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.2022 Aug 2;11(15):2384.
doi: 10.3390/cells11152384.

Serotonin Receptor 5-HT2A Regulates TrkB Receptor Function in Heteroreceptor Complexes

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Serotonin Receptor 5-HT2A Regulates TrkB Receptor Function in Heteroreceptor Complexes

Tatiana Ilchibaeva et al. Cells..

Abstract

Serotonin receptor 5-HT2A and tropomyosin receptor kinase B (TrkB) strongly contribute to neuroplasticity regulation and are implicated in numerous neuronal disorders. Here, we demonstrate a physical interaction between 5-HT2A and TrkB in vitro and in vivo using co-immunoprecipitation and biophysical and biochemical approaches. Heterodimerization decreased TrkB autophosphorylation, preventing its activation with agonist 7,8-DHF, even with low 5-HT2A receptor expression. A blockade of 5-HT2A receptor with the preferential antagonist ketanserin prevented the receptor-mediated downregulation of TrkB phosphorylation without restoring the TrkB response to its agonist 7,8-DHF in vitro. In adult mice, intraperitoneal ketanserin injection increased basal TrkB phosphorylation in the frontal cortex and hippocampus, which is in accordance with our findings demonstrating the prevalence of 5-HT2A-TrkB heteroreceptor complexes in these brain regions. An expression analysis revealed strong developmental regulation of 5-HT2A and TrkB expressions in the cortex, hippocampus, and especially the striatum, demonstrating that the balance between TrkB and 5-HT2A may shift in certain brain regions during postnatal development. Our data reveal the functional role of 5-HT2A-TrkB receptor heterodimerization and suggest that the regulated expression of 5-HT2A and TrkB is a molecular mechanism for the brain-region-specific modulation of TrkB functions during development and under pathophysiological conditions.

Keywords: 5-hydroxytryptamine 2A receptor; autophosphorylation; heteroreceptor; oligomerization; tropomyosin receptor kinase B.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Interaction between receptors 5-HT2A and TrkB in neuroblastoma N1E-115 cells. (A) Co-immunoprecipitation of recombinant HA-tagged 5-HT2A and GFP (mEGFP)-tagged TrkB. Using a mixture of cells expressing each individual protein (mix) or cells that co-expressed both proteins (co-transfection), we performed immunoprecipitation (IP) with an anti-GFP antibody, followed by Western blot (WB) analysis with anti-GFP (right) and anti-HA (left) antibodies. Top: Expression of proteins before IP (lysate w/o IP). Bottom: Expression of proteins after IP. Results are representative of at least three independent experiments. (B–D) Specific interaction between 5-HT2A-mTurquoise2 and TrkB-YPet. Cells co-expressing 5-HT2A-mTurquoise2 and TrkB-YPet were analyzed using the lux-FRET method after confocal microscopy. (B) Distributions of 5-HT2A-mTurquoise2 (donor) and TrkB-YPet (acceptor), and merged images quantified by linear unmixing of the fluorescence emission spectra. Scale bar, 5 µm. (C) Apparent FRET efficiency (EfDA). A representative cell is shown. (D) Quantification of FRET efficiency (EfDA) between 5-HT2A-mTurquoise2 and TrkB-YPet. ***p < 0.001 (one-way ANOVA).
Figure 2
Figure 2
Interaction between receptors 5-HT2A and TrkB in the mouse brain. (A) Co-localization of 5-HT2A and TrkB in the mouse brain. Brain slices from cortex (CRTX), striatum (STR), and hippocampus (HIPP) were subjected to immunohistochemistry for the detection of 5-HT2A (green) and TrkB (red), followed by confocal microscopy. Nuclei are shown in blue. Right: Merged images and a magnified view of representative neurons. (B) Co-immunoprecipitation between 5-HT2A and TrkB in brain lysates. Whole-brain homogenates were prepared from different brain areas and subjected to IP with an anti-5-HT2A receptor antibody, followed by WB analysis with anti-TrkB and anti-5-HT2A antibodies. AB: antibody; IP, immunoprecipitation; WB, Western blot; ● glycosylated TrkB. Results are representative of at least three independent experiments. (C) Detection of 5-HT2A–TrkB heteroreceptor complexes in mouse brain slices using proximity ligation assay (PLA). Representative images of PLA staining in cortex (CRTX), hippocampus (HIPP), and striatum (STR) are shown as a single Z-stack. Nuclei are shown in blue, and PLA blobs are shown in red. Negative control was performed omitting primary antibodies. Scale bar, 20 µm. (D) Quantification of the 5-HT2A-TrkB heterodimer density. Data are presented as means ± SEM (n = 6 mice). Each colored dot represents data obtained for one mouse. *p < 0.05, ***p < 0.001 (one-way ANOVA).
Figure 3
Figure 3
Heterodimerization does not affect basal Ca2+ levels in N1E-115 cells. Neuroblastoma cells were transfected with Ca2+ indicator GCaMP6f, either alone or together with 5-HT2A-mTurquoise2, TrkB-YPet, or both. Basal Ca2+ activity was calculated as F/Fmax. To saturate Ca2+ levels, 10 µM ionomycin was applied after 6 min of recording. The saturated Ca2+ signal was used as Fmax. Data are presented as means ± SEM (12 ≤n ≤ 17). ***p < 0.001 (Kruskal–Wallis test with Dunn’s multiple-comparison post hoc test).
Figure 4
Figure 4
Heterodimerization affects TrkB phosphorylation. (A,B) Analysis of TrkB phosphorylation. Neuroblastoma N1E-115 cells expressing either HA-tagged 5-HT2A (1 µg), GFP-tagged TrkB (1 µg), or co-expressing equal amounts of both receptors were treated for 30 min with either (A) the 5-HT2A receptor agonist 25CN-NBOH (1 μM) or (B) the TrkB agonist 7,8-DHF (500 nM). Cells were lysed, followed by SDS-PAGE and Western blot (WB) analysis using antibodies against either total TrkB or phosphorylated TrkB. The 5-HT2A receptor was detected in parallel. In WB, ß-tubulin was used as a loading control. Representative Western blots are shown. Lower panels show the quantification of TrkB phosphorylation, which was performed by densitometry and calculated as the ratio of total TrkB expression to the TrkB phosphorylation signal after adjustment for the general expression level. Bars show means ± SEM (n ≤ 8). ***p < 0.001 (two-way ANOVA). (C) Heterodimerization affects agonist-mediated TrkB phosphorylation. Neuroblastoma cells were co-transfected with 1 μg of cDNA encoding the GFP-tagged TrkB receptor, together with increasing concentrations of HA-tagged 5-HT2A receptor. Cells were treated for 30 min with either 500 nM 7,8-DHF or vehicle, followed by WB analysis. Lower panel: Quantification of TrkB phosphorylation. Bars show means ± SEM (n = 6). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA).
Figure 5
Figure 5
Expression profiles of TrkB and 5-HT2A receptors during postnatal development. (A,B) Expression levels of mRNAs encoding 5-HT2A (A) and TrkB (B) in the indicated brain regions were determined at different stages of postnatal development using real-time PCR. Gene expression is presented as the number of gene cDNA copies per 100 cDNA copies ofPolr2a, used as a calibration control. (C) Expression ratios between 5-HT2A and TrkB. Bars represent means ± SEM (5 ≤n ≤ 10). *p < 0.05; **p < 0.01 ***p < 0.001 (one-way ANOVA). (D) Heterodimerization exerts an inhibitory effect on agonist-independent TrkB autophosphorylation. Neuroblastoma cells were co-transfected with 1 μg of cDNA encoding the GFP-tagged TrkB receptor, together with increasing concentrations of HA-tagged 5-HT2A receptor, followed by Western blot (WB) analysis. Lower panel: Quantification of TrkB phosphorylation. Bars show means ± SEM (n = 6). ns: not significant,p = 0.06: an insignificant trend; **p < 0.01, ***p < 0.001 (one-way ANOVA).
Figure 6
Figure 6
Blockade of 5-HT2A with ketanserin reversed TrkB autophosphorylation in vitro and in vivo. (A) Neuroblastoma N1E-115 cells expressing HA-tagged 5-HT2A receptor, GFP-tagged TrkB, or co-expressing both receptors were treated with the 5-HT2A receptor antagonist ketanserin (1 μM) for 10 min, followed by treatment with either 7,8-DHF (500 nM) or vehicle for 30 min. Cell lysates were then subjected to SDS-PAGE and Western blot (WB) analysis with antibodies against phosphorylated or total TrkB. β-tubulin was used as a loading control. Representative WBs are shown. Bars show means ± SEM (n ≤ 8). *p < 0.05; ***p < 0.001 (two-way ANOVA). (B) Mice were intraperitoneally injected with ketanserin (1 mg/kg) or 25CN-NBOH (1 mg/kg), resulting in increased TrkB phosphorylation in the hippocampus and frontal cortex. At 10 min (for 25CN-NBOH) or 30 min (for ketanserin) after injection, the animals were euthanized, and brain lysates were prepared from the frontal cortex, hippocampus, and striatum. This was followed by SDS-PAGE and WB to detect phosphorylated and total TrkB. GAPDH was used as a loading control. Representative WBs are shown. Bars represent means ± SEM (5 ≤n ≤ 8) *p < 0.05; **p < 0.01 (two-way ANOVA).
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This research was supported by the DFG grants PO732 to Evgeni Ponimaskin, AZ994 to Andre Zeug and GU1521 to Daria Guseva and Russian Foundation for Basic Research grant 20-04-00253 to Tatiana V. Ilchibaeva. The animal maintenance was supported by the Basic Research Project FWNR-2022-0023.

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