Mutational analysis of the serotonin receptor 5HT2c in severe early-onset human obesity

Deletion of the serotonin receptor 5HT2c in mice results in increased food intake and obesity. We screened 95 individuals with severe early-onset obesity for mutations in the coding sequence of this gene. We found a novel missense variant c.1255A > G (Thr419Ala) in a single Caucasian subject that was not found in 192 Caucasian control subjects. In transiently-transfected COS cells, the Thr419Ala variant was indistinguishable from the wild-type receptor in its ability to generate inositol phosphate, although differences in coupling to other pathways were not excluded. Three previously unreported silent variants: IVS3 + 30G > A, IVS3 + 80C > G and IVS4 - 31A > G were found with prevalences of 11.5%, 0.5% and 17.9%, respectively. In conclusion, mutations in 5HT2c are unlikely to be a common cause of severe early-onset human obesity. The identification of several novel polymorphisms at this locus may aid future genetic epidemiological studies.     

The serotonin receptor SER-1 (5HT2ce) contributes to the regulation of locomotion in Caenorhabditis elegans

Serotonin (5-hydroxytryptamine: 5HT) is an important neuroactive substance in the model roundworm, Caenorhabditis elegans. Aside from having effects in feeding and egg-laying, 5HT inhibits motility and also modulates several locomotory behaviors, notably food-induced slowing and foraging. Recent evidence showed that a serotonergic 5HT2-like receptor named SER-1 (also known as 5HT2ce) was responsible for the effect of 5HT on egg-laying. Here we confirm this observation and show that SER-1 also plays an important role in locomotion. A mutant lacking SER-1 was found to be highly resistant to exogenous 5HT in the absence of food and this resistant phenotype was rescued by reintroducing the SER-1 gene in a mutant background. Pharmacological studies showed that the same antagonists that blocked the activity of recombinant SER-1 in vitro also inhibited the effect of 5HT on motility, suggesting the same receptor was responsible for both effects. When tested for locomotory behaviors, the SER-1 mutant was found to be moderately defective in food-induced slowing. In addition, the mutant changed direction more frequently than the wildtype when searching for food, suggesting that SER-1 may play a role in navigational control during foraging. Both these effects required the presence of MOD-1, a 5HT gated chloride channel, and the results indicate that SER-1 and MOD-1 modulate these behaviors through a common pathway. On the basis of expression analysis of a ser-1::GFP translational fusion, SER-1 is prominently located in central, integrating neurons of the head ganglia (RIA and RIC) but not the body wall musculature. The evidence suggests that SER-1 controls locomotion through indirect modulation of neuromuscular circuits and has effects both on speed and direction of movement.     

Association analysis of the catechol-o-methyltransferase (COMT), serotonin transporter (5-HTT) and serotonin 2A receptor (5HT2A) gene polymorphisms with obsessive-compulsive disorder

Family and twin studies have supported a strong genetic factor in the etiology of obsessive-compulsive disorder (OCD), although the precise mechanism of inheritance is unclear. Clinical and pharmacological studies have implicated the serotonergic and dopaminergic systems in disease pathogenesis. In this cross-sectional study, we have examined the allelic and genotypic frequencies of a Val-158-Met substitution in the COMT gene, a 44-base pair (bp) length variation in the regulatory region of the serotonin transporter gene ( 5-HTTLPR ) and the T102C and C516T variants in the serotonin receptor type 2A ( 5HT2A ) gene in 79 OCD patients and 202 control subjects. There were no observed differences in the frequencies of allele and genotype between patients and control groups for the COMT , the 5HTTLPR and the T102C 5HT2A gene polymorphisms. In contrast, a statistically significant difference between OCD patients and controls was observed on the genotypic distribution (χ² = 16.7, 2df, P  = 0.0002) and on the allelic frequencies (χ² = 15.8, 1df, P  = 0.00007) for the C516T 5HT2A gene polymorphism. The results suggest that the C516T variant of the 5HT2A gene may be one of the genetic risk factors for OCD in our sample. However, further studies using larger samples and family based methods are recommended to confirm these findings.     

Serotonin receptor genes 5HT1A and 5HT2A modify the relation between childhood temperament and adulthood hostility

We examined a modifying role of 5HT1A and 5HT2A receptors in the relation between childhood difficult temperament and adulthood hostility in 729 subjects derived from a population-based sample. Subjects were 3–12 years when their childhood temperaments consisting of hyperactivity, low sociability and negative emotionality (i.e. the difficult temperament), were assessed by their mothers. Their adulthood hostility comprising anger, cynicism and paranoia, was measured twice, 17 and 21 years later. It was found that the 5HT1A and 5HT2A receptors were not related to childhood temperament or to adult hostility, but they modified the association between childhood hyperactivity and adult hostility in men. Male carriers of T/T genotype of 5HTR2A who were rated hyperactive by their mothers expressed a high level of hostility, especially that of cynicism, in adulthood. For men with other genetic variants, such an association was not seen. This finding was consistent across the two follow-ups 4 years apart. Further research is needed to clarify whether mother-related hyperactivity adequately describes the temperament of the child or is a reflection of mother's hostile child-rearing attitudes.     

A-to-I editing of the 5HT2C receptor and behaviour.

Site-specific deamination of five adenosine residues in the pre-mRNA of the serotonin 2C receptor, 5HT2CR, alters the amino acid sequence of the encoded protein. Such RNA editing can produce 32 mRNA variants, encoding 24 protein isoforms that vary in biochemical and pharmacological properties. Because serotonin functions in the regulation of mood and behaviour, modulation of serotonin signalling by RNA editing may be relevant to such psychiatric disorders as anxiety and depression. Several recent human studies have reported changes in 5HT2CR editing in schizophrenia, major depression or suicide, but results are variable and not conclusive. Rodent studies have begun to examine effects of drug treatments and stress. Understanding the importance of 5HT2CR editing in mood and behaviour will be assisted by experiments designed to analyse multiple strains of mice, in different behavioural tests, with optimal evaluation of the time course of molecular changes.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Functional characterization of a novel serotonin receptor (5-HTap2) expressed in the CNS of Aplysia californica

Serotonin has been shown to be a neuromodulator in the Aplysia californica CNS. The diversity of serotonin actions is due to the existence of several different receptor subtypes. In this study we report the cloning of a full-length cDNA, coding for a novel serotonin receptor (5-HTap2). The receptor protein bears the characteristics of G protein-coupled receptors. It shares 68% and 34% of its amino acid sequence identity with the 5-HTlym receptor from Lymnaea stagnalis and the mammalian 5-HT1A receptor, respectively. When transfected in HEK 293 cells, 5-HTap2 was negatively coupled to adenylate cyclase. Ligand binding analysis indicated that the order of potencies of various drugs for the inhibition of [3H]LSD binding was: methiothepin > metergoline > 5-CT > PAPP > 5-HT > ketanserin > NAN-190 > 8-OH-DPAT > clozapine. RT-PCR amplification of RNA isolated from different tissues indicated that this receptor is expressed in the CNS and in bag cells. The expression of 5-HTap2 restricted to the CNS suggests an important role for this receptor in the modulation of neuronal functions in Aplysia. Moreover, the high expression of 5-HTap2 in the bag cells, associated with its pharmacological profile, suggests that this receptor may be implicated in modulating the afterdischarge during the egg-laying behavior.     

Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT_2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT_2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.     

Expression of the mouse serotonin receptor (5HT1c) cDNA in cultured insect cells

Baculovirus-mediated cloning and expression of the mouse serotonin receptor (5HT1c) cDNA in insect cells was proposed to obtain an alternative to an oocyte-based system, which is commonly employed in electrophysiological studies of ionic channels. A recombinant bacmid was constructed, and the 5HT1c cDNA transferred into the AcNPV genome to yield a recombinant baculovirus. Infected inset Sf9 cells produced recombinant 5HT1c.     

昆虫血清素受体蛋白的生物信息学分析

【目的】昆虫血清素(5-羟色胺)受体已知有5个亚型。本文旨在系统分析昆虫5-羟色胺受体亚型蛋白的结构和进化关系。【方法】首先对文献报道已明确亚型7种昆虫的5-羟色胺受体(23个亚型序列)进行生物信息学分析,然后采用多序列比对和进化树构建的方法对NCBI数据库中推测可能为昆虫5-羟色胺受体蛋白序列进行分析。【结果】发现47个推测是昆虫5-羟色胺受体的蛋白序列中,有40个蛋白序列属于昆虫5-羟色胺受体,其余7个未能确认的昆虫5-羟色胺受体的蛋白序列都具有7个跨膜区域,属于G蛋白偶联受体家族,但不一定为5-羟色胺受体。【结论】本文对昆虫5-羟色胺受体蛋白的系统进化树分析,间接地证明了本文确认的昆虫5-羟色胺受体亚型注释信息的准确性,发现分类上同属一个目的昆虫5-HT受体序列的亲缘性较近。本研究为昆虫5-羟色胺受体的结构和功能分析提供基础。     

Exploration of bivalent ligands targeting putative mu opioid receptor and chemokine receptor CCR5 dimerization

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR–CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5–MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5–MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation.     

Mutagenesis analysis of the serotonin 5-HT2C receptor and a Caenorhabditis elegans 5-HT2 homologue: conserved residues of helix 4 and helix 7 contribute to agonist-dependent activation of 5-HT2 receptors

An alignment of serotonin [5-hydroxytryptamine (5-HT)] G protein-coupled receptors identified a lysine at position 4.45 (helix 4) and a small polar residue (serine or cysteine) at 7.45 (helix 7) that occur exclusively in the 5-HT2 receptor family. Other serotonin receptors have a hydrophobic amino acid, typically a methionine, at 4.45 and an invariant asparagine at 7.45. The functional significance of these class-specific substitutions was tested by site-directed mutagenesis of two distantly related 5-HT2 receptors, Caenorhabditis elegans 5-HT2ce and rat 5-HT2C. Residues 4.45 and 7.45 were each mutated to a methionine and asparagine, respectively, or an alanine and the resulting constructs were tested for activity. A K4.45M mutation decreased serotonin-dependent activity (Emax) of the rat 5-HT2C receptor by 60% and that of the C. elegans homologue by 40%, as determined by a fluorometric plate-based calcium assay. The rat mutant also exhibited nearly sixfold higher agonist binding affinity and significantly lower constitutive activity compared with wildtype. Mutagenesis of S7.45 in the C. elegans receptor increased serotonin binding affinity by up to 25-fold and decreased Emax by up to 65%. The same mutations of the cognate C7.45 in rat 5-HT2C produced a smaller fourfold change in the affinity for serotonin and decreased agonist efficacy by up to 50%. Substitutions of S/C7.45 did not produce a significant change in the basal activity of either receptor. All mutants tested exhibited levels of receptor expression similar to the corresponding wildtype based on measurements of specific [3H]-mesulergine binding or flow cytometry analyses. Taken together, these results suggest that K4.45 and S/C7.45 play an important role in the conformational rearrangements leading to agonist-induced activation of 5-HT2 receptors.     

The role of 5‐HT2c receptor on corticosterone‐mediated food intake

Corticosterone plays an important role in feeding behavior. However, its mechanism remains unclear. Therefore, the present study aimed to investigate the effect of corticosterone on feeding behavior. In this study, cumulative food intake was increased by acute corticosterone administration in a dose‐dependent manner. Administration of the 5‐HT_2c receptor agonist m‐chlorophenylpiperazin (mCPP) reversed the effect of corticosterone on food intake. The anorectic effects of mCPP were also blocked by the 5‐HT_2c receptor antagonist RS102221 in corticosterone‐treated mice. Both corticosterone and mCPP increased c‐Fos expression in hypothalamic nuclei, but not the nucleus of the solitary tract. RS102221 inhibited c‐Fos expression induced by mCPP, but not corticosterone. In addition, mCPP had little effect on TH and POMC levels in the hypothalamus. Furthermore, mCPP antagonized decreasing effect of the leptin produced by corticosterone. Taken together, our findings suggest that 5‐HT_2c receptors and leptin may be involved in the effects of corticosterone‐induced hyperphagia.     

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology

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Opposing actions of 5HT1A and 5HT2-like serotonin receptors on modulations of the electric signal waveform in the electric fish Brachyhypopomus pinnicaudatus

Serotonin (5-HT) is an indirect modulator of the electric organ discharge (EOD) in the weakly electric gymnotiform fish, Brachyhypopomus pinnicaudatus. Injections of 5-HT enhance EOD waveform "masculinity", increasing both waveform amplitude and the duration of the second phase. This study investigated the pharmacological identity of 5-HT receptors that regulate the electric waveform and their effects on EOD amplitude and duration. We present evidence that two sets of serotonin receptors modulate the EOD in opposite directions. We found that the 5HT1AR agonist 8-OH-DPAT diminishes EOD duration and amplitude while the 5HT1AR antagonist WAY100635 increases these parameters. In contrast, the 5HT2R agonist alpha-Me-5-HT increases EOD amplitude but not duration, yet 5-HT-induced increases in EOD duration can be inhibited by blocking 5HT2A/2C-like receptors with ketanserin. These results show that 5-HT exerts bi-directional control of EOD modulations in B. pinnicaudatus via action at receptors similar to mammalian 5HT1A and 5HT2 receptors. The discordant amplitude and duration response suggests separate mechanisms for modulating these waveform parameters.     

Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor

The human adenosine A_2A receptor (A_2AR) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [θ]₂₂₂/[θ]₂₀₈ obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A_2AR at position M193, which is located in the fifth TM domain, noticeably alters A_2AR monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A_2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.     

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors

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5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.