Convergence of melatonin and serotonin (5-HT) signaling at MT2/5-HT2C receptor heteromers

Abstract

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2C receptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT2 and 5-HT2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT2/5-HT2C heteromers amplified the 5-HT-mediated Gq/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT2C protomer of MT2/5-HT2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows "biased signaling." These observations demonstrate the existence of functionally unique MT2/5-HT2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT2/5-HT2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.

Keywords: Cell Signaling; Depression; G Protein-coupled Receptor (GPCR); GPCR; Heteromerization; Melatonin; Molecular Pharmacology; Serotonin.

FIGURE 1.

Formation of MT₁/5-HT_2C and MT₂/5-HT_2C heteromers. A and B, co-immunoprecipitation of MT₁/5-HT_2C and MT₂/5-HT_2C heteromers. Lysates from HEK293T cells expressing the indicated receptors were immunoprecipitated (IP) with antibodies recognizing Myc and FLAG epitopes, and the presence of co-precipitated 5-HT_2C-YFP was detected with antibodies against GFP (lower Western blot (WB)). Expression of fusion proteins in lysates was assessed by immunoblotting with the indicated antibodies (upper Western blots). Data are representative of three experiments. C, BRET donor saturation curves were determined by co-transfecting a fixed amount of 5-HT_2C-Rluc in the presence of increasing amounts of the indicated YFP fusion proteins in HEK293 cells. Curves were normalized to BRET_max values. The saturation curves were obtained from three independent experiments performed in triplicates. D, immunofluorescence staining of permeabilized HeLa cells showing localization of 3xHA-5-HT_2C (FITC) and Myc-MT₂ (TRITC) expressed alone (upper and middle panels, respectively) or together (lower panels) (scale bar, 17 μm). Data are representative of three experiments. E, immunoprecipitation of melatonin receptors with 5-HT_2C receptors in pork choroid plexus; ¹²⁵I-labeled melatonin receptors were immunoprecipitated from solubilized membranes by anti-5-HT_2C antibodies (Ab) or control (Ctrl) rabbit sera (pool of five preimmune rabbit sera). Data represent the mean ± S.E. (error bars) of three independent experiments (*, p < 0.05). Representative results are shown for A, C, and D; similar results were obtained in two additional experiments.

FIGURE 2.

Effect of melatonin- and 5-HT-promoted activation of MT₂/5-HT_2C heteromers on the G_i/cAMP pathway. HEK293 cells expressing 5-HT_2C (▿), MT₂ (●), or 5-HT_2C and MT₂ (○) receptors were either treated with 2 μm forskolin (Fsk) and increasing concentrations of melatonin (MLT) (A) or increasing concentrations of 5-HT (B). Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates.

FIGURE 3.

5-HT-promoted potentiation of the G_q/PLC pathway by the MT₂/5-HT_2C heteromer. A, 5-HT-induced IP production was assessed in HEK293 cells expressing 5-HT_2C (▿), MT₂ (●), or 5-HT_2C and MT₂ (○) receptors. B–E, HEK293 cells expressing 5-HT_2C receptors alone or together with MT₂ receptors were treated with heterotrimeric G protein inhibitors (B), 5-HT_2C receptor ligands (D), or the melatonin receptor ligand S20928 (E) or co-expressed with the βARK_Cter Gβγ scavenger (C). F, cell surface expression of 5-HT_2C and MT₂ receptors in cells expressing 5-HT_2C in the absence and presence of MT₂ receptors measured by in-cell Western experiments (*, p < 0.05; n.s., not significantly different). Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates. Results that are statistically different compared with 5-HT alone for 5-HT_2C cells (●, p < 0.05; ●●, p < 0.01) and MT₂/5-HT_2C cells (**, p < 0.01; ***, p < 0.001) are indicated.

FIGURE 4.

Melatonin activates the G_q/PLC pathway in the MT₂/5-HT_2C heteromer. A, melatonin (MLT)-induced IP production was assessed in HEK293 cells expressing 5-HT_2C (▿), MT₂ (●), or 5-HT_2C and MT₂ (○) receptors. B, HEK293 cells co-expressing MT₂ receptors and M1 muscarinic receptors were stimulated with melatonin or acetylcholine, and IP production was measured. The influence of the βARK_Cter Gβγ scavenger (C) or heterotrimeric G protein inhibitors (D) on melatonin-promoted IP production of the MT₂/5-HT_2C heteromer is shown. Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates. Results that are statistically different compared with melatonin alone for MT₂/5-HT_2C cells (*, p < 0.05) are indicated.

FIGURE 5.

Transactivation of the G_q/PLC pathway by melatonin in the MT₂/5-HT_2C heteromer. A, melatonin (MLT)-induced IP production in the presence of 5-HT_2C ligands in HEK293 cells expressing MT₂ receptors alone or co-expressing 5-HT_2C receptors (*, p < 0.05: melatonin versus melatonin + SB206553 on heteromers). B, 5-HT-induced IP production in cells co-expressing the 5-HT_2C-S138N mutant and MT₂ receptor. C, melatonin-induced transactivation of 5-HT_2C wild-type and 5-HT_2C-S138N mutant receptors in the presence of MT₂. D, BRET donor saturation curves of HEK293 cells expressing MT₂-Rluc and 5-HT_2C-Rluc or 5-HT_2C-S138N-Rluc. Melatonin-induced (E) and 5-HT-induced (F) recruitment of β-arrestin 2 measured by BRET in HEK293 cells expressing 5-HT_2C (▿), MT₂ (●), or 5-HT_2C and MT₂ (○) receptors is shown. Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates. mBu, milli-BRET units.

FIGURE 6.

Biased ligands reveal unique functional profile of MT₂/5-HT_2C heteromers. A, cAMP production in HEK293 cells co-expressing MT₂ and 5-HT_2C receptors in the presence of 2 μm forskolin (Fsk) and different concentrations of melatonin (MLT) or 10 nm melatonin and different concentrations of 4-PPDOT, luzindole, or S20928. B, antagonistic effect of S20928 on melatonin-induced IP production in HEK293 cells expressing MT₂ alone or together with 5-HT_2C receptors. C, IP production in HEK293 cells co-expressing MT₂ and 5-HT_2C receptors in the presence of different concentrations of the indicated ligands. Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates.

FIGURE 7.

Effect of agomelatine on the MT₂/5-HT_2C heteromer signaling. Effect of melatonin (MLT) and agomelatine on cAMP (A) and IP (B) production in HEK293 cells co-expressing MT₂ and 5-HT_2C receptors. C, effect of agomelatine on melatonin-promoted IP production in cells expressing MT₂ receptors alone or together with 5-HT_2C receptors. D, effect of agomelatine on 5-HT-promoted IP production in cells expressing 5-HT_2C receptors alone or together with MT₂ receptors. Data represent the mean ± S.E. (error bars) of at least three independent experiments performed in triplicates. Results that are statistically different compared with melatonin (C) or 5-HT (D) alone for MT₂/5-HT_2C cells (*, p < 0.05) are indicated. Fsk, forskolin.