Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul 24;7(9):624-30.
doi: 10.1038/nchembio.623.

CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Eneko Urizar  1 , Hideaki Yano, Rachel Kolster, Céline Galés, Nevin Lambert, Jonathan A Javitch
Affiliations

CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Eneko Urizar et al. Nat Chem Biol. .

Abstract

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a G protein-coupled receptor (GPCR) protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling.

PubMed Disclaimer

Conflict of interest statement

Competing Financial Interests: Authors declare no competing financial interests.

Figures

Figure 1
Figure 1. D1R coexpression enhances NPA potency at D2R
HEK 293T cells transiently expressing wild type human D1R (a) or D2R (b) full length RLuc8 C-terminal fusions alone or in combination with unfused D2R (a) or D1R (b) respectively; together with Gαs-mVenus (a) or Gαi-mVenus (b) and unfused β1 and γ2 G protein subunits were harvested 48 h post-transfection, washed with PBS, centrifuged and resuspended in PBS. Cells were distributed in 96-well plates and preincubated with coelenterazine H for 1 min and then with increasing concentrations of dopamine for 1 min. BRET1 was performed as described in Materials and Methods and results were fit by non-linear regression to a sigmoidal dose-response relationship against the agonist concentration. The graphs are representative of 2-12 independent experiments performed with triplicate samples (error bars represent S.E.M), and summary data are presented in Table 1. The cartoons indicate the constructs expressed: D1R in red and D2R in black with the full length RLuc8 (blue) at the C terminus and the indicated Gα sensor (red for Gαs or black for Gαi with the β1 (in green) and γ2 (in light blue) subunits; the dotted arrow represents the energy transfer.
Figure 2
Figure 2. Characterization of homomeric receptors in G protein signaling complexes
HEK 293T cells coexpressing D1R-L1, D1R-L2 and Gαs-mVenus (red symbols and curves) or Gαi-mVenus (black symbols) (a-d) or D2R-L1, D2R-L2 and Gαi-mVenus (black symbols and curves) or Gαs-mVenus (red symbols) (e-h) were prepared as in Figure 1. In experiments in which antagonists (SCH23390, sulpiride) were used (b, f open symbols), the antagonists (1 μM) were preincubated for 15 mins at RT before the addition of the substrate and the tested agonist. BRET1 was measured as explained in Material and Methods and analyzed as for Figure 1. The graphs are representative of 2-12 independent experiments performed with triplicate samples (error bars represent S.E.M), and summary data are presented in Table 1.
Figure 3
Figure 3. Characterization of the heteromeric receptors in G protein signaling complexes
HEK 293T cells coexpressing D2R-L1, D1R-L2 and Gαs-mVenus (a-d) or Gαi-mVenus (e-h) were prepared as described in Figure 1. In experiments in which antagonists were used (open symbols), the antagonists (1 μM) were preincubated for 15 mins at RT before the addition of the substrate and the tested agonist. BRET1 was measured as explained in Material and Methods and analyzed as for Figure 1. The graphs are representative of 2-12 independent experiments performed with triplicate samples (error bars represent S.E.M), and summary data are presented in Table 1.
Figure 4
Figure 4. CODA-RET reveals functional selectivity of NPA at the D2R-D1R heteromer
HEK 293T cells coexpressing D1R-L1 and D1R-L2 or D2R-L1 and D1R-L2 together with Gαs-mVenus (a) or D2R-L1 and D2R-L2 or D2R-L1 and D1R-L2 together with Gαi-mVenus (b) were prepared as described in Figure 1. BRET1 was measured as explained in Material and Methods and analyzed as for Figure 1 and EC50 values were calculated after normalization (% of maximal BRET1 signal) and application of global sigmoidal dose-response fitting with shared values for basal, EC50 and Emax. The graphs are representative of at 2-12 independent experiments performed with triplicate samples (error bars represent S.E.M).
Figure 5
Figure 5
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Springael JY, Urizar E, Costagliola S, Vassart G, Parmentier M. Allosteric properties of G protein-coupled receptor oligomers. Pharmacology & Therapeutics. 2007;115:410–418. - PubMed
    1. Han Y, Moreira IS, Urizar E, Weinstein H, Javitch JA. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat Chem Biol. 2009;5:688–695. - PMC - PubMed
    1. Ferre S, et al. Building a new conceptual framework for receptor heteromers. Nat Chem Biol. 2009;5:131–134. - PMC - PubMed
    1. Albizu L, et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol. 2010;6:587–594. - PMC - PubMed
    1. Rivero-Muller A, et al. Rescue of defective G protein-coupled receptor function in vivo by intermolecular cooperation. Proc Natl Acad Sci U S A. 2010;107:2319–2324. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources