BACKGROUND
g-protein-coupled receptor  oligomerization is heavily supported by recent biochemical and structural data .
optical-based techniques are instrumental in studying the dynamics and organization of receptor complexes in living cells  <cit> .
for instance, total internal reflection fluorescence microscopy shows that  <dig> % of muscarinic m <dig> receptors exist as dimers  that undergo interconversion with monomers on a timescale of seconds  <cit> .
similarly, the β1-adrenergic receptors  are expressed as a mixture of monomers and dimers whereas β2-adrenergic receptors  have a tendency to form dimers and higher-order oligomers  <cit> .
moreover, the monomer-dimer equilibrium of the chemoattractant n-formyl peptide receptor at a physiological level of expression lies within a timescale of milliseconds  <cit> .
together, these studies in heterologous systems show that a given gpcr is present in a dynamic equilibrium between monomers, dimers, and higher-order oligomers.
studies in a broad spectrum of gpcrs  show that these receptors may form heteromers.
gpcr heteromers are defined as novel signaling units with functional properties different from homomers and they represent a completely new field of study  <cit> .
innovative crystallographic techniques have permitted researchers to obtain crystal structures of gpcr families a, b, c, and f, bound to either agonists, antagonists, inverse agonists or allosteric modulators; in the form of monomers or homo-oligomers; and in complex with a g protein or with a ß-arrestin  <cit> .
however, crystal structures of gpcr heteromers have not yet been obtained.
here, we propose a quaternary structure of a heteromer, taking into account the molecular stoichiometry and the interacting g proteins.
adenosine a1-a2a receptor  complexes constitute a paradigm in the gpcr heteromer field because a1r is coupled to gi and a2ar to gs; that is, they transduce opposite signals in cyclic adenosine monophosphate -dependent intracellular cascades.
first described as a concentration-sensing device in striatal glutamatergic neurons  <cit> , the a1r-a2ar heteromer is thought to function as a gs/gi-mediated switching mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release  <cit> .
the structural basis of this switch is key to understanding heteromer function and the biological advantage behind the gpcr heteromerization phenomenon.
here, we have devised the molecular architecture of the adenosine a1r-a2ar heteromer in complex with g proteins using a combination of microscope-based single-particle tracking, molecular modeling, and energy transfer assays in combination with molecular complementation.
the results point to a <dig> and a2a receptors organizing into a rhombus-shaped heterotetramer that couples to gi and gs.
the overall structure is very compact and provides interacting interfaces for gpcrs and for g proteins.
RESULTS
reciprocal restriction of adenosine receptor motion in the plasma membrane
to examine the dynamics of a1r-a2ar heteromers in the plasma membrane of a living cell, the motion of the receptors tagged with fluorescent proteins  was measured by real-time single-particle tracking  .
examples of fluorescent images and individual particle trajectories are shown in additional file 1: figure s <dig>  analysis of data corresponding to  <dig> a1r-gfp particles showed a linear relationship between the mean square displacement  versus time lag in the trajectories of up to  <dig> single fluorescent particles .
this is typical for brownian diffusion, indicating a lack of restrictions in a1r-gfp motion.
co-expression of a2ar-mcherry  led to a reduction in the lateral mobility of a1r-gfp, which became confined to plasma membrane regions of  <dig>  ±  <dig>  μm in diameter.
its diffusion coefficient decreased from  <dig>  ±  <dig>  μm2/s to  <dig>  ±  <dig>  μm2/s .
similarly, a1r-gfp also decreased the a2ar-mcherry diffusion coefficient from  <dig>  ±  <dig>  μm2/s to  <dig>  ±  <dig>  μm2/s  .
a2ar moved within a confinement zone of  <dig>  ±  <dig>  μm in diameter that was reduced to  <dig>  ±  <dig>  μm  when both receptors were co-expressed.
we conclude from these mobility comparisons that reciprocally restricted motion of the individual receptor particles must be due to a1r-a2ar receptor-receptor interactions.fig.
<dig> cell surface mobility of a1r-gfp and a2ar-mcherry.
individual trajectories of particles containing gfp fused to the c-terminus of a1r   or mcherry fused to the c-terminus of a2ar   on hek-293t cells expressing a1-gfp , a2a-mcherry  or both .
the trajectory and the fluorescence intensity of the individual particles were recorded over time using total internal reflection microscopy  and an electron multiplying charged-coupled device  camera recording.
receptor motion was determined by plotting  the mean square displacement  of a1-gfp  in the absence  or presence of a2a-mcherry , or a2a-mcherry  in the presence  or presence of a1-gfp .
data sets were fitted to mathematical models of free and confined diffusion for a1r and a2ar respectively.
g co-localization of a1-gfp and a2a-mcherry is observed .
scale bar:  <dig> nm.
h distribution of the fluorescence signal of a1-gfp  and a2a-mcherry  within co-localized receptors .
curves approximately delineate the number of monomers, dimers, or trimers within the co-localized complex.
i stoichiometry analysis performed for co-localized a1-gfp and a2a-mcherry receptor particles co-expressed in hek-293t cells .
green corresponds to a1-gfp and red to a2a-mcherry
stoichiometry of a <dig> and a2a receptor heterocomplexes
the stoichiometry of the fluorescent receptors on the cell surface can be calculated from the brightness distribution of the individual particles  <cit>  .
in cells expressing a1r-gfp, we found the majority of clusters to consist of either two  or four  receptors, and clusters with one or three receptors were scarce  .
in the case of a2ar-mcherry, the stoichiometry analysis showed that the clusters mostly expressed trimers , with dimers  and tetramers  the second and third most common populations .
remarkably, this stoichiometry for either a <dig> or a2a receptors was altered when the partner receptor was also expressed.
in cells co-expressing a1r-gfp and a2ar-mcherry, the dimer population increased  and became the predominant species .
in order to focus the analysis on heteromer complexes, we identified clusters containing both receptors .
in ~ <dig> analyzed co-localized clusters that consisted of a mixture of a1-gfp and a2a-cherry , we found a similar high amount of dimers of a1r  and a2ar .
trimers and tetramers of a1r, and monomers and tetramers of a2ar, were in the minority or negligible .
in summary, given that the percentage of dimers of either a1r-gfp or a2ar-mcherry in the yellow dots  was similar and high , the heterotetramer containing two a1rs and two a2ars must have been the most predominant species.
to our knowledge, this is the first stoichiometry data for a gpcr heteromer in living cells.
arrangement of g proteins interacting with a <dig> and a2a receptors
monomeric gpcrs are capable of activating g proteins  <cit> .
however, recent findings suggest that one gpcr homodimer bound to a single g protein may be a common functional unit  <cit> .
thus, an emerging question is how g proteins couple to gpcr heteromers.
because a1r selectively couples to gi and a2ar to gs  <cit> , the working hypothesis was that both gi and gs proteins may couple to the a1r-a2ar heterotetramer.
to test this hypothesis, we used bioluminescence resonance energy transfer  assays  <cit> .
in agreement with the spt experiments , homodimers and heterodimers were detected by bret assays in cells expressing a1r fused with renilla luciferase  or yellow fluorescent protein  , a2ar-rluc and a2ar-yfp , or a1r-rluc and a2ar-yfp .
neither a1r-rluc nor a2ar-yfp interacted with the ghrelin receptor 1a fused to yfp , used as a control as a protein unable to directly interact with these adenosine receptors .
in order to test the presence of the two g proteins in the heterotetramer, we transfected cells with minigenes that code for peptides blocking either gi or gs binding to gpcrs  <cit> .
in addition, cells were treated with pertussis or cholera toxins that catalyze adp-ribosylation of gi or gs.
clearly, treating cells with pertussis toxin, or expressing the minigene-coded peptide that blocks αi coupling, reduced the value of bretmax for a1r-a1r homodimers  and for a1r-a2ar heterodimers  but not for a2ar-a2ar homodimers .
this indicates that gi is coupled to a1r in both the homodimer and the heterodimer.
similarly, blocking gs-receptor interaction using cholera toxin or a minigene-coded peptide that blocks αs coupling reduced bretmax for a2ar-a2ar homodimers  and for a1r-a2ar heterodimers  but not for a1r-a1r homodimers .
interestingly, bret curves showed sensitivity to both cholera and pertussis toxins in cells expressing either a1r-rluc-a1r-yfp and a2ar  or a2ar-rluc-a2ar-yfp and a1r .
functionality of constructs and controls in cells expressing minigenes, and in cells expressing the ghrelin ghs1a receptor instead of one of the adenosine receptors, are shown in additional file 3: figure s <dig>  to further confirm that gi binds a2ar in the receptor heteromer, the energy transfer between rluc fused to the n-terminal domain of the α-subunit of gi  and a2ar-yfp was analyzed in cells co-expressing or not co-expressing a1r .
a hyperbolic bret curve was observed in the presence of a1r, but not in its absence, indicating that gi and gs are bound to their respective receptor homodimers within the a1r-a2ar heteromer.fig.
<dig> influence of g proteins on a1r and a2ar homodimerization and heterodimerization.
b bioluminescence resonance energy transfer  saturation curves were performed in hek-293t cells  <dig> h post-transfection with   <dig>  μg of cdna corresponding to a1r-rluc and increasing amounts of a1r-yfp  or ghs1a-yfp  as negative control , without  or with   <dig>  μg of cdna corresponding to a2ar;   <dig>  μg of cdna corresponding to a2ar-rluc and increasing amounts of a2ar-yfp  or ghs1a-yfp  as negative control , without  or with   <dig>  μg of cdna corresponding to a1r;   <dig>  μg of cdna corresponding to a1r-rluc and increasing amounts of a2ar-yfp ; and   <dig>  μg of cdna corresponding to a1r ,  <dig> μg of cdna corresponding to gi-rluc, and increasing amounts of a2ar-yfp .
in panels a, b, and e, cells were also transfected with  <dig>  μg of cdna corresponding to the gi-related  or gs-related  minigenes.
cells were treated for  <dig> h with medium , with  <dig> ng/ml of pertussis toxin , or with  <dig> ng/ml of cholera toxin  prior to bret determination.
to confirm similar donor expressions  while monitoring the increase in acceptor expression , the fluorescence and luminescence of each sample were measured before energy transfer data acquisition.
milibret unit  values are the mean ± standard error of the mean of four to six different experiments grouped as a function of the amount of bret acceptor.
in each panel  a cartoon depicts the proteins to which rluc and yfp were fused and the presence or not of partner receptors and/or gs or gi proteins
further, two complementary bret experiments were performed to determine the orientation of gi and gs within the a1r-a2ar heterocomplex.
first, rluc and yfp were respectively fused to the n-terminal domains of the α-subunit of gi  and gs  ; second, they were fused to the n-terminal domain of the γ-subunit  .
we observed significant energy transfer between γ-rluc and γ-yfp in cells co-expressing a1r and a2ar  but minimal amounts in negative-control cells .
in cells expressing either a1r or a2ar, the energy transfer between γ-rluc and γ-yfp was also low , suggesting that dimers but not tetramers were the most prevalent form of surface receptors in single-transfected cells.
these results in co-transfected cells corroborate the 2: <dig> stoichiometry obtained from analysis of the fluorescence in single particles and are consistent with gi and gs binding to these a1r-a2ar heterotetramers.fig.
<dig> gs and gi coupling to adenosine a1r-a2ar heterocomplexes.
bioluminescence resonance energy transfer  experiments were performed in hek-293t cells  <dig> h post-transfection with   <dig>  μg of cdna corresponding to a1r and  <dig>  μg of cdna corresponding to a2ar;   <dig>  μg of cdna corresponding to a1r or  <dig>  μg of cdna corresponding to a2ar and  <dig>  μg of cdna corresponding to growth hormone secretagogue receptor ghs1a;   <dig>  μg of cdna corresponding to a1r; or   <dig>  μg of cdna corresponding to a2ar.
cells were also transfected with  <dig> μg of cdna corresponding to the α-subunit of gi fused to rluc and increasing amounts of cdna corresponding to the α-subunit of gs fused to yfp  or  <dig>  μg of cdna corresponding to the γ-subunit fused to rluc and increasing amounts of cdna corresponding to the γ-subunit fused to yfp .
maximum milibret unit  values are the mean ± standard error of the mean of four different experiments.
a scheme showing the protein to which rluc and yfp were fused is provided .
***p <  <dig>  by one-way anova with post - hoc dunnett’s test
molecular model of gi and gs bound to the a1r-a2ar heterotetramer
to identify the orientation of the g protein in the receptor homodimer, we combined energy transfer assays between αs-rluc  and a2ar-yfp  with information on transmembrane  interfaces based on crystal structures of gpcrs  <cit> , which have been recently summarized  <cit> .
the observed high-energy transfer using αs-rluc and a2ar-yfp indicated close proximity between the n-tail of the α-subunit of gs and the c-tail of a2ar.
interestingly, rluc and yfp in the “monomeric” a2ar-gs complex  point toward distant positions in space .
therefore, the observed bret should occur between rluc in the g protein α-subunit and a second a2ar-yfp protomer.
among all described tm interfaces for receptor homodimerization , we propose the tm4/ <dig> interface, which is observed in the oligomeric structure of β1-ar  <cit>  and in structures derived from coarse-grained molecular dynamics  simulations  <cit> .
in fact, this is the only interface that favors bret between αs-rluc and a second a2ar-yfp protomer in a homodimer .
the homologous a1r homodimer was built using the same tm4/ <dig> interface as for a2ar .fig.
<dig> orientation of a g protein in a receptor homodimer.
bioluminescence resonance energy transfer  saturation experiments were performed in hek-293t cells transfected with  <dig> μg of cdna corresponding to the α-subunit of gs fused to rluc and increasing amounts of a2ar-yfp  cdna.
a bret measurements in cells pretreated for  <dig> h with medium  or with  <dig> ng/ml of cholera toxin .
both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions  while monitoring the increase in acceptor expression .
milibret unit  values are the mean ± standard error of the mean of four to five different experiments grouped as a function of the amount of bret acceptor.
a scheme of the placement of donor and acceptor bret moieties is provided .
b molecular model of the a2ar-gs complex.
rluc  is attached to the n-terminal αn helix of gs , and yfp  is attached to the c-terminal domain of a2ar  .
c arrangement of a2ar homodimers modeled via the tm4/ <dig> interface as observed in the oligomeric structure of β1-ar  <cit> .
the a2ar protomer bound to αs is shown in light green, whereas the second a2ar-yfp protomer is shown in dark green.
the molecular model in panel c , in contrast to the model shown in panel b , would favor the observed high-energy transfer  between αs-rluc and a2ar-yfp
the remaining possible tms able to form heteromeric interfaces are tm <dig> and tm5/ <dig> .
both are possible inter-gpcr interfaces as observed in the structure of the μ-opioid receptor   <cit> .
to discern between these two possibilities, a bimolecular fluorescence complementation strategy was undertaken.
for this purpose, the n-terminal fragment of rluc <dig> was fused to a1r  and its c-terminal domain to a2ar , which only upon complementation can act as a bret donor .
the bret acceptor protein was obtained upon complementation of the n-terminal fragment of yfp venus protein fused to a1r  and its c-terminal domain fused to a2ar .
when all four receptor constructs were transfected, we obtained a positive and saturable bret signal  that was not obtained for negative controls .
figure 5a, b shows that the hemi-donor  and the hemi-acceptor  moieties, placed at the c-terminus of the receptors, can only complement if a1r-a2ar heterodimerization occurs via the tm5/ <dig> interface.
the tm4/ <dig> interface for homodimerization and the tm5/ <dig> interface for heterodimerization give a rhombus-shaped tetramer organization .
remarkably, cell pre-incubation with either pertussis or cholera toxins decreased the bretmax by  <dig> % , further suggesting that both gs and gi proteins bind to the a1r-a2ar heterotetramer.fig.
<dig>  bioluminescence resonance energy transfer -aided construction of a model consisting of gi and gs bound to an a1r-a2ar heterotetramer.
a, b a1r-a2ar tetramer built using tm5/ <dig>  or tm <dig>  inter-receptor interfaces modeled as in the structure of the μ opioid receptor  <cit> .
tm helices  <dig>   <dig>  and  <dig>  involved in receptor dimerization, are highlighted in dark blue, light blue, and gray, respectively.
nrluc <dig> and crluc <dig> are shown in blue and nvenus and cvenus in dark yellow.
c bret and bimolecular fluorescence complementation experiments were performed in hek-293t cells transfected with  <dig>  μg of cdna corresponding to a1r-crluc <dig> and a2ar-nrluc <dig>  and  <dig>  μg of cdna corresponding to a1r-nvenus and a2ar-cvenus.
as the negative control, cells were transfected with  <dig> μg of cdna corresponding to nrluc <dig> and  <dig>  μg of cdna corresponding to a2ar-nrluc <dig>  a1r-nvenus, and a2ar-cvenus.
cells were treated for  <dig> h with medium ,  <dig> ng/ml of pertussis toxin , or  <dig> ng/ml of cholera toxin  prior to bret determination.
the relative amount of bret is given as in fig.
<dig> and values are the mean ± standard error of the mean of three different experiments.
student’s t-test showed statistically significant differences with respect to the control  and a significant effect in the presence of either toxin over bret in the absence of toxins .
a schematic representation at the top shows the protein to which the hemi luminescent or fluorescent proteins were fused.
d molecular model of the a1r-a2ar tetramer in complex with gi and gs.
a1r bound to gi is shown in red, gi-unbound a1r is shown in orange, a2ar bound to gs is shown in dark green, gs-unbound a2ar is shown in light green, and the α, β-, and γ-subunits of gi and gs are shown in dark gray, light gray, and purple, respectively.
transmembrane helices  <dig> and  <dig> are highlighted in light blue and gray, respectively
we next evaluated, using computational tools, whether the proposed a1r-a2ar heterotetramer could couple to both gi and gs proteins.
clearly, the external protomers of the proposed a1r-a2ar heterotetramer can bind to gi and gs proteins .
this model positions the α-subunits of gi and gs in close contact, facing the interior of the tetrameric complex, while the n-terminal α-helices of αi and αs point outside the complex.
the n-terminal α-helices of the γ-subunits are in close proximity, facing the inside , which explains the significant energy transfer observed between γ-rluc and γ-yfp .
the model provides experimental insights into the structural arrangement of heteromers consisting of two gpcrs and coupled to two g proteins, the possibility of which has recently been discussed  <cit> .
we used md simulations to study the stability of this complex.
additional file 7: figure s <dig> shows root-mean-square deviations  on protein α-carbons throughout the md simulation, as well as key intermolecular distances among protomers and g proteins.
clearly, both the a1r protomer bound to gi and the a1r protomer that does not interact with it maintained a close structural similarity  relative to the initial structures.
similar results were obtained for the a2ar protomers  .
the fact that rmsd values of the whole system, formed by the a1r-a2ar heterotetramer bound to gi and gs, are of the order of  <dig>  nm indicates that the initial structural model is maintained during the md simulation .
as a consequence, selected intermolecular distances among protomers and g proteins remain constant during the md simulation .
a key aspect in the assembly of the heterotetramer is the tm interfaces for homodimerization  and heterodimerization .
additional file 8: figure s8b shows rmsd values of the four-helix bundle forming the tm4/ <dig> and tm5/ <dig> interfaces, the initial and final snapshots of these bundles, and the evolution of the a1r-a2ar heterotetramer during the md simulation.
clearly, the rather small structural variations of these four-helix bundles, also reflected by rmsd < <dig>  nm, suggest a stable complex.
notably, the tm5/ <dig> four-helix bundle seems more stable than the tm4/ <dig> bundles, as shown by its lower rmsd value.
additional file 8: figure s8b, c depicts contact maps of the tm4/ <dig> and tm5/ <dig> interfaces, as well as the evolution of the network of hydrophobic interactions within these interfaces during the md simulation.
CONCLUSIONS
for more than a decade, experimental evidence has supported the occurrence of homo-oligomers and hetero-oligomers of gpcrs  <cit> .
however, our basic understanding of what makes heteromers different from homomers remains unknown.
our results, studying adenosine receptors as a model heteromer, point to three important new findings.
first, the predominant stoichiometry in cells expressing a1r-a2ar heteromers is 2:2; that is, a dimer of dimers .
second, two different heterotrimeric g proteins can couple to heteromers, the overall complex constituting a functional unit.
third, the molecular orientation within the heteromer complex affords various qualitatively different interfaces; the two more relevant are the inter-protomer heteromeric interface and the inter-g-protein interface.
presumably, the two interfaces provide the key characteristic of heteromers: the ability of one protomer/g-protein complex to influence the signaling of the other.
surely, allosteric effects occurring between heteroreceptors and between gs and gi proteins are due to conformational changes transmitted along the intimately interacting molecules in the complex.
in our controlled cell transfection system, which expressed a low density of receptors, minor species formed by monomers and trimers were found in addition to a predominance of tetramers in the plasma membrane, strongly supporting the occurrence of an in vivo dynamic distribution of receptors.
adenosine was, from an evolutionary point of view, one of the first extracellular regulators given that it is involved in energy and nucleic acid metabolisms.
adenosine a <dig> and a2a receptors are expressed in almost every mammalian organ and tissue.
in the heart, where adenosine plays a key role in both inotropic and chronotropic regulation, a1r-mediated cardioprotection did not occur in a2ar knockout mice, suggesting an interaction between a <dig> and a2a receptors.
in neurons, a <dig> and a2a receptors show co-localization, leading to inter-receptor interactions unveiled by pharmacological treatments.
for instance, okada et al.
<cit>  showed that camp-dependent protein kinase a plays a role in the regulation of hippocampal serotonin release mediated by both a <dig> and a2a receptors.
similarly, the control of γ-amino butyric acid transport in astrocytes was attributed to the expression of a1r-a2ar heteromers and to a specific mechanism by which the heteromer signals via gi or gs depending on the concentration of adenosine  <cit> .
the structural basis of the differential signaling by the heteromer/g-protein macromolecular complex likely implies communication at the receptor-receptor level but also between gs and gi.
because the binding of two g proteins to a heterodimer is not feasible due to steric clashes  <cit> , our finding that the a1r-a2ar heterotetramer may bind to both gs and gi provides a structural framework to interpret experimental data.

 g-protein-coupled receptor oligomerization is heavily supported by recent biochemical and structural data. optical-based techniques are instrumental in studying the dynamics and organization of receptor complexes in living cells. for instance, total internal reflection fluorescence microscopy shows that % of muscarinic m receptors exist as dimers that undergo interconversion with monomers on a timescale of seconds. similarly, the β1-adrenergic receptors are expressed as a mixture of monomers and dimers whereas β2-adrenergic receptors have a tendency to form dimers and higher-order oligomers. moreover, the monomer-dimer equilibrium of the chemoattractant n-formyl peptide receptor at a physiological level of expression lies within a timescale of milliseconds. together, these studies in heterologous systems show that a given gpcr is present in a dynamic equilibrium between monomers, dimers, and higher-order oligomers. studies in a broad spectrum of gpcrs show that these receptors may form heteromers. gpcr heteromers are defined as novel signaling units with functional properties different from homomers and they represent a completely new field of study. innovative crystallographic techniques have permitted researchers to obtain crystal structures of gpcr families a, b, c, and f, bound to either agonists, antagonists, inverse agonists or allosteric modulators; in the form of monomers or homo-oligomers; and in complex with a g protein or with a ß-arrestin. however, crystal structures of gpcr heteromers have not yet been obtained. here, we propose a quaternary structure of a heteromer, taking into account the molecular stoichiometry and the interacting g proteins. adenosine a1-a2a receptor complexes constitute a paradigm in the gpcr heteromer field because a1r is coupled to gi and a2ar to gs; that is, they transduce opposite signals in cyclic adenosine monophosphate -dependent intracellular cascades. first described as a concentration-sensing device in striatal glutamatergic neurons, the a1r-a2ar heteromer is thought to function as a gs/gi-mediated switching mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. the structural basis of this switch is key to understanding heteromer function and the biological advantage behind the gpcr heteromerization phenomenon. here, we have devised the molecular architecture of the adenosine a1r-a2ar heteromer in complex with g proteins using a combination of microscope-based single-particle tracking, molecular modeling, and energy transfer assays in combination with molecular complementation. the results point to a and a2a receptors organizing into a rhombus-shaped heterotetramer that couples to gi and gs. the overall structure is very compact and provides interacting interfaces for gpcrs and for g proteins.  reciprocal restriction of adenosine receptor motion in the plasma membrane to examine the dynamics of a1r-a2ar heteromers in the plasma membrane of a living cell, the motion of the receptors tagged with fluorescent proteins was measured by real-time single-particle tracking. examples of fluorescent images and individual particle trajectories are shown in additional file 1: figure s analysis of data corresponding to a1r-gfp particles showed a linear relationship between the mean square displacement versus time lag in the trajectories of up to single fluorescent particles. this is typical for brownian diffusion, indicating a lack of restrictions in a1r-gfp motion. co-expression of a2ar-mcherry led to a reduction in the lateral mobility of a1r-gfp, which became confined to plasma membrane regions of  ±  μm in diameter. its diffusion coefficient decreased from  ±  μm2/s to  ±  μm2/s. similarly, a1r-gfp also decreased the a2ar-mcherry diffusion coefficient from  ±  μm2/s to  ±  μm2/s. a2ar moved within a confinement zone of  ±  μm in diameter that was reduced to  ±  μm when both receptors were co-expressed. we conclude from these mobility comparisons that reciprocally restricted motion of the individual receptor particles must be due to a1r-a2ar receptor-receptor interactions.fig. cell surface mobility of a1r-gfp and a2ar-mcherry. individual trajectories of particles containing gfp fused to the c-terminus of a1r or mcherry fused to the c-terminus of a2ar on hek-293t cells expressing a1-gfp, a2a-mcherry or both. the trajectory and the fluorescence intensity of the individual particles were recorded over time using total internal reflection microscopy and an electron multiplying charged-coupled device camera recording. receptor motion was determined by plotting the mean square displacement of a1-gfp in the absence or presence of a2a-mcherry, or a2a-mcherry in the presence or presence of a1-gfp. data sets were fitted to mathematical models of free and confined diffusion for a1r and a2ar respectively. g co-localization of a1-gfp and a2a-mcherry is observed. scale bar: nm. h distribution of the fluorescence signal of a1-gfp and a2a-mcherry within co-localized receptors. curves approximately delineate the number of monomers, dimers, or trimers within the co-localized complex. i stoichiometry analysis performed for co-localized a1-gfp and a2a-mcherry receptor particles co-expressed in hek-293t cells. green corresponds to a1-gfp and red to a2a-mcherry stoichiometry of a and a2a receptor heterocomplexes the stoichiometry of the fluorescent receptors on the cell surface can be calculated from the brightness distribution of the individual particles. in cells expressing a1r-gfp, we found the majority of clusters to consist of either two or four receptors, and clusters with one or three receptors were scarce. in the case of a2ar-mcherry, the stoichiometry analysis showed that the clusters mostly expressed trimers, with dimers and tetramers the second and third most common populations. remarkably, this stoichiometry for either a or a2a receptors was altered when the partner receptor was also expressed. in cells co-expressing a1r-gfp and a2ar-mcherry, the dimer population increased and became the predominant species. in order to focus the analysis on heteromer complexes, we identified clusters containing both receptors. in ~ analyzed co-localized clusters that consisted of a mixture of a1-gfp and a2a-cherry, we found a similar high amount of dimers of a1r and a2ar. trimers and tetramers of a1r, and monomers and tetramers of a2ar, were in the minority or negligible. in summary, given that the percentage of dimers of either a1r-gfp or a2ar-mcherry in the yellow dots was similar and high, the heterotetramer containing two a1rs and two a2ars must have been the most predominant species. to our knowledge, this is the first stoichiometry data for a gpcr heteromer in living cells. arrangement of g proteins interacting with a and a2a receptors monomeric gpcrs are capable of activating g proteins. however, recent findings suggest that one gpcr homodimer bound to a single g protein may be a common functional unit. thus, an emerging question is how g proteins couple to gpcr heteromers. because a1r selectively couples to gi and a2ar to gs, the working hypothesis was that both gi and gs proteins may couple to the a1r-a2ar heterotetramer. to test this hypothesis, we used bioluminescence resonance energy transfer assays. in agreement with the spt experiments, homodimers and heterodimers were detected by bret assays in cells expressing a1r fused with renilla luciferase or yellow fluorescent protein, a2ar-rluc and a2ar-yfp, or a1r-rluc and a2ar-yfp. neither a1r-rluc nor a2ar-yfp interacted with the ghrelin receptor 1a fused to yfp, used as a control as a protein unable to directly interact with these adenosine receptors. in order to test the presence of the two g proteins in the heterotetramer, we transfected cells with minigenes that code for peptides blocking either gi or gs binding to gpcrs. in addition, cells were treated with pertussis or cholera toxins that catalyze adp-ribosylation of gi or gs. clearly, treating cells with pertussis toxin, or expressing the minigene-coded peptide that blocks αi coupling, reduced the value of bretmax for a1r-a1r homodimers and for a1r-a2ar heterodimers but not for a2ar-a2ar homodimers. this indicates that gi is coupled to a1r in both the homodimer and the heterodimer. similarly, blocking gs-receptor interaction using cholera toxin or a minigene-coded peptide that blocks αs coupling reduced bretmax for a2ar-a2ar homodimers and for a1r-a2ar heterodimers but not for a1r-a1r homodimers. interestingly, bret curves showed sensitivity to both cholera and pertussis toxins in cells expressing either a1r-rluc-a1r-yfp and a2ar or a2ar-rluc-a2ar-yfp and a1r. functionality of constructs and controls in cells expressing minigenes, and in cells expressing the ghrelin ghs1a receptor instead of one of the adenosine receptors, are shown in additional file 3: figure s to further confirm that gi binds a2ar in the receptor heteromer, the energy transfer between rluc fused to the n-terminal domain of the α-subunit of gi and a2ar-yfp was analyzed in cells co-expressing or not co-expressing a1r. a hyperbolic bret curve was observed in the presence of a1r, but not in its absence, indicating that gi and gs are bound to their respective receptor homodimers within the a1r-a2ar heteromer.fig. influence of g proteins on a1r and a2ar homodimerization and heterodimerization. b bioluminescence resonance energy transfer saturation curves were performed in hek-293t cells h post-transfection with μg of cdna corresponding to a1r-rluc and increasing amounts of a1r-yfp or ghs1a-yfp as negative control, without or with μg of cdna corresponding to a2ar; μg of cdna corresponding to a2ar-rluc and increasing amounts of a2ar-yfp or ghs1a-yfp as negative control, without or with μg of cdna corresponding to a1r; μg of cdna corresponding to a1r-rluc and increasing amounts of a2ar-yfp; and μg of cdna corresponding to a1r, μg of cdna corresponding to gi-rluc, and increasing amounts of a2ar-yfp. in panels a, b, and e, cells were also transfected with μg of cdna corresponding to the gi-related or gs-related minigenes. cells were treated for h with medium, with ng/ml of pertussis toxin, or with ng/ml of cholera toxin prior to bret determination. to confirm similar donor expressions while monitoring the increase in acceptor expression, the fluorescence and luminescence of each sample were measured before energy transfer data acquisition. milibret unit values are the mean ± standard error of the mean of four to six different experiments grouped as a function of the amount of bret acceptor. in each panel a cartoon depicts the proteins to which rluc and yfp were fused and the presence or not of partner receptors and/or gs or gi proteins further, two complementary bret experiments were performed to determine the orientation of gi and gs within the a1r-a2ar heterocomplex. first, rluc and yfp were respectively fused to the n-terminal domains of the α-subunit of gi and gs; second, they were fused to the n-terminal domain of the γ-subunit. we observed significant energy transfer between γ-rluc and γ-yfp in cells co-expressing a1r and a2ar but minimal amounts in negative-control cells. in cells expressing either a1r or a2ar, the energy transfer between γ-rluc and γ-yfp was also low, suggesting that dimers but not tetramers were the most prevalent form of surface receptors in single-transfected cells. these results in co-transfected cells corroborate the 2: stoichiometry obtained from analysis of the fluorescence in single particles and are consistent with gi and gs binding to these a1r-a2ar heterotetramers.fig. gs and gi coupling to adenosine a1r-a2ar heterocomplexes. bioluminescence resonance energy transfer experiments were performed in hek-293t cells h post-transfection with μg of cdna corresponding to a1r and μg of cdna corresponding to a2ar; μg of cdna corresponding to a1r or μg of cdna corresponding to a2ar and μg of cdna corresponding to growth hormone secretagogue receptor ghs1a; μg of cdna corresponding to a1r; or μg of cdna corresponding to a2ar. cells were also transfected with μg of cdna corresponding to the α-subunit of gi fused to rluc and increasing amounts of cdna corresponding to the α-subunit of gs fused to yfp or μg of cdna corresponding to the γ-subunit fused to rluc and increasing amounts of cdna corresponding to the γ-subunit fused to yfp. maximum milibret unit values are the mean ± standard error of the mean of four different experiments. a scheme showing the protein to which rluc and yfp were fused is provided. ***p <  by one-way anova with post - hoc dunnett’s test molecular model of gi and gs bound to the a1r-a2ar heterotetramer to identify the orientation of the g protein in the receptor homodimer, we combined energy transfer assays between αs-rluc and a2ar-yfp with information on transmembrane interfaces based on crystal structures of gpcrs, which have been recently summarized. the observed high-energy transfer using αs-rluc and a2ar-yfp indicated close proximity between the n-tail of the α-subunit of gs and the c-tail of a2ar. interestingly, rluc and yfp in the “monomeric” a2ar-gs complex point toward distant positions in space. therefore, the observed bret should occur between rluc in the g protein α-subunit and a second a2ar-yfp protomer. among all described tm interfaces for receptor homodimerization, we propose the tm4/ interface, which is observed in the oligomeric structure of β1-ar and in structures derived from coarse-grained molecular dynamics simulations. in fact, this is the only interface that favors bret between αs-rluc and a second a2ar-yfp protomer in a homodimer. the homologous a1r homodimer was built using the same tm4/ interface as for a2ar.fig. orientation of a g protein in a receptor homodimer. bioluminescence resonance energy transfer saturation experiments were performed in hek-293t cells transfected with μg of cdna corresponding to the α-subunit of gs fused to rluc and increasing amounts of a2ar-yfp cdna. a bret measurements in cells pretreated for h with medium or with ng/ml of cholera toxin. both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions while monitoring the increase in acceptor expression. milibret unit values are the mean ± standard error of the mean of four to five different experiments grouped as a function of the amount of bret acceptor. a scheme of the placement of donor and acceptor bret moieties is provided. b molecular model of the a2ar-gs complex. rluc is attached to the n-terminal αn helix of gs, and yfp is attached to the c-terminal domain of a2ar. c arrangement of a2ar homodimers modeled via the tm4/ interface as observed in the oligomeric structure of β1-ar. the a2ar protomer bound to αs is shown in light green, whereas the second a2ar-yfp protomer is shown in dark green. the molecular model in panel c, in contrast to the model shown in panel b, would favor the observed high-energy transfer between αs-rluc and a2ar-yfp the remaining possible tms able to form heteromeric interfaces are tm and tm5/. both are possible inter-gpcr interfaces as observed in the structure of the μ-opioid receptor. to discern between these two possibilities, a bimolecular fluorescence complementation strategy was undertaken. for this purpose, the n-terminal fragment of rluc was fused to a1r and its c-terminal domain to a2ar, which only upon complementation can act as a bret donor. the bret acceptor protein was obtained upon complementation of the n-terminal fragment of yfp venus protein fused to a1r and its c-terminal domain fused to a2ar. when all four receptor constructs were transfected, we obtained a positive and saturable bret signal that was not obtained for negative controls. figure 5a, b shows that the hemi-donor and the hemi-acceptor moieties, placed at the c-terminus of the receptors, can only complement if a1r-a2ar heterodimerization occurs via the tm5/ interface. the tm4/ interface for homodimerization and the tm5/ interface for heterodimerization give a rhombus-shaped tetramer organization. remarkably, cell pre-incubation with either pertussis or cholera toxins decreased the bretmax by %, further suggesting that both gs and gi proteins bind to the a1r-a2ar heterotetramer.fig. bioluminescence resonance energy transfer -aided construction of a model consisting of gi and gs bound to an a1r-a2ar heterotetramer. a, b a1r-a2ar tetramer built using tm5/ or tm inter-receptor interfaces modeled as in the structure of the μ opioid receptor. tm helices and involved in receptor dimerization, are highlighted in dark blue, light blue, and gray, respectively. nrluc and crluc are shown in blue and nvenus and cvenus in dark yellow. c bret and bimolecular fluorescence complementation experiments were performed in hek-293t cells transfected with μg of cdna corresponding to a1r-crluc and a2ar-nrluc and μg of cdna corresponding to a1r-nvenus and a2ar-cvenus. as the negative control, cells were transfected with μg of cdna corresponding to nrluc and μg of cdna corresponding to a2ar-nrluc a1r-nvenus, and a2ar-cvenus. cells were treated for h with medium, ng/ml of pertussis toxin, or ng/ml of cholera toxin prior to bret determination. the relative amount of bret is given as in fig.  and values are the mean ± standard error of the mean of three different experiments. student’s t-test showed statistically significant differences with respect to the control and a significant effect in the presence of either toxin over bret in the absence of toxins. a schematic representation at the top shows the protein to which the hemi luminescent or fluorescent proteins were fused. d molecular model of the a1r-a2ar tetramer in complex with gi and gs. a1r bound to gi is shown in red, gi-unbound a1r is shown in orange, a2ar bound to gs is shown in dark green, gs-unbound a2ar is shown in light green, and the α, β-, and γ-subunits of gi and gs are shown in dark gray, light gray, and purple, respectively. transmembrane helices and are highlighted in light blue and gray, respectively we next evaluated, using computational tools, whether the proposed a1r-a2ar heterotetramer could couple to both gi and gs proteins. clearly, the external protomers of the proposed a1r-a2ar heterotetramer can bind to gi and gs proteins. this model positions the α-subunits of gi and gs in close contact, facing the interior of the tetrameric complex, while the n-terminal α-helices of αi and αs point outside the complex. the n-terminal α-helices of the γ-subunits are in close proximity, facing the inside, which explains the significant energy transfer observed between γ-rluc and γ-yfp. the model provides experimental insights into the structural arrangement of heteromers consisting of two gpcrs and coupled to two g proteins, the possibility of which has recently been discussed. we used md simulations to study the stability of this complex. additional file 7: figure s shows root-mean-square deviations on protein α-carbons throughout the md simulation, as well as key intermolecular distances among protomers and g proteins. clearly, both the a1r protomer bound to gi and the a1r protomer that does not interact with it maintained a close structural similarity relative to the initial structures. similar results were obtained for the a2ar protomers. the fact that rmsd values of the whole system, formed by the a1r-a2ar heterotetramer bound to gi and gs, are of the order of nm indicates that the initial structural model is maintained during the md simulation. as a consequence, selected intermolecular distances among protomers and g proteins remain constant during the md simulation. a key aspect in the assembly of the heterotetramer is the tm interfaces for homodimerization and heterodimerization. additional file 8: figure s8b shows rmsd values of the four-helix bundle forming the tm4/ and tm5/ interfaces, the initial and final snapshots of these bundles, and the evolution of the a1r-a2ar heterotetramer during the md simulation. clearly, the rather small structural variations of these four-helix bundles, also reflected by rmsd < nm, suggest a stable complex. notably, the tm5/ four-helix bundle seems more stable than the tm4/ bundles, as shown by its lower rmsd value. additional file 8: figure s8b, c depicts contact maps of the tm4/ and tm5/ interfaces, as well as the evolution of the network of hydrophobic interactions within these interfaces during the md simulation.  for more than a decade, experimental evidence has supported the occurrence of homo-oligomers and hetero-oligomers of gpcrs. however, our basic understanding of what makes heteromers different from homomers remains unknown. our results, studying adenosine receptors as a model heteromer, point to three important new findings. first, the predominant stoichiometry in cells expressing a1r-a2ar heteromers is 2:2; that is, a dimer of dimers. second, two different heterotrimeric g proteins can couple to heteromers, the overall complex constituting a functional unit. third, the molecular orientation within the heteromer complex affords various qualitatively different interfaces; the two more relevant are the inter-protomer heteromeric interface and the inter-g-protein interface. presumably, the two interfaces provide the key characteristic of heteromers: the ability of one protomer/g-protein complex to influence the signaling of the other. surely, allosteric effects occurring between heteroreceptors and between gs and gi proteins are due to conformational changes transmitted along the intimately interacting molecules in the complex. in our controlled cell transfection system, which expressed a low density of receptors, minor species formed by monomers and trimers were found in addition to a predominance of tetramers in the plasma membrane, strongly supporting the occurrence of an in vivo dynamic distribution of receptors. adenosine was, from an evolutionary point of view, one of the first extracellular regulators given that it is involved in energy and nucleic acid metabolisms. adenosine a and a2a receptors are expressed in almost every mammalian organ and tissue. in the heart, where adenosine plays a key role in both inotropic and chronotropic regulation, a1r-mediated cardioprotection did not occur in a2ar knockout mice, suggesting an interaction between a and a2a receptors. in neurons, a and a2a receptors show co-localization, leading to inter-receptor interactions unveiled by pharmacological treatments. for instance, okada et al. showed that camp-dependent protein kinase a plays a role in the regulation of hippocampal serotonin release mediated by both a and a2a receptors. similarly, the control of γ-amino butyric acid transport in astrocytes was attributed to the expression of a1r-a2ar heteromers and to a specific mechanism by which the heteromer signals via gi or gs depending on the concentration of adenosine. the structural basis of the differential signaling by the heteromer/g-protein macromolecular complex likely implies communication at the receptor-receptor level but also between gs and gi. because the binding of two g proteins to a heterodimer is not feasible due to steric clashes, our finding that the a1r-a2ar heterotetramer may bind to both gs and gi provides a structural framework to interpret experimental data. 
the combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric g proteins. these novel results constitute an important advance in understanding the molecular intricacies involved in gpcr function. using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and g-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the gpcr tetramer in complex with two g proteins. g-protein-coupled receptors, in the form of monomers or homodimers that bind heterotrimeric g proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. gpcrheterotetramerheterotrimeric g proteinsingle-particle trackingbretmolecular modelinghttp://dx.doi.org/ /501100007136secretaría de estado de investigación, desarrollo e innovaciónsaf2010-18472saf2011-23813canela enric i. mccormick peter j. issue-copyright-statement© the author 2016 we used single-particle tracking experiments in cells expressing functional adenosine a1-a2a receptors fused to fluorescent proteins to show the loss of brownian movement of the a receptor in the presence of the a2a receptor, and a preponderance of cell surface 2: receptor heteromers.
 g-protein-coupled receptors, in the form of monomers or homodimers that bind heterotrimeric g proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. different gpcrs may also interact to form heteromers that are novel signaling units. despite the exponential growth in the number of solved gpcr crystal structures, the structural properties of heteromers remain unknown.  we used single-particle tracking experiments in cells expressing functional adenosine a1-a2a receptors fused to fluorescent proteins to show the loss of brownian movement of the a receptor in the presence of the a2a receptor, and a preponderance of cell surface 2: receptor heteromers. using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and g-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the gpcr tetramer in complex with two g proteins.  the combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric g proteins. these novel results constitute an important advance in understanding the molecular intricacies involved in gpcr function. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gpcrheterotetramerheterotrimeric g proteinsingle-particle trackingbretmolecular modelinghttp://dx.doi.org/ /501100007136secretaría de estado de investigación, desarrollo e innovaciónsaf2010-18472saf2011-23813canela enric i. mccormick peter j. issue-copyright-statement© the author 2016 