**4.3 Interpretation of BRET results – Possible drawbacks**

88 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

**relevance** 

Implicated in decreasing airway smooth muscle relaxation during

asthma.

for pain

Baclofen is an antispasm drug

**Oligomer name Organism** *In vivo* **evidence Potential clinical** 

*Homo sapiens, Mus musculus* 

*Homo sapiens, Rattus norvegicus*

*Homo sapiens Mus musculus* 

*Rattus* 

*norvegicus, Mus musculus* 

*Homo sapiens, Rattus norvegicus*

*Homo sapiens* evidence for physical association in

smooth muscle)

*Homo sapiens* identification of a specific functional property in native tissue

*Mus musculus* evidence for physical association in

*Rattus norvegicus* evidence for physical association in

*Mus musculus* evidence for physical association in

*Rattus norvegicus* evidence for physical association in

native tissue or primary cells

property in native tissue

native tissue or primary cells

native tissue or primary cells

native tissue or primary cells, identification of a specific functional

evidence for physical association in native tissue or primary cells

property in native tissue

evidence for physical association in native tissue or primary cells

*Mus musculus* colocalization in spinal cord tissue-specific agonist

colocalize in brain GABAB1 agonist

native tissue or primary cells

evidence for physical association in native tissue or primary cells, identification of a specific functional property in native tissue (airway

identification of a specific functional

Adrenergic 2A receptor - Opioid μ receptor oligomer (**2A-adrenoreceptor –** 

**opioid μ**)

Adrenergic 2 - Prostaglandin EP1 receptor oligomer (**2 adrenoreceptor - EP1**)

Cannabinoid CB1 - Dopamine D2 oligomer (**CB1 - D2**)

Chemokine CCR2- CXCR4 receptor oligomer (**CCR2 -** 

Dopamine D1 - Histamine H3 receptor oligomer (**D1 - H3**)

Dopamine D2 - Histamine H3 receptor oligomer (**D2 - H3**)

Opioid δ - Opioid κ receptor oligomer (**δ –** 

Opioid δ - Opioid μ receptor oligomer

**Family C 7TMRs**  γ-aminobutiric acid GABAb receptor oligomer (**GABAB1 -** 

**Family A/C 7TMRs**  Adenosine A2A - Metabotropic glutamate 5 (mGLU 5)

Dopamine D1 - Opioid μ receptor oligomer

**CXCR4**)

(**D1 – μ**)

**κ**)

(**δ – μ**)

**GABAB2**)

oligomer (**A2A - mGLU5**)

Dopamine D2 - Metabotropic glutamate 5 (mGLU 5) oligomer (**D2 mGLU5**)

BRET signal indicates that molecules of the same (or two different) receptors are at maximum distance of 100 Å (that equals 10 nm) or more accurately that the donor and acceptor moieties are within this distance. The efficiency of energy transfer depends on the relative orientation of the donor and acceptor and the distance between them (Zacharias et al., 2000), so that absolute distances can not be measured. Experimentally determined Förster distance R0 (distance at which the energy transfer efficiency is 50%) for BRET1 and BRET2 is 4.4 nm and 7.5 nm, respectively (Dacres et al., 2010). 7TMR transmembrane core spans ~40 Å across the intracellular surface (Palczewski et al., 2000), which makes BRET suitable to the study of dimerization. However, certain facts need to be considered when interpreting BRET results. Firstly, the size of 27 kDa fluorescent proteins and 34 kDa *Renilla luciferase* is comparable to that of the transmembrane core of 7TMRs (diameter ∼40 Å). These proteins are usually attached to the receptor C-terminus, which in different 7TMRs varies in length from 25 to 150 amino acids. Polypeptides of this length in extended conformation can cover 80−480 Å. Thus, a BRET signal indicates that the donor and acceptor moieties are at distance less than 10 nm, which may occur when receptors form structurally defined dimer or when they are far >500 Å apart (reviewed by (Gurevich & Gurevich, 2008a)). The use of acceptor and donor molecules genetically fused to 7TMRs can alter the functionality of the receptor; fusion proteins can also be expressed in the intracellular compartments, thus making difficult to demonstrate that the RET results from a direct interaction of proteins at the cell surface (Ferre & Franco, 2010). The use of fusion proteins can therefore be a major limitation for this application. Secondly, quantitative BRET measurements are limited by the quality of the signal and noise level. Fluorescent proteins and luciferase yield background signals arising from incompletely processed proteins inside the cell and high cell autofluorescence in the spectral region used (Gurevich & Gurevich, 2008a). Thirdly, so called bystander BRET results from frequent encounters between overexpressed receptors and has no physical meaning (Kenworthy & Edidin, 1998; Mercier et al., 2002). BRET assays should therefore be able to discriminate between genuine dimerization compared to random collision due to over-expression. To determine specify of BRET signal the following experiments has been proposed: negative control with a non-interacting receptor or protein, BRET saturation and competition assays and experiments that observe ligand-promoted changes in BRET (Achour et al., 2011; Ayoub & Pfleger, 2010; Ferre & Franco, 2010). Additionally, interpretation of BRET data also requires quantitative analysis of the results, which was so far done only in a small number of studies (Ayoub et al., 2002; Mercier et al., 2002; Vrecl et al., 2006). The theoretical background of the assays described below provides some guidelines for the appropriate interpretation and quantitative evolution of BRET results.

#### **5. Mathematical models to quantitatively assess the oligomerization state of studied receptors**

#### **5.1 Basic assumptions**

Bioluminescent resonance energy transfer takes place at 1-10 nm distances between molecules thus allowing study of protein-protein interaction. It is a quite robust tool but still some care should be taken with interpretation of the results. Resonance energy transfer is described by the Förster equation for energy transfer efficiency *E* (Förster, 1959):

$$E = \frac{R\_0^6}{R\_0^6 + r^6} \tag{1}$$

Quantitative Assessment of Seven Transmembrane Receptors

saturation and competition assays (Breit et al., 2004).

**5.2 BRET dilution assay** 

0

**5.3 BRET saturation assay** 

should be used.

50

100

150

*BRET* (mBU)

200

250

300

350

(7TMRs) Oligomerization by Bioluminescence Resonance Energy Transfer (BRET) Technology 91

This is a simplest control experiment to check for oligomerisation. Resonant energy transfer takes place if the distance between donor and acceptor molecules is in the range of Förster radius *R*0. Molecules can get close enough for BRET also by random collisions (bystander BRET) if their density is high enough (Kenworthy & Edidin, 1998; Mercier et al., 2002). Excluding random collisions there should be no concentration dependence for coupled

where [*D*] and [*A*] are donor and acceptor concentrations. With lowering the concentration of both receptors simultaneously (dilution) the *BRET* signal approaches *BRET*0 which is the real oligomerisation signal (Fig. 1). Dilution assay is used to set the concentration range for

0 2 4 6 810

 random collisions oligomerization signal

oligomerization + random collisions

[*A*]+[*D*]

Fig. 1. BRET dilution assay. Theoretical BRET concentration curves for receptors forming monomers or oligomers. A constant ratio between acceptor and donor concentrations

Saturation assay involves expressing a constant amount of donor-tagged receptor with an increasing amounts of acceptor-tagged receptor. Theoretically, *BRET* signal should increase with increasing amounts of acceptor until all donor molecules are interacting with acceptor

*BRET BRET k D A* <sup>0</sup> (4)

donor and acceptor molecules. In practice we can approximate the *BRET* signal as:

where *r* is a distance between donor and acceptor, Förster radius *R*0 depends on spectral overlap and dipole orientations yielding *R*0 values of 4.4 nm for BRET1and 7.5 nm for BRET2 (Dacres et al., 2010). *E* is an important parameter in interpretation of the BRET assays used for oligomerisation studies. If the BRET luminometer is properly calibrated then *E* can be calculated from the *BRET*max signal obtained when all donor molecules are accompanied by acceptor molecules:

$$E = \frac{BRET\_{\text{max}}}{BRET\_{\text{max}} + 1} \tag{2}$$

Calibration should take into account differences in the detector quantum efficiencies at donor and acceptor emission wavelengths and the proportion of the detected emission spectra of both markers. Knowing a Förster radius for certain type of BRET technology used and energy transfer efficiency *E* we can estimate the distance between the donor and acceptor marker species in the protein complex.

Calculations in presented BRET assays are derived from Veatch and Stryer article (Veatch & Stryer, 1977) covering FRET experiments with Gramicidin dimers. In FRET experiments the 28 *Q*/*Q*0 is a measurement parameter representing the ratio between not-transmitted energy *Q* and total energy *Q*0. Vaecht and Stryer equations have been adopted for BRET experiments where we measure the ratio between transmitted *T* and not-transmitted energy *Q*:

$$BRET = \frac{T}{Q} = \frac{Q\_0}{Q} - 1\tag{3}$$

Single BRET measurements do not give unambiguous proof that receptors form oligomers because the signal can be a consequence of random collisions. To get better indication of the oligomerisation state several quantitative assays were developed.

#### **5.2 BRET dilution assay**

90 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

& Pfleger, 2010; Ferre & Franco, 2010). Additionally, interpretation of BRET data also requires quantitative analysis of the results, which was so far done only in a small number of studies (Ayoub et al., 2002; Mercier et al., 2002; Vrecl et al., 2006). The theoretical background of the assays described below provides some guidelines for the appropriate interpretation and

**5. Mathematical models to quantitatively assess the oligomerization state of** 

Bioluminescent resonance energy transfer takes place at 1-10 nm distances between molecules thus allowing study of protein-protein interaction. It is a quite robust tool but still some care should be taken with interpretation of the results. Resonance energy transfer is

> 6 0 6 6 0 *<sup>R</sup> <sup>E</sup> R r*

> > max max 1

*BRET <sup>E</sup>*

Calibration should take into account differences in the detector quantum efficiencies at donor and acceptor emission wavelengths and the proportion of the detected emission spectra of both markers. Knowing a Förster radius for certain type of BRET technology used and energy transfer efficiency *E* we can estimate the distance between the donor and

Calculations in presented BRET assays are derived from Veatch and Stryer article (Veatch & Stryer, 1977) covering FRET experiments with Gramicidin dimers. In FRET experiments the 28 *Q*/*Q*0 is a measurement parameter representing the ratio between not-transmitted energy *Q* and total energy *Q*0. Vaecht and Stryer equations have been adopted for BRET experiments

<sup>0</sup> <sup>1</sup> *<sup>T</sup> <sup>Q</sup> BRET*

Single BRET measurements do not give unambiguous proof that receptors form oligomers because the signal can be a consequence of random collisions. To get better indication of the

where we measure the ratio between transmitted *T* and not-transmitted energy *Q*:

oligomerisation state several quantitative assays were developed.

where *r* is a distance between donor and acceptor, Förster radius *R*0 depends on spectral overlap and dipole orientations yielding *R*0 values of 4.4 nm for BRET1and 7.5 nm for BRET2 (Dacres et al., 2010). *E* is an important parameter in interpretation of the BRET assays used for oligomerisation studies. If the BRET luminometer is properly calibrated then *E* can be calculated from the *BRET*max signal obtained when all donor molecules are accompanied by

(1)

*BRET* (2)

*Q Q* (3)

described by the Förster equation for energy transfer efficiency *E* (Förster, 1959):

quantitative evolution of BRET results.

**studied receptors 5.1 Basic assumptions** 

acceptor molecules:

acceptor marker species in the protein complex.

This is a simplest control experiment to check for oligomerisation. Resonant energy transfer takes place if the distance between donor and acceptor molecules is in the range of Förster radius *R*0. Molecules can get close enough for BRET also by random collisions (bystander BRET) if their density is high enough (Kenworthy & Edidin, 1998; Mercier et al., 2002). Excluding random collisions there should be no concentration dependence for coupled donor and acceptor molecules. In practice we can approximate the *BRET* signal as:

$$BRET = BRET\_0 + k\left(\left[D\right] + \left[A\right]\right) \tag{4}$$

where [*D*] and [*A*] are donor and acceptor concentrations. With lowering the concentration of both receptors simultaneously (dilution) the *BRET* signal approaches *BRET*0 which is the real oligomerisation signal (Fig. 1). Dilution assay is used to set the concentration range for saturation and competition assays (Breit et al., 2004).

Fig. 1. BRET dilution assay. Theoretical BRET concentration curves for receptors forming monomers or oligomers. A constant ratio between acceptor and donor concentrations should be used.

#### **5.3 BRET saturation assay**

Saturation assay involves expressing a constant amount of donor-tagged receptor with an increasing amounts of acceptor-tagged receptor. Theoretically, *BRET* signal should increase with increasing amounts of acceptor until all donor molecules are interacting with acceptor molecules. Therefore, a saturation level is achieved beyond which a further elevation of the amount of acceptor does not increase the *BRET* signal, thereby reaching a maximal *BRET*  level (*BRET*max) (Achour et al., 2011; Ayoub & Pfleger, 2010; Hamdan et al., 2006; Mercier et al., 2002). By using a saturation assay it is possible to obtain the oligomerisation state of homologous receptors. BRET saturation curve is derived from Veatch and Stryer model:

$$BRET = \frac{T}{Q} = \frac{E[AD]}{2\left[DD\right] + (1 - E)\left[AD\right]}\tag{5}$$

Quantitative Assessment of Seven Transmembrane Receptors

0

competition curve for dimers:

50

100

150

*BRET* (mBU)

200

250

300

(7TMRs) Oligomerization by Bioluminescence Resonance Energy Transfer (BRET) Technology 93

 Dimer Trimer Tetramer

 Monomer - low receptor conc. Monomer - high receptor conc.

0 2 4 6 810

[*A*]/[*D*]

Fig. 2. BRET saturation assay. Theoretical curves for oligomer formation are plotted as a function of ratio of receptors tagged with acceptor [*A*] and donor [*D*] molecules. In the case

where C represents untagged competitor. If all receptors form dimers and association constants are the same for AA, AD, DD, CD, AC and CC dimers we obtain BRET

> 

*A D A C D D*

1 (1 )

*E*

Usually in BRET saturation experiments high acceptor to donor concentration ratio is used because the variation in this ratio do not influence the *BRET* signal as much as for [*A*]/[*D*]=1. In general the interaction with the untagged receptors causes the reduction of *BRET* signal following a hyperbolic curve (Figure 3). We can very well distinguish if the oligomerisation is present, but the exact oligomerisation state is difficult to assess. Competition assay is more suited for the study of hetero-oligomers where different kind of untagged receptor is competing with the homo-oligomers. The saturation curve is shallower

*E*

(9)

of monomers the BRET signal is created due to random collisions.

*BRET*

if there is a low affinity for hetero-dimer formation compared to homo-dimers

*BRET*<sup>50</sup>

*BRET*max

where [*AD*] are acceptor-donor and [*DD*] donor-donor dimer concentrations. If all receptors form dimers and association constants are the same for AA, AD and DD we obtain BRET saturation curve for dimers:

$$BRET = \frac{E\frac{[A]}{[D]}}{1 + (1 - E)\frac{[A]}{[D]}}\tag{6}$$

For higher oligomers a general BRET saturation curve can be derived (Vrecl et al., 2006):

$$\frac{BRET}{BRET\_{\text{max}}} = 1 - \frac{1}{E + (1 - E)\left(1 + \frac{[A]}{[D]}\right)^N} \tag{7}$$

where *N*=1 for dimer, *N*=2 for trimer and *N*=3 for tetramer. Theoretical BRET saturation curves are presented in Fig. 2. *BRET* for higher oligomers shows faster saturation. For comparison the monomer *BRET* signal which corresponds to random collisions is presented. If receptor concentration is very high then random collisions can generate saturation curve similar to that of the dimers. Thus a dilution experiment should be done first to distinguish random collisions from the oligomerisation.

In heterologous saturation assay different receptors are used as donors and acceptors. In this case saturation curve is influenced by the affinities for homo-dimer and hetero-dimer formation. In practice we can observe a right-shift of the saturation curve where the association constant for hetero-dimers is smaller than that of the homo-dimers yielding higher *BRET*50 values.

#### **5.4 BRET competition assay**

In an attempt to further confirm the existence of oligomer complexes, competition assay can be performed. In this assay the concentration of untagged receptor is increased over a constant concentrations of donor and acceptor tagged receptors (Achour et al., 2011; Vrecl et al., 2006). It is expected that the *BRET* signal would decrease if untagged receptors compete with the tagged receptors for the binding in complexes. Following the Veatch and Stryer approach we obtain *BRET* signal:

$$BRET = \frac{T}{Q} = \frac{E[AD]}{2[DD] + (1 - E)[AD] + [CD]} \tag{8}$$

molecules. Therefore, a saturation level is achieved beyond which a further elevation of the amount of acceptor does not increase the *BRET* signal, thereby reaching a maximal *BRET*  level (*BRET*max) (Achour et al., 2011; Ayoub & Pfleger, 2010; Hamdan et al., 2006; Mercier et al., 2002). By using a saturation assay it is possible to obtain the oligomerisation state of homologous receptors. BRET saturation curve is derived from Veatch and Stryer model:

*T E AD*

where [*AD*] are acceptor-donor and [*DD*] donor-donor dimer concentrations. If all receptors form dimers and association constants are the same for AA, AD and DD we obtain BRET

*Q DD E AD*

 

*A D A D*

1 (1 )

*E*

1 1

*<sup>N</sup> <sup>A</sup> D*

*E*

 max <sup>1</sup> <sup>1</sup>

where *N*=1 for dimer, *N*=2 for trimer and *N*=3 for tetramer. Theoretical BRET saturation curves are presented in Fig. 2. *BRET* for higher oligomers shows faster saturation. For comparison the monomer *BRET* signal which corresponds to random collisions is presented. If receptor concentration is very high then random collisions can generate saturation curve similar to that of the dimers. Thus a dilution experiment should be done first to distinguish

In heterologous saturation assay different receptors are used as donors and acceptors. In this case saturation curve is influenced by the affinities for homo-dimer and hetero-dimer formation. In practice we can observe a right-shift of the saturation curve where the association constant for hetero-dimers is smaller than that of the homo-dimers yielding

In an attempt to further confirm the existence of oligomer complexes, competition assay can be performed. In this assay the concentration of untagged receptor is increased over a constant concentrations of donor and acceptor tagged receptors (Achour et al., 2011; Vrecl et al., 2006). It is expected that the *BRET* signal would decrease if untagged receptors compete with the tagged receptors for the binding in complexes. Following the Veatch and Stryer

*T E AD*

*Q DD E AD CD*

 2 (1 )

*BRET*

*BRET*

random collisions from the oligomerisation.

higher *BRET*50 values.

**5.4 BRET competition assay** 

approach we obtain *BRET* signal:

*BRET*

*BRET*

For higher oligomers a general BRET saturation curve can be derived (Vrecl et al., 2006):

*BRET E E* 

saturation curve for dimers:

 2 (1 )

(5)

(6)

(7)

(8)

Fig. 2. BRET saturation assay. Theoretical curves for oligomer formation are plotted as a function of ratio of receptors tagged with acceptor [*A*] and donor [*D*] molecules. In the case of monomers the BRET signal is created due to random collisions.

where C represents untagged competitor. If all receptors form dimers and association constants are the same for AA, AD, DD, CD, AC and CC dimers we obtain BRET competition curve for dimers:

$$BRET = \frac{E\frac{[A]}{[D]}}{1 + (1 - E)\frac{[A]}{[D]} + \frac{[C]}{[D]}}\tag{9}$$

Usually in BRET saturation experiments high acceptor to donor concentration ratio is used because the variation in this ratio do not influence the *BRET* signal as much as for [*A*]/[*D*]=1. In general the interaction with the untagged receptors causes the reduction of *BRET* signal following a hyperbolic curve (Figure 3). We can very well distinguish if the oligomerisation is present, but the exact oligomerisation state is difficult to assess. Competition assay is more suited for the study of hetero-oligomers where different kind of untagged receptor is competing with the homo-oligomers. The saturation curve is shallower if there is a low affinity for hetero-dimer formation compared to homo-dimers

Quantitative Assessment of Seven Transmembrane Receptors

odimer (Urizar et al., 2011).

Western blot or ELISA assays.

*Med* 7, 1003-1009.

**8. Acknowledgment** 

21528.

12-008).

**9. References** 

**7. Conclusions** 

(7TMRs) Oligomerization by Bioluminescence Resonance Energy Transfer (BRET) Technology 95

ligand-dependent manner is fused to a YFP. Ligand-induced BRET signal indicates that activation of untagged receptor or the heteromer results in recruitment of YFP-tagged protein to the heteromer. Recently developed complemented donor-acceptor resonance energy transfer (CODA-RET) method combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer. CODA-RET quantify the BRET between a receptor hetero-dimer and a subunit of the heterotrimeric G-protein. It eliminates a contribution from homodimeric signaling and enables analyzing the effect of drugs on a defined 7TMR heter-

BRET-based techniques are extremely powerful, provided that they are conducted with the appropriate controls and correctly interpreted. Quantitative BRET assays allow us to support the ability of receptor for homo-dimer and hetero-dimer. Homologous saturation assay provide us with the oligomerisation state of receptors. Data interpretation is more difficult for hetero-oligomers and the mixtures of monomer, dimer and higher oligomer populations. For the quantitative approach we also need to know the relative concentrations of all receptors used in the experiment, which can be obtained from radioligand binding,

We acknowledge funding from the Slovenian Research Agency (program P4-0053) and Slovenian-Danish collaboration grants (BI-DK/06-07-007, BI-DK/07-09-002 and BI-DK/11-

AbdAlla S, Lother H, el Massiery A, Quitterer U. (2001) Increased AT(1) receptor

Achour L, KM, Jockers R, Marullo S. (2011) Using quantitative BRET to assess G proteincoupled receptor homo- and heterodimerization. *Methods Mol Biol*, 756: 183-200. Angers S, Salahpour A, Joly E et al. (2000) Detection of beta 2-adrenergic receptor

Avissar S, Amitai G, Sokolovsky M. (1983) Oligomeric structure of muscarinic receptors is

Ayoub MA, Couturier C, Lucas-Meunier E et al. (2002) Monitoring of ligand-independent

(BRET). *Proc Natl Acad Sci U S A*, 97: 3684-3689.

affinity agonist states. *Proc Natl Acad Sci U S A*, 80: 156-159.

heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. *Nat* 

dimerization in living cells using bioluminescence resonance energy transfer

shown by photoaffinity labeling: subunit assembly may explain high- and low-

dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. *J Biol Chem*, 277: 21522-

Fig. 3. BRET competition assay. In homologous assay the same receptor is used as a competitor, whereas in heterologous assay different receptor is used. For the latter case a hetero-dimer with lower association constant than that of the homo-dimer is presented.

#### **6. Other BRET-based approaches to identify 7TMR hetero-dimerization**

To overcome certain limitations of the classical BRET assays described above, some other BRET-based approaches have been developed to study 7TMR oligomerization/ heterodimerization. Sequential-BRET-FRET (SRET) enables identification of oligomers formed by three different proteins. In SRET, the oxidation of the RLuc substrate by an RLuc-fusion protein triggers the excitation of the acceptor GFP2 by BRET2 and subsequent energy transfer to the acceptor YFP by FRET. Combination of bimolecular fluorescence complementation (BiFC) and BRET techniques is based on the ability to produce a fluorescent complex from non-fluorescent constituents if a protein-protein interaction occurs. Two receptors are fused at their C-termini with either N-terminal or C-terminal fragments of YFP, respectively, and receptor hetero-dimerization causes YFP reconstitution. Then, if there is hetero-trimerization, BRET can be obtained when the cells also co-express the third receptor fused to Rluc (reviewed by (Ferré & Franco, 2010)). GPCR-Heteromer Identification Technology (GPCR-HIT) utilizes BRET and ligand-dependent recruitment of a 7TMR-specific interaction partners (such as a β-arrestin, PKC or G-protein) to enable 7TMR heteromer discovery and characterization (Mustafa & Pfleger, 2011; See et al., 2011). In this set up, only one receptor subtype is fused to Rluc and the second receptor subtype is untagged. A third protein capable of interacting specifically with one or both receptors in a ligand-dependent manner is fused to a YFP. Ligand-induced BRET signal indicates that activation of untagged receptor or the heteromer results in recruitment of YFP-tagged protein to the heteromer. Recently developed complemented donor-acceptor resonance energy transfer (CODA-RET) method combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer. CODA-RET quantify the BRET between a receptor hetero-dimer and a subunit of the heterotrimeric G-protein. It eliminates a contribution from homodimeric signaling and enables analyzing the effect of drugs on a defined 7TMR heterodimer (Urizar et al., 2011).
