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
. 2009 May;157(1):64-75.
doi: 10.1111/j.1476-5381.2009.00152.x.

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Carla Ferrada  1 , Estefanía Moreno, Vicent Casadó, Gerold Bongers, Antoni Cortés, Josefa Mallol, Enric I Canela, Rob Leurs, Sergi Ferré, Carme Lluís, Rafael Franco
Affiliations

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Carla Ferrada et al. Br J Pharmacol. 2009 May.

Abstract

Background and purpose: Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1-H3 receptor heteromers and their biochemical characteristics.

Experimental approach: D1-H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors.

Key results: Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to G(s) proteins and leading to increases in cAMP, did not couple to G(s) but to G(i) in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor.

Conclusions and implications: D1-H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G(s)-independent and G(i)-dependent manner. An antagonist of one of the receptor units in the D1-H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Heteromerization of functional D1 and H3 receptors. (A) Confocal microscopy images of HEK-293 cells expressing D1 receptor–RLuc (0.1 µg plasmid) and H3 receptor–YFP (0.1 µg plasmid). Proteins were identified by fluorescence or by immunocytochemistry. D1 receptor–RLuc immunoreactivity in shown in blue (a), H3 receptor–YFP fluorescence in shown in green (b) and co-localization of D1 receptor–RLuc and H3 receptor–YFP is shown in light blue (c). (B) Functionality of D1 receptor–RLuc (D1R–RLuc) and H3 receptor–YFP (H3R–YFP) constructs. HEK-293 cells transfected with 5 µg of cDNA corresponding to D1 receptors or D1 receptor–RLuc were stimulated with the D1 receptor agonist SKF 81297 (10 µmol·L−1), and HEK-293 cells transfected with 5 µg of cDNA corresponding to H3 receptors or H3 receptor–YFP were treated with 10 µmol·L−1 forskolin plus the H3 receptor agonist RAMH (0.1 µmol·L−1). Results (mean ± SEM; n= 2–4) are expressed as percentage over basal (upper panel) or over forskolin (FK) alone (lower panel); significantly different compared with the basal for D1 receptors and D1 receptor–RLuc or compared with forskolin alone for H3 receptors or H3 receptor–YFP, (non-paired Student's t-test: *P < 0.05, **P < 0.01 and ***P < 0.001). (C) Correlation between 2.1 nmol·L−1[3H]SCH 23390 binding and luminiscence expression (upper panel) or 1.9 nmol·L−1[3H]RAMH binding and fluorescence expression (lower panel) in HEK-293 cell transfected with increasing amounts of cDNA for D1 receptor–RLuc (upper panel) or H3 receptor–YFP (lower panel) (D) D1–H3 receptor heteromerization in HEK-293 cells. BRET experiments were performed with HEK-293 cells co-expressing D1 receptor–RLuc and H3 receptor–YFP, D1 receptor–RLuc and CB1 receptor–YFP, 5HT2B receptor–RLuc and H3 receptor–YFP or D1 receptor–RLuc and H4 receptor–YFP constructs. Co-transfections were performed with increasing amounts of plasmid–YFP (0.5–9 µg cDNA) whereas the plasmid–RLuc construct was maintained constant (250 ng cDNA). Both fluorescence and luminiscence of each sample were measured before every experiment to confirm similar donor expressions (about 250 000 luminescent units) while monitoring the increase acceptor expression (5000–80 000 fluorescent units). The relative amount of BRET is given as the ratio between the fluorescence of the acceptor and the luciferase activity of the donor. YFP0 corresponds to the fluorescence value of cells expressing the donor alone. BRET data are expressed as means ± SD of 3–13 different experiments grouped as a function of the amount of BRET acceptor. [3H]RAMH, [3H]R-α-methyl histamine; BRET, Bioluminescence Resonance Energy Transfer; HEK, human embryonic kidney; RLuc, Renilla luciferase; Veh, vehicle.
Figure 2
Figure 2
Crosstalk between H3 receptors and D1 receptors in HEK-293 cells. HEK-293 cells transiently expressing H3 receptors (HEK-H3) or D1 receptors (HEK-D1) (A) or both (HEK-D1H3) (B) were treated for 2 min with the H3 receptor agonist RAMH (1 µmol·L−1) or with the D1 receptor agonist SKF 81297 (1 µmol·L−1, SKF), in the presence or in the absence of the H3 receptor antagonist thioperamide (10 µmol·L−1, Thiop) or the D1 receptor antagonist SCH 23390 (10 µmol·L−1, SCH), and ERK1/2 phosphorylation (P-ERK) was determined as indicated in Methods. A representative Western blot is shown in each panel. The immunoreactive bands from three independent experiments were quantified, and values represent the mean ± SEM of fold increase of phosphorylation over the basal levels found in untreated cells. Significant differences with respect to the treatment with vehicle, were calculated by Student's t-test for unpaired samples (*P < 0.05 and **P < 0.01). ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; RAMH, R-α-methyl histamine; Veh, vehicle.
Figure 3
Figure 3
ERK1/2 phosphorylation (P-ERK) via the D1–H3 receptor heteromer in human neuroblastoma cells. SK-N-MC cells expressing H3 receptors (SK-N-MC/H3) or D1 receptors (SK-N-MC/D1) or both (SK-N-MC/D1H3) were treated with the H3 receptor agonist, RAMH (1 µmol·L−1), or with the D1 receptor agonist, SKF 81297 (1 µmol·L−1, SKF) alone or in combination, in the presence or in the absence of the H3 receptor antagonist, thioperamide (10 µmol·L−1, Thiop) or the D1 receptor antagonist, SCH 23390 (10 µmol·L−1, SCH). ERK1/2 phosphorylation was determined as indicated in Methods after 2 min of agonist treatment (A, B and C). In (D) a time–course response of ERK1/2 phosphorylation induced by 1 µmol·L−1 SKF 81297 or 1 µmol·L−1 RAMH in SK-N-MC/D1H3 cells is shown. A representative Western blot is shown in each panel. The immunoreactive bands from three to four experiments were quantified, and values represent the mean ± SEM of fold increase of phosphorylation over the basal levels found in untreated cells. Significant differences were calculated by Student's t-test for unpaired samples (*P < 0.05, **P < 0.01, ***P < 0.001). ERK, extracellular signal-regulated kinase; RAMH, R-α-methyl histamine; Veh, vehicle.
Figure 4
Figure 4
Effect of receptor antagonists on ERK1/2 phosphorylation (P-ERK) via the D1–H3 receptor heteromer in human neuroblastoma cells. SK-N-MC cells expressing H3 receptors and D1 receptors (SK-N-MC/D1H3) were treated with the H3 receptor agonist, RAMH (1 µmol·L−1), or the D1 receptor agonist, SKF 81297 (1 µmol·L−1. SKF), in the presence or in the absence of the H3 receptor antagonist, thioperamide (10 µmol·L−1, Thiop) or the D1 receptor antagonist, SCH 23390 (10 µmol·L−1, SCH). ERK1/2 phosphorylation was determined as indicated in Methods after 2 min of agonist treatment. A representative Western blot is shown. The immunoreactive bands from four experiments were quantified, and values represent the mean ± SEM of percentage of phosphorylation of agonist-treated cells. Significant differences were calculated by Student's t-test for unpaired samples (**P < 0.01, ***P < 0.001). ERK, extracellular signal-regulated kinase; RAMH, R-α-methyl histamine.
Figure 5
Figure 5
cAMP production by D1–H3 receptor heteromer in human neuroblastoma cells. SK-N-MC cells expressing (A) H3 receptors (SK-N-MC/H3) or (B) D1 receptors (SK-N-MC/D1) or (C) both (SK-N-MC/D1H3) were treated or not with 10 µmol·L−1 forskolin (FK) and the H3 receptor agonist, RAMH (0.1 µmol·L−1), and/or the D1 receptor agonist, SKF 81297 (1 µmol·L−1, SKF). The effect of the H3 receptor antagonist, thioperamide (10 µmol·L−1, Thiop) or the D1 receptor antagonist, SCH 23390 (10 µmol·L−1, SCH) was also assayed. cAMP levels were determined as indicated in Methods. Results are expressed as fold increase over basal levels obtained in untreated cells (mean ± SEM of three to five experiments). Significant differences were calculated by Student's t-test for unpaired samples (*P < 0.05, **P < 0.01). RAMH, R-α-methyl histamine; Veh, vehicle.
Figure 6
Figure 6
Effect of PTX and CTX on SKF- or RAMH-induced ERK1/2 phosphorylation (P-ERK). SK-N-MC cells expressing H3 receptors and D1 receptors (SK-N-MC/D1H3) were treated with PTX (100 ng·mL−1) for 16 h or with CTX (1 µg·mL−1) for 30 min prior to the addition of the H3 receptor agonist, RAMH (1 µmol·L−1), or the D1 receptor agonist, SKF 81297 (1 µmol·L−1, SKF). ERK1/2 phosphorylation was determined as indicated in Methods. A representative Western blot is shown. The immunoreactive bands from four experiments were quantified, and values represent the mean ± SEM of fold increase of phosphorylation over basal levels found in untreated cells. Significant differences were calculated by Student's t-test for unpaired samples (*P < 0.05 and **P < 0.01). CTX, cholera toxin; ERK, extracellular signal-regulated kinase; RAMH, R-α-methyl histamine; PTX, Pertussis toxin; Veh, vehicle.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agnati LF, Ferré S, Lluis C, Franco R, Fuxe K. Molecular mechanisms and therapeutical implications of intramembrane receptor/receptor interactions among heptahelical receptors with examples from the striatopallidal GABA neurons. Pharmacol Rev. 2003;55:509–550. - PubMed
    1. Agnati LF, Fuxe K, Ferré S. How receptor mosaics decode transmitter signals. Possible relevance of cooperativity. Trends Biochem Sci. 2005;30:188–193. - PubMed
    1. Alexander SPH, Mathie A, Peters JA. Guide to Receptors and Channels (GRAC) Br J Pharmacol. (3rd) 2008;153(Suppl.)(2):S1–S209. edn (2008 revision. - PMC - PubMed
    1. Anichtchik OV, Peitsaro N, Rinne JO, Kalimo H, Panula P. Distribution and modulation of histamine H(3) receptors in basal ganglia and frontal cortex of healthy controls and patients with Parkinson. s disease. Neurobiol Dis. 2001;8:707–716. - PubMed
    1. Arias-Montano JA, Floran B, Garcia M, Aceves J, Young JM. Histamine H(3) receptor-mediated inhibition of depolarization-induced, dopamine D(1) receptor-dependent release of [(3)H]-gamma-aminobutryic acid from rat striatal slices. Br J Pharmacol. 2001;33:165–171. - PMC - PubMed

Publication types

MeSH terms

Substances