In this paper, we present evidence that activation of 5-hydroxytryptamine 2B (5-HT2B) receptors by serotonin (5-HT) leads to cell-cycle progression through retinoblastoma protein hyperphosphorylation ...and through activation of both cyclin D1/cdk4 and cyclin E/cdk2 kinases by a mechanism that depends on induction of cyclin D1 and cyclin E protein levels. The induction of cyclin D1 expression, but not that of cyclin E, is under mitogen-activated protein kinase (MAPK) control, indicating an independent regulation of these two cyclins in the 5-HT2B receptor mitogenesis. Moreover, by using the specific platelet-derived growth factor receptor (PDGFR) inhibitor AG 1296 or by overexpressing a kinase-mutant PDGFR, we show that PDGFR kinase activity is essential for 5-HT2B-triggered MAPK/cyclin D1, but not cyclin E, signaling pathways. 5-HT2B receptor activation also increases activity of the Src family kinase, c-Src, Fyn, and c-Yes. Strikingly, c-Src, but not Fyn or c-Yes, is the crucial molecule between the Gqprotein-coupled 5-HT2B receptor and the cell-cycle regulators. Inhibition of c-Src activity by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidi ne (PP1) or depletion of c-Src is sufficient to abolish the 5-HT-induced (i) PDGFR tyrosine kinase phosphorylation and MAPK activation, (ii) cyclin D1 and cyclin E expression levels, and (iii) thymidine incorporation. This paper elucidates a model of 5-HT2B receptor mitogenesis in which c-Src acts alone to control cyclin E induction and in concert with the receptor tyrosine kinase PDGFR to induce cyclin D1 expression via the MAPK/ERK pathway.
New genetic models that target the serotonin system show that transient alterations in serotonin homeostasis cause permanent changes to adult behaviour and modify the fine wiring of brain ...connections. These findings have revived a long-standing interest in the developmental role of serotonin. Molecular genetic approaches are now showing us that different serotonin receptors, acting at different developmental stages, modulate different developmental processes such as neurogenesis, apoptosis, axon branching and dendritogenesis. Our understanding of the specification of the serotonergic phenotype is improving. In addition, studies have revealed that serotonergic traits are dissociable, as there are populations of neurons that contain serotonin but do not synthesize it.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The serotonin receptor subtypes 2 comprise 5-HT2A, 5-HT2B, and 5-HT2C, which are Gαq-coupled receptors and display distinct pharmacological properties. Although co-expressed in some brain regions and ...involved in various neurological disorders, their functional interactions have not yet been studied. We report that 5-HT2 receptors can form homo- and heterodimers when expressed alone or co-expressed in transfected cells. Co-immunoprecipitation and bioluminescence resonance energy transfer studies confirmed that 5-HT2C receptors interact with either 5-HT2A or 5-HT2B receptors. Although heterodimerization with 5-HT2C receptors does not alter 5-HT2C Gαq-dependent inositol phosphate signaling, 5-HT2A or 5-HT2B receptor-mediated signaling was totally blunted. This feature can be explained by a dominance of 5-HT2C on 5-HT2A and 5-HT2B receptor binding; in 5-HT2C-containing heterodimers, ligands bind and activate the 5-HT2C protomer exclusively. This dominant effect on the associated protomer was also observed in neurons, supporting the physiological relevance of 5-HT2 receptor heterodimerization in vivo. Accordingly, exogenous expression of an inactive form of the 5-HT2C receptor in the locus ceruleus is associated with decreased 5-HT2A-dependent noradrenergic transmission. These data demonstrate that 5-HT2 receptors can form functionally asymmetric heterodimers in vitro and in vivo that must be considered when analyzing the physiological or pathophysiological roles of serotonin in tissues where 5-HT2 receptors are co-expressed.
Taking advantage of three cellular systems, we established that 5-HT2B receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line andMastomys natalensis carcinoid cells, ...which naturally express the 5-HT2B receptor, as well as in transfected LMTK− fibroblasts, stimulation of the 5-HT2Breceptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT2Breceptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT2B receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT2B peptide in the three cell types but also in LMTK− fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT2B/iNOS coupling mechanisms appear more complex because neutralization of endogenous Gα13 by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT2B receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.
During embryogenesis, serotonin has been reported to be involved in craniofacial and cardiovascular morphogenesis. The detailed molecular mechanisms underlying these functions, however remain ...unknown. From mouse and human species, we have recently reported the cloning of 5-HT2B receptors which share signal transduction pathways with other 5-HT2 receptor subtypes (5-HT2A and 5-HT2C). In addition to phospholipase C stimulation, it appears that these three subtypes of receptor transduce a common serotonin-induced mitogenic activity, which could be important for cell differentiation and proliferation. We have first investigated the expression of 5-HT2 receptor mRNAs in the mouse embryo. Interestingly, a peak of 5-HT2B receptor mRNA expression was detected 8â9 days postcoitum, whereas there was only low level 5-HT2A and no 5-HT2C receptor mRNA expression at this stage. Expression of this receptor was confirmed by binding assays using a 5-HT2-specific ligand which revealed a peak of binding to membrane preparations from 9 days postcoitum embryos. In addition, whole mount in situ hybridisation and immunohistochemistry on similar stage embryos detected 5-HT2B expression in neural crest cells, heart myocardium and somites. The requirement for functional 5-HT2B receptors between 8 and 9 days postcoitum is supported by culture of embryos exposed to 5-HT2-specific ligands; 5-HT2B high-affinity antagonist such as ritanserin, induced morphological defects in the cephalic region, heart and neural tube. These antagonistic treatments interfere with cranial neural crest cell migration, induce their apoptosis, and are responsible for abnormal sarcomeric organisation of the subepicardial layer and for the absence of the trabecular cell layer in the ventricular myocardium. This report indicates for the first time that 5-HT2B receptors are actively mediating the action of serotonin on embryonic morphogenesis, probably by preventing the differentiation of cranial neural crest cells and myocardial precursor cells.
The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking the serotonin transporter and rely on long-term adaptations of ...pre- and post-synaptic receptors. We show here that long-term behavioral and neurogenic SSRI effects are abolished after either genetic or pharmacological inactivation of 5-HT2B receptors. Conversely, direct agonist stimulation of 5-HT2B receptors induces an SSRI-like response in behavioral and neurogenic assays. Moreover, the observation that (i) this receptor is expressed by raphe serotonergic neurons, (ii) the SSRI-induced increase in hippocampal extracellular serotonin concentration is strongly reduced in the absence of functional 5-HT2B receptors and (iii) a selective 5-HT2B agonist mimics SSRI responses, supports a positive regulation of serotonergic neurons by 5-HT2B receptors. The 5-HT2B receptor appears, therefore, to positively modulate serotonergic activity and to be required for the therapeutic actions of SSRIs. Consequently, the 5-HT2B receptor should be considered as a new tractable target in the combat against depression.
From a single gene Htr2 (pink) located within the Psmd1 gene (grey) in pre-vertebrates (Ciona), and according to published models, we propose that the whole genome duplication (WGD), which arrived at ...the divergence between chordates and jawless fishes, generated one Htr2a (Exons-blue boxes), and one Htr2b (pink) within the Psmd1 gene (grey). A second whole genome duplication that aroused before the appearance of cartilaginous fish and all tetrapods would have generated one Htr2a, one Htr2c (orange), and one Htr2b within exons of Psmd1 and likely a lost copy (X). A later whole genome duplication that took place before the appearance of teleost fish would have generated two Htr2a, two Htr2c (orange), and one Htr2b within exons of Psmd1 and likely a lost copy (X).
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Serotonin is a neurotransmitter widely conserved from ancient organisms lacking nervous systems through man, and its presence precedes the appearance of nervous systems on both developmental and evolutionary time scales. Serotonin receptor subtypes diversified approximately at the time period during which vertebrates diverged from invertebrates. The biological and clinical importance of serotonin receptors, may benefit from studies on their evolution. Although potentially informative about their pathophysiological functions, reviews on this topic are sparse. Several observations support basic functions mediated by serotonin, both in periphery and central nervous system. In particular, 5-HT2B receptors have been implicated in embryonic development, including cell proliferation, survival, and/or differentiation, in either neural crest cell derivatives, myeloid cell lineage, or heart embryogenesis. In this review, we collected existing data about the genomic association between the RPN2 proteasome subunit gene Psmd1 and the 5-HT2B receptor gene Htr2b. We discuss about the possibility that, during genome duplications, a single copy of this pair of genes has been conserved, suggesting a strong selective pressure. Many basic physiological functions in which serotonin system is involved could be linked to the early association of these two genes in pre-vertebrates. Their evolutionary association highlights the possibility that the 5-HT2B receptor gene, Htr2b, is the common ancestor of 5-HT2A/2B/2C-receptor subfamily. Disentangling these possibilities could bring new understanding of the respective importance of these receptors in pathophysiology of serotonin.
Although potentially implicated in several physiological functions, few functional mutations have been identified in the human
5-hydroxytryptamine (HT) 2B receptor gene. A heterozygous mutation R393X ...in the 5-HT 2B receptor was recently identified in a patient diagnosed with pulmonary hypertension after intake of the anorexigenic dexfenfluramine.
Although reported to generate a lack of function, this C terminus-truncated 5-HT 2B receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative
modifications in transduction of the R393X-mutated 5-HT 2B receptor. In stably transfected cells, we confirmed the loss of inositol 1,4,5-trisphosphate stimulation caused by the G αq uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of
nitric-oxide synthase stimulation. However, the truncated R393X receptor presented 1) a strong gain of efficacy in cell proliferation
as assessed by mitogen-activated protein kinase activity and thymidine incorporation, 2) a preferential coupling to G α13 as shown by blocking antiserum, and 3) an apparent lack of internalization upon agonist stimulation as observed by confocal
microscopy. This work demonstrates that, in the 5-HT 2B receptor, the C terminus, including the palmitoylation and phosphorylation sites, is absolutely required for proper transduction
and internalization. For the first time, we show that the lack of C terminus can generate a switch of coupling to G α13 , a reduced NO synthase activation, and an increase in cell proliferation. All these modifications are relevant in pathophysiological
vasoconstriction.
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