Analysis of the Serotonergic System in a Mouse Model of Rett Syndrome Reveals Unusual Upregulation of Serotonin Receptor 5b.

Mutations in the transcription factor methyl-CpG-binding-protein 2 (MeCP2) cause a delayed-onset neurodevelopmental disorder known as Rett syndrome (RTT). Although alteration in serotonin levels have been reported in RTT patients, the molecular mechanisms underlying these defects are not well understood. Therefore, we chose to investigate the serotonergic system in hippocampus and brainstem of male Mecp2-/y knock-out mice in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The serotonergic system in mouse is comprised of 16 genes, whose mRNA expression profile was analyzed by quantitative RT-PCR. Mecp2-/y mice are an established animal model for RTT displaying most of the cognitive and physical impairments of human patients and the selected areas receive significant modulation through serotonin. Using anatomically and functional characterized areas, we found region-specific differential expression between wild type and Mecp2-/y mice at post-natal day 40. In brainstem, we found five genes to be dysregulated, while in hippocampus, two genes were dysregulated. The one gene dysregulated in both brain regions was dopamine decarboxylase, but of special interest is the serotonin receptor 5b (5-ht5b), which showed 75-fold dysregulation in brainstem of Mecp2-/y mice. This dysregulation was not due to upregulation, but due to failure of down-regulation in Mecp2-/y mice during development. Detailed analysis of 5-ht5b revealed a receptor that localizes to endosomes and interacts with Gαi proteins.

Computational and experimental analysis of the transmembrane domain 4/5 dimerization interface of the serotonin 5-HT(1A) receptor.

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT(1A) receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Förster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp175(4.64), Tyr198(5.41), Arg151(4.40), and Arg152(4.41)) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175(4.64) in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT(1A) receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.

5-HT7R/G12 signaling regulates neuronal morphology and function in an age-dependent manner.

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT(7) receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of Gα(12)-deficient mice reveals a critical role of G(12)-protein for 5-HT(7) receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT(7)R/G(12) signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT(7)/G(12) signaling are abolished. Moreover, inhibition of 5-HT(7) receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT(7) and Gα(12)-proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT(7) receptor and Gα(12)-protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking.

Serotonin receptors 5-HT(1A) and 5-HT(7) are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT(1A) and 5-HT(7) receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT(1A) or 5-HT(7) receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT(7)-5-HT(7) homodimers, followed by the 5-HT(7)-5-HT(1A) heterodimers and 5-HT(1A)-5-HT(1A) homodimers. Functionally, heterodimerization decreases 5-HT(1A)-receptor-mediated activation of G(i) protein without affecting 5-HT(7)-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT(1A) receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT(1A) receptor internalization and also enhances the ability of the 5-HT(1A) receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT(7) receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT(1A)-5-HT(7) heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

Constitutive Gs-mediated, but not G12-mediated, activity of the 5-hydroxytryptamine 5-HT7(a) receptor is modulated by the palmitoylation of its C-terminal domain.

The 5-HT(7) receptor is the most recently described member of the serotonin receptor family. This receptor is mainly expressed in the thalamus, hypothalamus as well as in the hippocampus and cortex. In the present study, we demonstrate that the mouse 5-hydroxytryptamine 5-HT(7(a)) receptor undergoes post-translational modification by the palmitate, which is covalently attached to the protein through a thioester-type bond. Analysis of protein-bound fatty acids revealed that the 5-HT(7(a)) receptor predominantly contains palmitic acid. Labelling experiments performed in the presence of agonists show that the 5-HT(7(a)) receptor is dynamically palmitoylated in an agonist-dependent manner and that previously synthesized receptors may be subjected to repeated cycles of palmitoylation/depalmitoylation. Mutation analysis revealed that cysteine residues 404 and 438/441 located in the C-terminal receptor domain are the main palmitoylation sites responsible for the attachment of 90% of the receptor-bound palmitate. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(7(a)) receptors were indistinguishable from the wild-type for their ability to interact with G(s)- and G(12)-proteins after agonist stimulation. However, mutation of the proximal palmitoylation site Cys404-Ser (either alone or in combination with Cys438/441-Ser) significantly increased the agonist-independent, G(s)-mediated constitutive 5-HT(7(a)) receptor activity, while the activation of Galpha(12)-protein was not affected. This demonstrates a functional importance of 5-HT(7(a)) dynamic palmitoylation for the fine tuning of receptor-mediated signaling.

Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors.

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.

Localization of the mouse 5-hydroxytryptamine(1A) receptor in lipid microdomains depends on its palmitoylation and is involved in receptor-mediated signaling.

In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine(1A) receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged alpha(i3) subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G(i)-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Galpha(i) protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca(2+) concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Galpha(i3)-CFP relocation and changes in [Ca(2+)](i) upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.