Serotonin receptor 2
Every living cell must detect, and respond appropriately to, external signals. Thus, the functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. A method has been devised by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HTDro7 and 5HTDro1A]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor has been shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results have uncovered a new role for cGMP signaling in tubules and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists (Kerr, 2004).
Ectopic expression of the rat ANP receptor (GC-A) in tubules was achieved under control of both principal cell and stellate cell GAL4 drivers and a heat-shock (hs) promoter. Expression of the GC-A transgene was confirmed by RT-PCR. Expression of GC-A in tubules confers sensitivity to ANP, with resultant production of cGMP. Measurement of cAMP levels in tubules that express GC-A in principal cells (using the c42 GAL4 driver), stellate cells (using the c742 driver), or ubiquitously, show that cGMP levels are stimulated neither by GC-A receptor, nor by ANP, alone. ANP raises cGMP in a dose-dependent manner in c42-GC-A and c724-GC-A tubules, with a nearly 4-fold maximal increase in cGMP levels in c42-GC-A tubules and a 2-fold cGMP increase in c724-GC-A tubules. Since stellate cells make up only a small fraction of the tubule volume, the apparently lower fold increase in stellate cell cGMP probably reflects a larger absolute rise in cGMP in these cells. The EC50 for ANP in both principal and stellate cells is similar, 10-8 M (Kerr, 2004).
Similar increases in fluid transport are observed upon stimulation with ANP, when GC-A is expressed either in only principal cells or in stellate cells. Although cGMP signaling has been shown in principal cells, a diuretic role for cGMP in stellate cells had not been demonstrated before. The effects of cGMP in principal and stellate cells are additive; when GC-A is expressed ubiquitously under hs control, maximal secretion rates are higher than when it is targeted to either cell alone (Kerr, 2004).
cGMP signals in principal cells may activate CNG-type calcium channels, resulting in calcium increase and fluid transport. Targeted expression of the calcium reporter, aequorin, was used to measure changes in intracellular calcium ([Ca2+]i) in GC-A tubules. In tubule principal cells, a biphasic elevation of [Ca2+]i is observed upon ANP stimulation, followed by a sustained secondary rise in [Ca2+]i. By contrast, no change in stellate cell [Ca2+]i was observed upon ANP challenge. Thus, major cellular targets for cGMP in principal cells are CNG channels. Stellate cells, however, must contain uncharacterized cGMP-activated targets that modulate fluid transport (Kerr, 2004).
ANP-mediated cGMP signaling and increased fluid transport are a result of specific ligand-receptor interactions, since the ANP antagonist, anantin, abolishes ANP stimulation of both cGMP and fluid transport in hs-GC-A tubules in a dose-dependent manner (Kerr, 2004).
Pilot experiments have shown that Drosophila tubules are insensitive to 5HT; there is thus scope to modulate second messengers by ectopic expression of the cognate GPCRs. The Drosophila 5HT7Dro receptor raises cAMP levels in cultured cells by activating adenylate cyclase. When 5HT7Dro is expressed in either principal or stellate cells using the appropriate GAL4 drivers, 5HT induces dose-dependent production of cAMP. EC50 for 5HT in both lines was 10−7 M. Similarly, the heat shock construct elicits elevation of [cAMP] upon 5HT treatment. Controls (non-heat-shocked hs-5HT7Dro and heat-shocked wild-type or parental lines) show no response to 5HT. Furthermore, there is no detectable impact on cGMP levels when 5HT7Dro is driven either in principal cells, stellate cells, or ubiquitously (Kerr, 2004).
As would be expected from the literature, increased [cAMP] in principal cells stimulates fluid transport. However, as for cGMP, a previously undocumented diuretic role of cAMP in stellate cells was uncovered, and the maximal rates of 5HT-induced fluid secretion observed with c42-5HT7Dro and c724-5HT7Dro tubules appear to be additive: the sum of maximal rates is approximately equal to the maximal 5HT-induced rate observed in heat-shocked hs-5HT7Dro tubules. It was thus hypothesized that, if stellate cells have the machinery to respond functionally to cAMP and cGMP, then they are also likely to have the machinery to produce the signals (Kerr, 2004).
As with GC-A, the possibility that [Ca2+]i may be altered upon activation of 5HT7Dro was investigated. In principal cells of 5HT7Dro/c42-aeq tubules, 5HT stimulation results in a biphasic elevation of [Ca2+]i, similar to that seen in GC-A/c42-aeq tubules. However, no [Ca2+]i rise was observed in 5HT-stimulated 5HT7Dro/c710-aeq tubules. These results are consistent with both cAMP and cGMP acting on a CNG channel that is expressed only in principal, and not in stellate, cells (Kerr, 2004).
It proved possible to reproduce the known pharmacology of this 5HT receptor. The antagonist (+)-butaclamol almost completely attenuated 5HT-stimulated production of cAMP in a dose-dependent manner, with an IC50 of 2.5 × 10−8 M. This agrees precisely with values obtained for the receptor in cell lines. A maximal dose of (+)-butaclamol (10−5 M) also reduced 5HT-stimulated fluid transport in hs-5HT7Dro tubules (Kerr, 2004).
Another Drosophila 5HT receptor, 5HT1ADro, is known to mobilize intracellular calcium. Expression of 5HT1ADro in either principal or stellate cells results in 5HT-induced calcium responses. As would be expected from the actions of capa and leucokinin (ligands known to act through [Ca2+]i), 5HT also stimulated fluid transport, when 5HT1ADro was expressed in either principal cells or stellate cells, and no effect was seen without a GAL4 driver. Although a comprehensive dose-response curve was not performed, 5HT-stimulated fluid transport was inhibited by yohimbine. The effective concentration for yohimbine in tubules (10−5 M) is comparable with the Ki (18 μM) obtained for this receptor in cell lines (Kerr, 2004).
The 5HT1ADro receptor has been previously shown to inhibit adenylate cyclase in vitro. Accordingly, cAMP and cGMP were measured in 5HT-stimulated c42-5HT1ADro and c724-5HT1ADro tubules. Activation of the 5HT1ADro receptor in tubules does not affect cyclic nucleotide levels, and thus the stimulatory effects of 5HT on fluid transport are solely due to stimulation of increased [Ca2+]i (Kerr, 2004).
Although receptor guanylate cyclases (like GC-A) are self-sufficient, the strategy of modulating second messengers by ectopic expression of GPCRs depends on the expression of cognate G proteins in the target cell type. A priori, it was expected that most G proteins would be widely expressed, but this was confirmed in tubules by RT-PCR with primers against all known G protein α, β, and γ subunits. As predicted, all the Gα subunit genes found in Drosophila are expressed in tubules. Tubules also express the full complement of genes encoding the Gγ subunit; the only G protein subunit that does not appear to be expressed in tubules is Gβ76C (Kerr, 2004).
Although a particular target cell type might not express cognate G proteins, an RT-PCR strategy compared with measurement of second messengers should be informative. As well as the apoaequorin calcium reporter, others are now available, and successful cAMP and cGMP measurements in the tiny (160-cell) tubule suggest that radioimmunoassay is sufficiently sensitive for most Drosophila tissues of interest (Kerr, 2004).
Obviously, the target cell must not normally express receptors for, or respond to, the ligand of choice. In the case of Drosophila, this condition is satisfied; there is no atrial natriuretic peptide-like sequence encoded within the Drosophila genome. For 5HT receptors or other GPCRs, more caution must be exercised; nonetheless, standard experimental controls would quickly identify any problems. In the case of 5HTDro1A, expression has only been documented in some cells of the embryonic nervous system; 5HTDro7 expression has been found in cells of the embryonic ventral midline, and in adult head, but not body. It is thus likely that these constructs will be useful in most Drosophila nonnervous tissues (Kerr, 2004).
This study demonstrates successful ectopic expression of vertebrate and Drosophila receptors in Malpighian tubules and quantifies the effects of such expression on signal transduction pathways and physiological output. There are clear results from this approach: (1) the interactions between 3 second messenger pathways were studied in unprecedented detail, in two cell types, in an organotypic context; (2) the results validated all that was known about signaling through known neuropeptides in the tubule; (3) a diuretic role for cAMP and cGMP in stellate cells was demonstrated, inviting the intriguing question as to which extracellular ligands normally activate these pathways; (4) this conserved pharmacology of a vertebrate receptor (rat GC-A) expressed in a model organism illustrates another possibility for functional genomics, i.e., that novel genes could be characterized relatively cheaply and easily by ectopic expression in a model organism where detailed organotypic-phenotypic analysis is possible. The Drosophila Malpighian tubule is an ideal such 'test bed' for genes where organotypic analysis may be important for normal function (Kerr, 2004).
The utility of this experimental system is more general, allowing sensitive and specific intervention in second messenger signaling in any Drosophila tissue for which there exists a GAL4 driver. Combined with the ready availability of real-time calcium reporters in Drosophila and the possibility of measuring cAMP and cGMP similarly, using transgenic FRET reporters, this simple model organism now has an impressive genetic toolbox for cell signaling studies (Kerr, 2004).
Cloning and characterization of 5-HT2B receptors
Serotonin [5-hydroxytryptamine (5-HT)] modulates feeding activity, egg-laying, and mating behavior in the free-living nematode, Caenorhabditis elegans. A novel receptor cDNA has been cloned from C. elegans (5-HT2Ce) that has high sequence homology with 5-HT2 receptors from other species. When transiently expressed in COS-7 cells, 5-HT2Ce exhibits 5-HT binding activity and activates Ca2+-mediated signaling in a manner analogous to other 5-HT2 receptors. However, 5-HT2Ce displays unusual pharmacological properties, which resemble both 5-HT2 and 5-HT1-like receptors but do not correlate well with any of the known 5-HT2 subtypes. Two splice variants of 5-HT2Ce that differ by 48 N-terminal amino acids were identified. The two isoforms have virtually identical binding and signaling properties but differ in their levels of mRNA expression, with the longer variant being four times more abundant than the shorter species in all developmental stages tested. Taken together, these results describe two variants of a novel C. elegans 5-HT receptor, which has some of the properties of the 5-HT2 family but whose pharmacological profile does not conform to any known class of receptor (Hamdan, 1999).
A G-protein-coupled receptor (5-HT2Lym) resembling members of the 5-HT2 receptor subfamily was cloned from the mollusc Lymnaea stagnalis. Serotonin induces a concentration-dependent increase in intracellular inositol phosphates in HEK293 cells expressing this receptor. 5-HT2Lym differs from mammalian 5-HT2 receptors by the presence of a large amino-terminal region. This large domain appears to preclude an adequate level of expression of 5-HT2Lym in HEK293. Therefore, a cDNA encoding an amino-terminally truncated receptor (delta N-5-HT2Lym) was constructed that appears to be much better expressed in HEK293 cells. delta N-5-HT2Lym-expressing cells exhibit a serotonin-induced stimulation of phosphatidylinositol bisphosphate hydrolysis and a high-affinity binding of the 5-HT2-selective antagonist [3H]mesulergine. Inhibition of this binding by several 5-HT2 antagonists and agonists reveals a pharmacological profile most closely resembling those of 5HT2Dro, 5-HT2B and 5-HT2C (Gerhardt, 1996).
The novel serotonin receptor 5-HT2B shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from 5-HT2B cDNA stably transfected LMTK- cell line, corresponds to a new 5-HT2-like receptor named 5-HT2B, although some differences exist between the mouse and rat pharmacology. A similar pharmacological profile is detected on the immortalized teratocarcinoma-derived cell line 1C11 upon 2 days of serotoninergic differenciation by cAMP. In both cell lines, the analysis 125I-DOI binding reveals the presence of a single class of sites, the affinity of which is one order of magnitude lower than the one reported for the 5-HT2A receptor. This demonstrates that the 5-HT2B receptor is functionally expressed before the complete serotoninergic differentiation of 1C11 cells. These observations are in good agreement with the presence of 5-HT2B mRNA in early mouse embryonic development. Furthermore, the major sites of 5-HT2B mRNA embryonic expression are in the heart and in the neural fold, before the closure of the neural tube. Therefore, this receptor could account, at least in part, for the trophic functions attributed to the 5-HT2-like receptors (Choi, 1994a).
The human serotonin 5-HT2B receptor, isolated from a human liver cDNA library, was transfected in COS-1 cells. Its pharmacological profile shows divergence with serotonin 5-HT2B receptors of other species. In particular, although strong correlation is observed between the human and the rat 5-HT2B receptor pharmacology, the correlation is almost as significant for the mouse 5-HT2B and the human 5-HT1D receptor agonists. The major sites of expression of its mRNA are in the human liver and kidney, with detectable expression in lung and heart. Therefore, this human 5-HT2B receptor could account for functions attributed to the peripheral 5-HT1D/5-HT2-like receptors, especially in the cardiovascular system. Thus, its detailed original pharmacology is of prime importance for therapeutic drug development (Choi, 1994b).
The recently characterized 5-HT2B subfamily of serotonin receptors has now been reported from three different species: human, rat and mouse. Their genomic structures include 2 introns present at identical positions. Despite this similarity, their respective protein sequences show some diversities. In addition, the pharmacology of these receptors is distantly related, and their sites of expression vary among species. Thus, it appears difficult at present to unambiguously classify these receptors into the same subfamily, raising the possibility of the existence of other 5-HT2B-like receptors, yet to be discovered (Choi, 1996b).
The 5-HT2B receptor is particularly interesting since it may be involved in diseases such as migraine. The isolation of a human 5-HT2B receptor clone from a cDNA library derived from SH-SY5Y cells is described. Although the receptor sequence was only 80% homologous to the rat sequence, the exon-intron distribution is conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels are found in the brain. After expression in HEK 293 cells, activation of the receptor stimulates the production of phosphatidylinositol. The pharmacology of this functional response correlates well with that of the rodent receptor (Schmuck, 1994).
5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by the culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin, suggesting autoreceptor functions for 5-HT2B receptors (Choi, 1998).
Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors has identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk- and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displays similar agonist affinities as the wild-type receptor. In Ltk- cells, the T149A 5-HT1A receptor inhibits cAMP accumulation by 30%, when compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) is observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor fails to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibits basal cAMP and partially inhibits Gs-stimulated cAMP accumulation, as compared with wild-type receptor (57 +/- 14% versus 86 +/- 2%). In contrast, the T149A 5-HT1A receptor mutant fails to block the influx of calcium induced by calcium channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibits the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by betagamma subunits of Gi2) and to inhibition of calcium channel activation (mediated by betagamma subunits of Go) but plays a minor role in coupling to alpha1-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors (Lembo, 1997).
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes [5-HT2A, 5-HT(2B) and 5-HT(2C)] that are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Transcripts encoding the 2C subtype of serotonin receptor [5-HT(2C)R] undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily (Burns, 1997).
Serotonin-2 receptor antagonists, like ritanserin, greatly enhance deep slow wave sleep (SWS-2) and low-frequency EEG power in humans and rodents. 5-HT2A and 5-HT2C receptors may be involved in these effects, but the role of the 5-HT2B receptor is still unclear. To investigate the role of the 5-HT2B receptor in regulation of the sleep-wake cycle, the subtype-selective antagonist SB-215505 was administered to Sprague-Dawley rats at light onset (beginning of passive phase). EEG, EMG and motor activity were recorded during the subsequent 8 h. SB-215505 dose-dependently increased wakefulness (W) at the expense of the intermediate stage of sleep, paradoxical sleep (PS) and SWS-2 in the first hour. Parallel to increased W, significantly increased motor activity was found. Spectral analysis of the EEG in W showed a dose-dependent decrease in power density in the 3-8 Hz frequency range (maximum effect at 6 Hz). In light slow wave sleep and SWS-2, the drug reduced low-frequency (<8 Hz) EEG power, suggesting decreased sleep intensity after SB-215505 treatment. In PS, the drug dose-dependently decreased EEG power solely in the theta (6-9 Hz) band, primarily affecting the peak power value (7 Hz). The well-known SWS-2 enhancing effect of 5-HT2 receptor antagonists is mediated by 5-HT2A and/or 5-HT2C receptors. In contrast, blockade of 5-HT2B receptors increases motor activity and W along with decreased theta activity during W and PS. Activation of 5-HT2B receptors may contribute to initiation of sleep and to theta generation during W and PS under physiological conditions (Kantor, 2004).
Serotonin plays key roles in sleep-wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. This study investigated the relative contribution of 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT2A+/+) and knock-out (5-HT2A-/-) mice that do not express 5-HT2A receptors. Acute blockade of 5-HT2A receptors induced an increase in NREMS in 5-HT2A+/+ mice, but not 5-HT2A-/- mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT2A+/+ mice, 5-HT2B receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT2A-/- mice, indicating a lower sensitivity of 5-HT2B receptors in mutants, with no change in 5-HT2B mRNA. Blockade of 5-HT2C receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT2A+/+ mice but not in 5-HT2A-/- mice. Whole-body plethysmographic recordings indicated that 5-HT2A receptor blockade in 5-HT2A+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT2A-/- mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. These results demonstrate that 5-HT exerts a 5-HT2B-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT2A receptors. Moreover, 5-HT2A gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT2A receptors in 5-HT2A+/+ mice (Popa, 2005).
Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. This study used recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or 'primed' by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations approximately 10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. These results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders (Bhattacharyya, 2006)
The family of serotonin 5-HT2 receptors stimulates the phospholipase C second messenger pathway via the alpha subunit of the Gq GTP-binding protein. Agonist stimulation of the 5-HT2B receptor subtype stably expressed in the mouse fibroblast LMTK- cell line causes a rapid and transient activation of the proto-oncogene product p21ras, as measured by an increase in GTP-bound Ras in response to serotonin. Furthermore, 5-HT2B receptor stimulation activates p42mapk/p44mapk (ERK2/ERK1) mitogen-activated protein kinases, as assayed by phosphorylation of myelin basic protein. Antibodies against p21ras, Galphaq, -beta, or -gamma2 subunits of the GTP-binding protein inhibit MAP kinase-dependent phosphorylation. The MAP kinase activation is correlated with a stimulation of cell division by serotonin. In addition to this mitogenic action, transforming activity of serotonin is mediated by the 5-HT2B receptor since its expression in LMTK- cells is absolutely required for foci formation and for these foci to form tumors in nude mice. Expression of the 5-HT2B receptor was detected in spontaneous human and Mastomys natalensis carcinoid tumors and, similar to the 5-HT2B receptor transfected cells, the Mastomys tumor cells are also responsive to serotonin with similar coupling to p21ras activation (Launay, 1996).
Serotonin (5-HT) is a potent mitogen in many cells types, an action that is frequently mediated through pertussis toxin-sensitive G proteins. Pharmacological inhibitors and dominant negative signaling constructs have been used to delineate elements that participate in the activation of MAPK, a growth-associated mitogen-activated protein kinase, by human G protein-coupled 5-HT1A receptor transfected into CHO-K1 cells in a stable manner. The activation pathway does not directly involve phorbol ester-sensitive protein kinase C types, but does require (1) pertussis toxin-sensitive G protein beta gamma-subunits; (2) a staurosporine- and genistein-sensitive protein kinase; (3) phosphoinositide-3'-kinase activity; (5) activation of Sos in a multimolecular complex that contains p46Shc, and p52Shc, and Grb2; (5) the GTPase p21Ras, and (6) the protein kinase p74Raf-1. These data demonstrate that the 5-HT1A receptor mediates MAPK activity by convergence upon a common activation pathway that is shared with receptor tyrosine kinases (Garnovskaya, 1996).
Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation is attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation. This is because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src, but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase have no effect on 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis is required for 5-HT1A receptor-mediated Erk1/2 activation, a role for CAM in receptor endocytosis is postulated. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuates 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevents agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. It is concluded that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects CAM's role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2 (Della Rocca, 1999).
5-HT2B receptors and lipid signaling
Serotonin receptor 2 Evolutionary homologs part 2/3 | part 3/3
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