked to A
poss-ible explanation would be that the difference in folding
rates between the GFP fluorophore (or luciferase
cata-lytic site) and the binding sites of specific GPCRs
varies from one receptor to another It follows that the
amount of properly folded binding site⁄ folded
fluoro-phore would be an intrinsic property of the individual
GPCR-fusion construct Alternatively, it is certainly
the case that different, but individually well studied,
ligands can generate different Bmaxvalues for the same
GPCR in the same membrane preparation [50] and
thus that the proportion of receptor molecule recognized
by the radioligand may differ for different receptors
Functional complementation
If the coexpression of two nonequivalent and
nonfunc-tional mutants of a GPCR is both able and required
to reconstitute receptor ligand-binding and⁄ or
func-tion, this can provide evidence in favour of direct
pro-tein–protein interactions and quaternary structure for
the active receptor Indeed, coexpression of two forms
of the angiotensin AT1 receptor that were unable to
bind angiotensin II or related ligands due to point
mutations in either transmembrane III or
transmem-brane region V restored ligand-binding [51] Such an
approach has also been used to explore mechanisms of
dimerization Theoretical models of GPCR
dimeriza-tion include both ‘contact’ and ‘domain-swap’ dimers
[52] Class A GPCRs can, at least to some degree,
re-assemble from coexpressed fragments The most
widely used strategy in this regard has been to split
GPCR sequences in two by cleavage within the third
intracellular loop Such experiments have resulted in
the N-terminus and transmembrane regions I–V and
transmembrane regions VI–VII and the C-terminal tail
being considered as units that are able to fold
inde-pendently (reference [53] in this series of reviews)
Indeed, generation of chimeric GPCRs based on this
general strategy provided some of the most elegant
early data in favour of molecular cross-talk between
GPCRs [54] Using the histamine H1 receptor as a
model, Bakker et al [41] showed that although single
point mutations in both transmembrane region III and
transmembrane region VI prevented binding of
antag-onist radioligands (including [3H]mepyramine),
coex-pression of the two mutants resulted in reconstitution
of [3H]mepyramine binding sites with the anticipated
pharmacological characteristics Conceptually this
should not be possible for a contact dimer in which
transmembrane domains are not exchanged but simply appose each other Although these studies provided support for domain swapping in the histamine H1 receptor, the Bmax obtained for [3H]mepyramine in the coexpression studies was only a small fraction of that obtained when the wild-type histamine H1 receptor was expressed [41] This may reflect that domain-swap and contact dimers are not mutually exclusive but can coexist, although domain-swapping may be energetic-ally less favourable The energetics of domain-swap-ping for a src homology 3 domain protein has been discussed recently [55] Domain-swapping may also contribute to GPCR heterodimerization and would be expected, almost as a matter of course, to alter the details of ligand pharmacology This is yet to be explored in significant detail but it is certainly true that, for example, coexpression of pairs of opioid receptor subtypes has been reported to result in the generation of novel pharmacology of ligand-binding and function [56,57] and this is also noted for other GPCR pairs [58] Although these changes could be explained by allosteric interactions between the pro-tomers of a contact dimer they could reflect domain-swap dimer interactions
In addition to the restoration of ligand-binding, studies that have used pairs of nonfunctional mutants
to restore GPCR signalling have produced data consis-tent with GPCR–GPCR interactions By generating mutants of the luteinizing hormone receptor that were either unable to bind ligand or unable to signal, although able to bind the agonist, Lee et al [59] were able to reconstitute agonist-mediated regulation of cAMP levels following coexpression of the two mutants The luteinizing hormone receptor, as with other GPCRs with related ligands, has an extended N-terminal region involved in ligand-binding As such, Lee et al [59] were able to consider the N-terminal exodomain and the seven transmembrane element endodomain as distinct entities in a manner equivalent
to the extracellular and transmembrane elements of class C GPCRs that have allowed elegant chimeric receptor approaches to understand the mechanism of signal transduction through obligate hetero-dimers [7] Interestingly, the pairs of complementary mutants des-cribed above have allowed demonstration of signal transduction through both transmembrane bundles when such mutant receptors do interact, supporting the notion of transactivation within receptor dimers [60] As a modification of the idea of pairs of exo- and endodomains in a single GPCR, Carrillo et al [61] extended the use of GPCR-G protein fusion proteins [62] to study both homo- and hetero dimerization of GPCRs Mutations were introduced to generate pairs
Trang 9with potential complementary function One of the
pairs had a mutation in the GPCR element of the
fusion that allowed ligand-binding but not G protein
activation, whereas the second fusion had a mutation
in the G protein element that prevented
agonist-media-ted guanine nucleotide exchange Although both were
nonfunctional when expressed individually,
coexpres-sion resulted in reconstitution of agonist-mediated
binding of [35S]GTPcS As [3H]antagonist binding was
unaltered by the mutations, then assays were
per-formed in conditions in which the total number of
GPCR-G protein fusion polypeptides were known and
this allowed estimates of the fraction of the constructs
that produced functional dimers [61] Intermolecular
interactions between the fusion proteins were
respon-sible for the generation of activity, rather than
activa-tion of endogenously expressed G proteins, as similar
results were produced when the pairs of constructs
were expressed in mouse embryo fibroblasts lacking
expression of the appropriate G proteins [61] One
potential caveat to this approach has been provided by
the work of Molinari et al [63] who used a similar
strategy to explore functions of fusions between the
DOP opioid receptor and Gao These workers reported
that the reconstitution of function might simply reflect
the higher concentration of G protein provided in the
membranes by the 1 : 1 GPCR : G protein
stoichio-metry of the fusion proteins allowing the G protein
fused to one receptor to interact with the G protein of
another receptor-G protein fusion that may or may
not be part of the same oligomer This is certainly an
important issue to consider and control However,
Carrillo et al [61] showed that providing extra,
non-GPCR-linked, G protein through attachment to a
truncated GPCR did not permit interaction with the
full length fusion proteins, ruling out the possibility that
the functional recovery resulted from membrane
crowd-ing and random collisions between fusion proteins
Ligand-binding studies
Classic monophasic ligand-binding isotherms as
gen-erally observed for the binding of antagonists is
con-sistent with the ligand-binding to a single class of
noninteracting sites As such, cooperative
characteris-tics of the binding of a antagonist is not compatible
with such a model and may be used to infer
pro-tein–protein interactions between GPCR monomers
This model has been most actively explored for the
M2 muscarinic acetylcholine receptor At least in
cer-tain conditions different 3H-labelled antagonists at
this GPCR display significantly different Bmax values
[64] Assuming that such effects do not reflect trivial
issues such as incorrect measurements of the specific activities of the two ligands, or the binding studies being performed under nonequilibrium conditions, such results are not compatible with the concept of
a single population of noninteracting sites Mathe-matical analysis of this data has been interpreted to suggest that the M2 muscarinic acetylcholine receptor may exist as at least a tetramer and that ligands can bind to the receptor sites in a cooperative manner [64] Related approaches have also been applied to the dop-amine D2receptor expressed in CHO cells Here, when
Na+was eliminated from binding studies, the observed
Bmax for [3H]raclopride was markedly lower than for [3H]spiperone, although this was not observed in the presence of Na+[50] Equally, in the absence of Na+, raclopride appeared to display negative cooperativity
on both its own binding and that of [3H]spiperone These results were modelled based on a GPCR dimer Although of considerable interest, these results may reflect altered interactions with other receptor partners such as the G proteins rather than the existence of dimerization This is particularly important considering that compounds considered as antagonists may display distinct inverse efficacy in the presence or absence of
Na+[65] and that ligand-binding studies using radiola-belled inverse agonists may lead to a different apparent
Bmax as a function of the spontaneous interaction with
G proteins [66] Despite this caveat, as long as they are performed in detail with appropriate controls, such ligand-binding studies offer means to examine GPCR protein–protein interactions in native tissues and with-out the need to manipulate the sequence of GPCRs to add tags required for detection in other types of assays
Conclusions The range of approaches that have been applied to assess the quaternary structure of class A GPCRs is both large and diverse Use of these techniques has led
to a general appreciation that GPCRs can certainly exist as dimers and⁄ or higher order oligomers and that such interactions may be central to delivery of cer-tainly some GPCRs to the cell surface and to their function However, given the nature of each of the widely used approaches it is still possible that observed interactions require the intermediacy and participation
of GPCR-interacting accessory or scaffolding proteins Indeed a very recent study [67] has suggested that MOP and DOP opioid receptor heterodimerization may require G protein interactions It is important that a significant range of different approaches is used
to explore such topics for any particular GPCR,
Trang 10because none of those currently in widespread use are
entirely convincing in isolation Key questions that
require further technical advances include the
propor-tion of any specific GPCR that is present as a
mono-mer⁄ dimer ⁄ oligomer and the significance of this for
function Equally, although methods are being
devel-oped and used to explore the relative affinities of
inter-actions between different GPCRs, an issue that is of
particular importance to the likely relevance of
poten-tial GPCR heterodimers in physiology, these are not in
widespread use at this time Also further
documenta-tion of the existence of distinct pharmacology and
functions for GPCR heterodimers should certainly lead
to the development of ligands with selective affinity
and function for such heterodimer pairings and thus
provide ultimate tools to probe the role of
heterodime-rization in vivo
Acknowledgements
Studies on GPCR dimerization in the Milligan and
Bouvier labs are supported by the Biotechnology and
Biosciences Research Council, the Medical Research
Council and the Wellcome Trust to Graeme Milligan
and the Canadian Institute for Health Research and
the Heart and Stroke Foundation of Canada to Michel
Bouvier M Bouvier holds the Canada Research Chair
in Signal Transduction and Molecular Pharmacology
References
1 Salahpour A, Angers S & Bouvier M (2000)
Functional significance of oligomerization of
G-pro-tein-coupled receptors Trends Endocrinol Metab 11,
163–168
2 Woolf PJ & Linderman JJ (2003) Self organization of
membrane proteins via dimerization Biophys Chem 104,
217–227
3 Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin
MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C,
Huang LY, et al (1998) GABA (B) receptors function
as a heteromeric assembly of the subunits GABA (B)
R1 and GABA (B) R2 Nature 396, 674–679
4 White JH, Wise A, Main MJ, Green A, Fraser NJ,
Dis-ney GH, Barnes AA, Emson P, Foord SM & Marshall
FH (1998) Heterodimerization is required for the
forma-tion of a funcforma-tional GABA (B) receptor Nature 396,
679–682
5 Kaupmann K, Huggel K, Heid J, Flor PJ, Bischoff S,
Mickel SJ, McMaster G, Angst C, Bittiger H, Froestl W
& Bettler B (1997) Expression cloning of GABA (B)
receptors uncovers similarity to metabotropic glutamate
receptors Nature 386, 239–246
6 Li X, Staszewski L, Xu H, Durick K, Zoller M & Adler
E (2002) Human receptors for sweet and umami taste Proc Natl Acad Sci USA 99, 4692–4696
7 Pin J-P, Kniazeff J, Liu J, Binet V, Goudet C, Rondard
P & Pre´zeau L (2005) Allosteric functioning of dimeric Class C G-protein coupled receptors FEBS J 272, 2947–2955
8 Hague C, Uberti MA, Chen Z, Hall RA & Minneman
KP (2004a) Cell surface expression of alpha1D-adrener-gic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors J Biol Chem 279, 15541– 15549
9 Hague C, Uberti MA, Chen Z, Bush CF, Jones SV, Ressler KJ, Hall RA & Minneman KP (2004b) Olfac-tory receptor surface expression is driven by association with the beta2-adrenergic receptor Proc Natl Acad Sci USA 101, 13672–13676
10 Bouvier M (2001) Oligomerization of G-protein-coupled transmitter receptors Nat Rev Neurosci 2, 274–286
11 George SR, O’Dowd BF & Lee SP (2002) G-protein-coupled receptor oligomerization and its potential for drug discovery Nature Rev Drug Discovery 1, 808–820
12 Milligan G (2004) G protein-coupled receptor dimeriza-tion: function and ligand pharmacology Mol Pharmacol
66, 1–7
13 Issafras H, Angers S, Bulenger S, Blanpain C, Parmen-tier M et al (2002) Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors J Biol Chem 277, 34666–34673
14 Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub
MA, Taulan M, Jockers R, Barberis C & Bouvier M (2003) Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during bio-synthesis Mol Endocrinol 17, 677–691
15 Salahpour A, Angers S, Mercier JF, Lagace M, Marullo
S & Bouvier M (2004) Homodimerization of the beta 2-adrenergic receptor as a pre-requisite for cell surface targeting J Biol Chem 279, 33390–33397
16 Baneres JL & Parello J (2003) Structure-based analysis of GPCR function: evidence for a novel pentameric assem-bly between the dimeric leukotriene B4 receptor BLT1 and the G-protein J Mol Biol 329, 815–829
17 Angers S, Salahpour A & Bouvier M (2002) Dimeriza-tion: an emerging concept for G protein-coupled recep-tor ontogeny and function Annu Rev Pharmacol Toxicol 42, 409–435
18 Herbert TE, Moffett S, Morello J-P, Loisel TP, Bichet
DG, Barret C & Bouvier M (1996) A peptide derived from a b2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation
J Biol Chem 271, 16384–16392
19 Nimchinsky EA, Hof PR, Janssen WG, Morrison JH & Schmauss C (1997) Expression of dopamine D3 receptor